Alcohol abuse and high fat diet induced liver diseases have been the most prevalent chronic liver diseases and the leading reasons for liver transplantation around the world. Cannabidiol (CBD) is a botanical component extracted from marijuana plants without psychoactive impact. In our previous reports, CBD can prevent fatty liver induced by Lieber DeCarlie ethanol diet or non-alcoholic fatty liver disease (NAFLD) induced by high fat high cholesterol diet. The current study is a further study on whether CBD can alleviate liver injuries induced by ethanol plus high fat high cholesterol diet (EHFD), which is a model simulating heavy alcohol drinkers in a western diet. A mice liver injury model induced by EHFD for 8 weeks was applied to explore the protective properties of CBD and the underlying mechanisms. We found that CBD prevented liver steatosis as well as oxidative stress induced by EHFD. CBD treatment inhibited macrophage recruitment, and suppressed activation of NFκB-NLRP3-pyroptosis pathway in mouse livers. The hepatoprotective property of CBD in the current model might be a result of inhibition of inflammation via alleviating activation of hepatic NFκB-NLRP3 inflammasome-pyroptosis pathway by CBD. Food industry news, voices and jobs. Optimized for your mobile phone. Cannabidiol improves brain and liver function in a fulminant hepatic failure-induced model of hepatic encephalopathy in mice Hepatic encephalopathy is a neuropsychiatric disorder of complex
CBD Alleviates Liver Injuries in Alcoholics With High-Fat High-Cholesterol Diet Through Regulating NLRP3 Inflammasome–Pyroptosis Pathway
Alcohol abuse and high-fat diet–induced liver diseases have been the most prevalent chronic liver diseases and the leading reasons for liver transplantation around the world. Cannabidiol (CBD) is a botanical component extracted from marijuana plants without psychoactive impact. In our previous reports, we found that CBD can prevent fatty liver induced by Lieber–DeCarli ethanol diet or non-alcoholic fatty liver disease (NAFLD) induced by high-fat high-cholesterol diet. The current work is a further study on whether CBD can alleviate liver injuries induced by ethanol plus high-fat high-cholesterol diet (EHFD), which is a model simulating heavy alcohol drinkers in a Western diet. A mice liver injury model induced by EHFD for 8 weeks was applied to explore the protective properties of CBD and the underlying mechanisms. We found that CBD prevented liver steatosis and oxidative stress induced by EHFD. CBD treatment inhibited macrophage recruitment and suppressed activation of NFκB–NLRP3–pyroptosis pathway in mice livers. The hepatoprotective property of CBD in the current model might be a result of inhibition of inflammation via alleviating activation of the hepatic NFκB–NLRP3 inflammasome–pyroptosis pathway by CBD.
Prolonged heavy consumption of alcohol-induced liver injuries accounts for the increasing morbidity and mortality rates worldwide (Xu et al., 2017). These liver injuries are characterized by a spectrum of progressing liver disorders, including simple fatty liver, alcoholic hepatitis, cirrhosis, and hepatocellular carcinoma (Seitz et al., 2018). Individuals abusing alcohol are usually accompanied with a superfluous calorie intake which will raise their predisposition of developing a metabolic disorder such as obesity, metabolic syndrome, and type 2 diabetes mellitus (T2DM) (Boyle et al., 2018). It has been recognized for many years that chronic heavy alcohol drinking can have synergistic or exacerbating effects on obesity and metabolic syndrome, which can further promote the formation of fatty Rinella (2015a) liver and aggravate the progression of ALD (Boyle et al., 2018) (Rinella, 2015b). Haein et al. proved that significant hepatic damage can be caused by alcohol administration for four weeks with increased hepatic lipid droplets and inflammatory cytokine levels and can be aggravated with the combination of high-fat diet (Kim et al., 2016). In the present study, we applied chronic administration of alcohol with a high-fat high-cholesterol diet (EHFD) to induce hepatic injury in mice.
Inflammation plays an important role in the formation and progress of liver damage induced either by alcohol or high-fat diet. Pyroptosis is an inflammatory process of caspase-1–dependent programmed cell death. In pyroptosis, the inflammasome and some other signals are activators of caspases. In general, pyroptosis starts with inflammasome formation recognizing various exogenous and endogenous signals, including LPS and ATP, and caspase-1 is subsequently activated. The activation of caspase-1 by the NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome leads to the cleavage of pro-inflammatory cytokine interleukin-1 beta (IL-1β) and pro-interleukin 18 (IL-18) and the production of mature IL-1β and IL-18. In addition, activated caspase-1 cleaves the pyroptotic substrate gasdermin D (GSDMD) and forms the membrane pores and induces pyroptosis, allowing the release of IL-1β and IL-18.
In canonical pyroptosis, inflammasome formation is an initiation step for pyroptosis activation. The inflammasome is an inflammatory signal platform, which can react with exogenous and endogenous signals and initiate a series of inflammatory responses (Fabio et al., 2002). The inflammasome is a cytoplasmic complex, mainly composed of three different functional proteins: sensor proteins, including a nucleotide-binding oligomerization domain (NOD)–like receptors (NLRs) family members; the adapter protein comprised apoptosis-associated speck-like protein containing a casp recruitment domain (ASC)— the effective protein pro-caspase-1 (Fabio et al., 2002; Benetti et al., 2013). Among the inflammasomes, studies have highlighted the importance of the NLRP3 inflammasome in many inflammatory disorders (Benetti et al., 2013), such as ALD, obesity (Mastrocola et al., 2018), type 2 diabetes (Luo et al., 2017), non-alcoholic fatty liver disease (NAFLD) (Wan et al., 2016; Cabrera et al., 2017), and metabolic syndrome (Mastrocola et al., 2018). The NLRP3 inflammasome can be activated by endogenous or exogenous factors, including pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), pore-forming toxins, and environmental irritants (Fabio et al., 2002). Nuclear factor κB (NF-κB) has been demonstrated as one of key factors activating the NLRP3 inflammasome. Upon activation of the NLRP3 inflammasome, caspase-1 is activated. Activated caspase-1 would cleave pro-IL-1β into mature IL-1β, resulting in release of IL-1β, inflammation, and cell and tissue damage (Fabio et al., 2002). In addition, activated caspase-1 cleaves GSDMD and forms membrane pores, which is a key step for pyroptosis, allowing the release of IL-1β and IL-18.
Excessive activation of inflammatory caspases is implicated in the pathogenesis of alcoholic liver disease and NASH, and pyroptosis is the dominant response following this activation (Jorgensen and Miao, 2015). Heo et al. proved that pyroptosis occurs in hepatocytes with alcohol exposure as NLRP3, ASC, and caspase-1 were all upregulated with ethanol (Heo et al., 2019). Numerous studies have shown that pyroptosis is an inflammatory link between simple steatosis and NASH, since no NLRP3 activation was observed in simple steatosis without inflammation, while NLRP3 activation in NASH has been shown in both human and animal models (Csak et al., 2011; Beier and Banales, 2018). Moreover, researchers had proved that the pyroptosis-inducing fragment GSDMD expression was higher in NASH and that IL-1β released following pyroptosis is the driver of liver inflammation and fibrosis (Tilg and Moschen, 2011; Shi et al., 2015).
Cannabidiol (CBD), a non-psychomimetic compound extracted from Cannabis sativa, exerts a broad range of pharmacological properties, including cardiac and neural protection, antioxidant and anti-inflammatory capabilities, and anti-epileptic potential (Yang et al., 2014; Wang et al., 2017; Huang et al., 2019). Although CBD can cross the blood–brain barrier, it does not cause psychoactive effects that lack abuse potential. CBD has been shown to reduce the inflammation in cultured human sebocytes and human skin organ culture through coupling to A2A adenosine receptor–dependent upregulation of tribbles homolog 3 (TRIB3) and inhibition of NF-κB signaling (Oláh et al., 2014). Moreover, the intervention of CBD delayed the onset of type 1 diabetes in non-obese diabetic mice and alleviated pancreas inflammation (Lehmann et al., 2016). It has also been shown that the administration of CBD targeted NF-κB and NLRP3 inflammasome pathways in macrophages, which could be a novel treatment for NASH (Mridha et al., 2017). In addition, CBD treatment suppressed hepatic lipid accumulation and attenuated oxidative stress and inflammatory response induced by excess energy diet Huang et al. (2019) or alcohol intake (Wang et al., 2017). Recent studies have shown that CBD was able to reduce accumulation of intracellular lipid levels (Silvestri et al., 2015) and cell death (Mukhopadhyay et al., 2011) in dose- and time-dependent manners. It is still elusive whether CBD can protect liver injury induced by EHFD, and the related mechanisms are unclear. Here, we aim to explore the potentiality of CBD for mitigating liver inflammation induced by EHFD, as well as the mechanism therein, focusing on the NLRP3 inflammasome–pyroptosis pathway in macrophages.
Materials and Methods
Cannabidiol (CBD) was from Tocris Bioscience (Ellisville, MO, United States, 1570/10), with purity ≥98% (HPLC). TRIzol was supplied by Invitrogen (Carlsbad, CA, United States). The PrimeScript RT Reagent Kit with a gDNA eraser and SYBR Premix Ex TaqII kit were from TaKaRa (Tokyo, Japan). Lysis buffer for Western blot and a BCA protein assay kit were purchased from Beyotime Biotechnology Company (Shanghai, China). Anti-pIκBα (9246s), anti-IκBα (4812s), anti-pNF-κB p65 (3033s), anti-NF-κB p65 (8242s), anti-ASC (#67824), and anti-IL-1β (#12242) were from Cell Signaling Technology. Anti-NLRP3 (ab214185) and anti-tubulin (ab6160) were obtained from Abcam. Anti–pro-caspase-1 (22915-1-AP) and anti-GSDMD (20770-1-AP) were obtained from Proteintech Group Inc. (United States). Anti-GAPDH (sc25778), anti–caspase-1 p20 (sc25778), and all secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States).
Animal Experimental Protocol
Six-week-old male C57B/6J mice were supplied by the Sun Yat-sen University Animal Center (Guangzhou, China). Animal experiments were approved by the Animal Experimentation Ethics Committee of Sun Yat-sen University, complied with the ARRIVE guidelines and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All mice were kept at a specific pathogen‐free environment (temperature 20–22°C, humidity 60%) with 12-hour light/dark cycle and free access to food and water. After two weeks of acclimatization, the mice were randomly assigned to the following three groups: 1) the control group, received a standard chow diet (5% fat w/w); 2) the EHFD group, received a high-fat high-cholesterol diet (containing 17% fat and supplemented with 1.25% cholesterol and 0.5% cholate) which was obtained from Trophic Animal Feed High-tech Co. Ltd. (China) Gäbele et al. (2011), with ad libitum access to alcohol in drinking water with increasing concentrations of alcohol (1% v/v of alcohol for the first 2 days, 2% from day 3 to day 7, 4% for the second week, and 5% for another 6 weeks); and 3) the EHFD + CBD group, on the basis of EHFD group, mice were gavaged with 5 mg/kg BW CBD solution per day for 8 weeks. The control and EHFD mice were given saline at equivalent volumes. The animal experiment protocol is shown in Figure 1A. The mice were euthanized at the age of 16 weeks after they were fasted for 16 h, and the serum and liver tissue were harvested for subsequent measurement.
FIGURE 1. CBD treatment ameliorated steatohepatitis induced by EHFD diet. Mice were fed ethanol plus high-fat high cholesterol diet for 8 weeks with or without CBD treatment. Control mice were fed a standard chow diet. The animal experiment protocol (A). Representative H&E staining of liver sections (magnification ×200) (B). Levels of hepatic TG (C) and TC (D). Levels of serum ALT (E) and AST (F). Values represent mean ± SEM (n = 6–8/group). *p < 0.05 vs the control group; #p < 0.05 vs the EHFD group.
The serum was collected after 3000×g centrifugation at 4°C for 15 min. Levels of serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), hepatic triglyceride (TG), and hepatic total cholesterol (TC) were determined by corresponding kits (Jiancheng Biotech, Nanjing, China) according to the manufacturer protocol. Hepatic malondialdehyde (MDA) levels, glutathione/glutathione disulfide (GSH/GSSG) levels, and activity of superoxide dismutase (SOD) were determined by using ELISA kits (Beyotime, Shanghai, China). The values were normalized by protein content.
Histology and Immunohistochemical Analysis
Western Blot Analysis
The proteins extracted from the liver tissues were analyzed by Western blot. The liver tissues were homogenized using RIPA (radioimmunoprecipitation assay) lysis buffer supplemented with protease and phosphatase inhibitors. The proteins were separated by 8–12% SDS-PAGE, followed by transferring to a 0.22/0.45-mm polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, United States). The membranes were pre-blocked with 5% (w/v) fat-free milk in phosphate-buffered saline containing 0.05% (v/v) Tween-20 (TBST). The membrane was incubated at 4°C overnight with specific primary antibodies and subsequently incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. The ECL Reagent Kit (Thermo) was used to determine signals. Primary antibodies included anti-pIκBα, anti-IκBα, anti-NF-κB p65, anti-pNF-κB p65, anti-NLRP3, anti-ASC, anti–pro-caspase-1, anti–caspase-1 p20, anti-GSDMD, anti-IL-1β, anti-GAPDH, and anti-tubulin. The band intensities were analyzed by ImageJ software.
Quantitative Real-Time Polymerase Chain Reaction
Total RNAs were extracted from livers with TRIzol reagent and reverse-transcribed into cDNA with a PrimeScript RT Reagent Kit. Gene expression was determined by quantitative real-time PCR with an SYBR Premix Ex Taq II kit and signaling detected by a Vii7 system (ABI, Carlsbad, CA, United States). Expression of each target gene was normalized to the internal standard (β-actin) by using the 2 −ΔΔCt comparative method. Primer sequences are listed in Table 1.
TABLE 1. Primer sequences for quantitative real-time polymerase chain reaction.
All statistical analyses were performed with SPSS 20.0, and a p value < 0.05 was considered statistically significant. Data were expressed as means ± SE analyzed by Student’s t-test (for two groups) or one-way ANOVA (for more than two groups), followed by the Bonferroni test.
CBD Treatment Ameliorated Steatohepatitis Induced by EHFD Diet
As shown in Figure 1B, EHFD diet resulted in marked hepatocellular lipid accumulation with obvious inflammatory cell infiltration in mice liver as compared with those in control, while CBD treatment markedly decreased lipid droplets and inflammatory lesions induced by EHFD in H&E-stained liver sections. Correspondingly, we found that the levels of TG and TC in the liver increased in the EHFD group (Figures 1C, D), with significantly increased serum ALT and AST levels (Figures 1E, F), indicating the development of steatohepatitis and the damage of liver cells in EHFD treatment. CBD treatment significantly inhibited hepatic steatohepatitis and liver injury induced by EHFD diet.
CBD Treatment Alleviated Oxidative Stress Induced by EHFD Diet
Excess of oxidative stress contributes to cellular injury and the development of non-alcoholic and alcoholic liver disease. To evaluate the effect of CBD on oxidative stress, we measured the activity of enzymes that maintain redox homeostasis and the level of lipid peroxidation product in mice liver. Figures 2A, B show that the mice in the EHFD group displayed significantly increased contents of MDA and a decreased ratio of GSH/GSSG, whereas these alterations were inhibited in the presence of CBD. Moreover, EHFD decreased hepatic activity of SOD, which was slightly increased by CBD treatment, with no statistical significance compared to the EHFD group (Figure 2C).
FIGURE 2. CBD treatment alleviated oxidative stress induced by EHFD diet. Mice were fed ethanol plus high-fat high cholesterol diet for 8 weeks with or without CBD treatment. Control mice were fed a standard chow diet. Levels of hepatic MDA (A). The ratio of hepatic GSH/GSSG (B). Activity of hepatic SOD (C). Values represent mean ± SEM (n = 6–8/group). *p < 0.05 vs the control group; #p < 0.05 vs the EHFD group.
Inflammation Induced by EHFD Diet Was Ameliorated by CBD Treatment
The H&E-stained liver sections revealed that CBD treatment decreased hepatic inflammation in EHFD mice. To further verify the anti-inflammatory effect of CBD, we assessed the expression of CD68 (macrophage marker) in mice liver. Immunohistochemistry staining of liver sections indicated that number of CD68-positive cells was increased in the EHFD group, suggesting an increased inflammatory cell infiltration. The expression of CD68 was blunted in the CBD group (Figure 3A). Consistently, gene expressions of the inflammatory markers such as F4/80, IL-1β, monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor alpha (TNF-α) were increased in the EHFD group, while these gene expressions were significantly reduced with CBD treatment (Figures 3B–E). These results indicated that administration of CBD alleviated the inflammatory response induced by EHFD.
FIGURE 3. Inflammation induced by EHFD diet was ameliorated by CBD treatment. Mice were fed ethanol plus high-fat high cholesterol diet for 8 weeks with or without CBD treatment. Control mice were fed standard chow diet. Immunohistochemistry staining was performed to determine CD68 + macrophages (magnification ×200 and ×400) (A). The mRNA expressions of F4/80 (B), IL-1β (C), MCP-1 (D), and TNF-α (E) were analyzed by qPCR. Values represent mean ± SEM (n = 6–8/group). *p < 0.05 vs the control group; #p < 0.05 vs the EHFD group.
CBD Treatment Inhibited the Activation of NF-κB Pathway Induced by EHFD Treatment
NF-κB is a key transcription factor that controls the expression of pro-inflammatory genes. To determine the possible involvement of the NF-κB pathway in the anti-inflammatory effect of CBD, we examined IκBα and NF-κB in the liver samples by Western blot. As shown in Figure 4, IkBa was decreased due to its phosphorylation in the EHFD group, EHFD treatment increased the expressions of phosphorylated IκBα and NF-κB, and the ratios of pIκBα/IκBα and pNF-κB/NF-κB increased, indicating the activation of the NF-κB pathway by EHFD. CBD treatment decreased the expressions of pIκBα, pNF-κB, the ratios of pIκBα/IκBα, and pNF-κB/NF-κB, showing the inhibition effect of CBD on NF-κB activation, suggesting the effect of CBD on EHFD-induced inflammation is associated with the activation of the NF-κB pathway.
FIGURE 4. CBD treatment inhibited NF-κB pathway activation induced by the EHFD diet. Mice were fed ethanol plus high-fat high-cholesterol diet for 8 weeks with or without CBD treatment. Control mice were fed a standard chow diet. Western blot analyses of pIκBα, IκBα, pNF-κB p65, and NF-κB p65 (A). Quantification of expressions of IκBα, pIκBα, and pNF-κB p65 (B). Values represent mean ± SEM (n = 6–8/group). *p < 0.05 vs the control group; #p < 0.05 vs the EHFD group.
CBD Treatment Suppressed NLRP3 Inflammasome Activation and Pyroptosis in EHFD-Fed Mice
NLRP3 inflammasome activation may lead to GSDMD-driven pyroptosis, which plays a key role in the occurrence and development of liver diseases (Guo et al., 2019). We explored whether CBD could attenuate NLRP3 inflammasome activation and the subsequent pyroptosis in livers. As shown in Figures 5A, B, EHFD diet significantly increased NLRP3, ASC, and caspase-1 p20 protein levels, which indicates NLRP3 inflammasome activation, while their expressions were significantly decreased by CBD treatment.
FIGURE 5. CBD treatment suppressed NLRP3 inflammasome activation and pyroptosis in EHFD-fed mice. Mice were fed ethanol plus high-fat high-cholesterol diet for 8 weeks with or without CBD treatment. Control mice were fed a standard chow diet. Western blot analyses of NLRP3, ASC, pro-caspase-1, and caspase-1 p20 (A). Quantification of expressions of NLRP3, ASC, and caspase-1 p20 (B). Western blot analyses of GSDMD and cleaved GSDMD (C). Quantification of relative expressions of cleaved GSDMD (D). Western blot analyses of IL-1β and pro-IL-1β (E). Quantification of expression of IL-1β (F). *p < 0.05 vs the control group; #p < 0.05 vs the EHFD group.
As certain inflammasomes trigger the inflammatory form of cell death, we then assessed the cleavage of GSDMD, which is the executor of cell pyroptosis. As shown in Figures 5C, D, the expression of cleaved GSDMD in the EHFD group was approximately 1.4 times higher than that in the control group. CBD treatment significantly lowered the protein expression of cleaved GSDMD in livers. Meanwhile, the protein levels of downstream inflammatory cytokine IL-1β were reduced by CBD treatment (Figures 5E, F). Thus, our results suggested a novel mechanism of CBD attenuating liver inflammatory response by inhibiting the NLRP3 inflammasome–pyroptosis pathway.
Metabolism-related liver diseases have been severe public health burdens worldwide. The early stage of liver diseases is reversible through alcohol abstinence, nutritional intervention, or medical treatment (Louvet and Mathurin, 2015). Recently, hepatoprotective property of CBD has been reported in several research studies both in the ALD mice model and cell model (Yang et al., 2014; Wang et al., 2017; Huang et al., 2019). In this study, we employed a liver-damaged mouse model induced by EHFD, which was used to imitate the alcoholics with Western dietary pattern (Gäbele et al., 2011). Our results demonstrated that CBD exerted a beneficial effect in preventing liver injury induced by EHFD diet; 1) CBD reduced lipid accumulation, liver injury, and oxidative stress induced by EHFD diet; 2) CBD attenuated inflammation, by suppressing activation of NF-κB and activation of the NLRP3 inflammasome; and 3) CBD inhibited cleaved GSDMD formation induced by EHFD treatment. Herein, our research is the first to have examined the function of CBD, which protected the liver from injury and inflammation induced by EHFD diet, by regulating the NF-κB–NLRP3 inflammasome–pyroptosis pathway.
Oxidative stress plays a key part in the pathogenesis of liver injuries by alcohol or high-fat diet (Xu et al., 2017). Acute or chronic alcohol drinking or high-fat diet may induce oxidative stress by increasing reactive oxygen species (ROS) production or decreasing antioxidant enzyme activities (Wei et al., 2019), resulting in liver injury. Much evidence indicated that CBD can possess antioxidant properties. We have reported previously that CBD can prevent fatty liver induced by alcohol via inhibiting oxidative stress and activating the autophagy pathway (Yang et al., 2014). In line with these reports, we found that CBD lessened EHFD-induced oxidative stress, which was shown by the increasing levels of MDA, the decreasing ratio of GSH/GSSG, and the reduction of the activity of SOD. The increased ratio of GSH/GSSG may contribute to the protection of livers from oxidative stress induced by EHFD. These indicated that part of the hepatoprotective effect of CBD derives from its antioxidative effect.
Chronic alcohol abuse or long-term high-fat diet can lead to augmentation of enteric permeability, hepatocyte injury, and activation of immune cells, resulting in the release of LPS, pro-inflammatory chemokines, and DAMPs (Kawaratani et al., 2017). Consequently, these factors may stimulate the inflammatory response, which is responsible for the progression of liver injuries. Inflammation in the liver has been proven to be a major contributor to the progression of liver disease. Importantly, it has been reported that CBD exerted a noteworthy anti-inflammatory effect in many disorders. Moreover, our laboratory and others previously found that CBD alleviated liver injury of NAFLD by suppressing NF-κB and the NLRP3 inflammasome (Mridha et al., 2017; Huang et al., 2019). In this study, besides inhibiting liver injury, we also found that CBD treatment prominently alleviated the inflammation induced by EHFD shown by counterbalancing the increment of macrophages in the liver induced by EHFD diet. Elevated infiltration of macrophages into the liver is an established pathological alteration of inflammation Zhang et al. (2018), resulting in the release of various cytokines, such as TNF-α, IL-1β, and MCP-1, which could exacerbate liver damage (Xiang et al., 2015). In line with this, we found that CBD treatment actually ameliorated the expression of TNF-α, IL-1β, and MCP-1 in the livers of EHFD-treated mice.
Although these detrimental factors can stimulate inflammatory response through diverse pathways, the most important one of these pathways is characterized by the activation of NF-κB. While these agonists combine with different receptors (Boaru et al., 2015), such as tumor necrosis factor receptor, toll-like receptors (TLRs) (Xiang et al., 2015), IL-1β receptor, and the cytosolic pattern recognition receptor (NOD2), the IκB kinase (IKK) subsequently is catalyzed into an active form, as well as NF-κB (Liu et al., 2017). Unsurprisingly, EHFD feeding caused the activation of IKK and NF-κB. However, CBD visibly inhibited these alterations. Of the NF-κB pathway network, the activation of the NLRP3 inflammasome is a major contributor to chronic liver diseases (Wu et al., 2017). Upgradation of NLRP3 by active NF-κB (signal 1), the so-called priming step, is the first and indispensable process in the activation of the NLRP3 inflammasome (Volt et al., 2016). Followed by the stimulation of signal 2, ACS is recruited and pro-caspase-1 self-catalyzed into caspase-1 (Volt et al., 2016), the mature form. Subsequently, caspase-1 cleaved pro-IL-1β into mature IL-1β and GSDMD into cleaved GSDMD, resulting in inflammation and injury (Fabio et al., 2002; TingTing et al., 2010). In accordance with the activation of NF-κB, the NLRP3 inflammasome and GSDMD were also activated by EHFD diet, CBD treatment expectedly downgraded the level of NLRP3 and ASC and reduced the activation of pro-caspase-1 and mature IL-1β.
In this study, we employed a liver damage model induced by ethanol combined with high-fat diet, simulating the social drinking action and impactful Western diet pattern. In this study, we only examined the effect and explored the mechanism in the mice model but did not confirm the molecular mechanism in hepatocytes. Further cell experiments with certain gene knockdown in the NF-κB–NLRP3 inflammasome–pyroptosis pathway are warranted to confirm the molecular mechanisms.
The current study indicates that CBD protects the liver against EHFD-induced liver inflammatory reactions, potentially via inhibiting NLRP3 inflammasome activation and pyroptosis. Future investigation toward elucidating the mechanisms underlying pyroptosis will benefit our understanding of the beneficial effects of CBD.
Data Availability Statement
The original contributions presented in the study are included in the article/supplementary material; further inquiries can be directed to the corresponding authors.
The animal study was reviewed and approved by the Animal Experimentation Ethics Committee of Sun Yat-sen University. Written informed consent was obtained from the owners for the participation of their animals in this study.
Study concept and design: ZT, ZZ, and LY. Conduction of study: XJ, YG, YH, YZ, NP, and JL. Data collection and analysis: XJ, YG, and YH. Critical review and revision of the manuscript for important intellectual content: all authors.
This work was supported by the National Natural Science Foundation of China (81872613 to LY) and Tip-top Scientific and Technical Innovative Youth Talents of Guangdong special support program (2016TQ03R517 to LY) and the National Natural Science Foundation of China (No. 81872069 to ZZ), Guangzhou Science and Technology program (No. 201803010038 to ZZ).
Conflict of Interest
ZT was employed by Guangdong Zhaotai Zinkernagel Biotech Co. Ltd.
The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors, and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
The authors thank all participants who participated in this study.
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Keywords: cannabidiol, alcohol liver disease, inflammation, NF-κB, nuclear factor kappa B, NLRP3 inflammasome, pyroptosis
Citation: Jiang X, Gu Y, Huang Y, Zhou Y, Pang N, Luo J, Tang Z, Zhang Z and Yang L (2021) CBD Alleviates Liver Injuries in Alcoholics With High-Fat High-Cholesterol Diet Through Regulating NLRP3 Inflammasome–Pyroptosis Pathway. Front. Pharmacol. 12:724747. doi: 10.3389/fphar.2021.724747
Received: 14 June 2021; Accepted: 04 August 2021;
Published: 22 September 2021.
Songtao Li, Zhejiang Chinese Medical University, China
Guang Yang, Jinan University, China
Yongke Lu, Marshall University, United States
Ping Yao, Huazhong University of Science and Technology, China
Copyright © 2021 Jiang, Gu, Huang, Zhou, Pang, Luo, Tang, Zhang and Yang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Study: CBD does not cause liver damage
Despite CBD’s popularity and increasing ubiquitousness, questions still linger regarding the safety and long-term impact of using the hemp and cannabis-derived substance.
Since hemp is now legal and CBD is becoming more mainstream, the FDA has come under pressure to clarify its stance on the ingredient so manufacturers eager to capitalize on the growing demand can develop products that can be sold.
A summer 2019 study involving mice suggested that there may be a link between CBD and liver damage. Critics pushed for a human study, like Validcare’s, saying there are differences between how CBD functions in mice versus humans, and most people would not ingest the level of CBD provided to rodents in the study.
The results of the study are likely good news for players in the CBD industry who may have been fearing heavy restrictions, should the results have raised red flags.
There are still plenty of other health-related questions about CBD to tackle. These stem from the relative newness of the ingredient and the lack of long-term research on side effects or how it may interact with medications. As a result, the FDA has cracked down on CBD-containing products making health claims or statements about what the ingredient can accomplish.
Consumers are showing increasing eagerness to add CBD to their daily regimens, with 40% saying in March 2019 that they would try CBD, according to a study by High Yield Insights. The ingredient got a boost during the COVID-19 pandemic, as consumers sought ways to relax, ease tension and improve sleep quality.
The lack of FDA regulations around CBD has not stopped companies from planning and creating products. These include completely new offerings and reimagined versions of existing ones. Unilever-owned ice cream maker Ben & Jerry’s was the first to hint at the possibility of adding CBD to some products after hemp cultivation was legalized in the 2018 Farm Bill. Soon after, Mondelez’s CEO announced that the company is exploring CBD snacks . Smoothie maker Bolthouse Farms has also expressed interest in developing a CBD-infused line of its drinks, but progress has been slow due to regulatory ambiguities , said the company’s vice president of marketing.
Drink makers seem to be leading the charge when it comes to getting CBD products on store shelves. Ocean Spray’s Lighthouse incubator launched a line of sparkling CBD water called CarryOn, while Constellation Brands’ Canopy Growth launched its own bubbly CBD beverage, Quatreau . Truss CBD USA, a partnership between Molson Coors Beverage Company and Canadian cannabis grower Hexo, has also debuted a sparkling CBD drink called Veryvell in the U.S.
With the number of states legalizing cannabis increasing and a new administration that appears more favorable toward the substance, momentum around CBD is gaining speed. Manufacturers have little reason to stop making CBD products to satiate growing consumer demand, but the FDA has a critical role to play in determining how many of them will come to market.
Cannabidiol improves brain and liver function in a fulminant hepatic failure-induced model of hepatic encephalopathy in mice
Hepatic encephalopathy is a neuropsychiatric disorder of complex pathogenesis caused by acute or chronic liver failure. We investigated the effects of cannabidiol, a non-psychoactive constituent of Cannabis sativa with anti-inflammatory properties that activates the 5-hydroxytryptamine receptor 5-HT1A, on brain and liver functions in a model of hepatic encephalopathy associated with fulminant hepatic failure induced in mice by thioacetamide.
Female Sabra mice were injected with either saline or thioacetamide and were treated with either vehicle or cannabidiol. Neurological and motor functions were evaluated 2 and 3 days, respectively, after induction of hepatic failure, after which brains and livers were removed for histopathological analysis and blood was drawn for analysis of plasma liver enzymes. In a separate group of animals, cognitive function was tested after 8 days and brain 5-HT levels were measured 12 days after induction of hepatic failure.
Neurological and cognitive functions were severely impaired in thioacetamide-treated mice and were restored by cannabidiol. Similarly, decreased motor activity in thioacetamide-treated mice was partially restored by cannabidiol. Increased plasma levels of ammonia, bilirubin and liver enzymes, as well as enhanced 5-HT levels in thioacetamide-treated mice were normalized following cannabidiol administration. Likewise, astrogliosis in the brains of thioacetamide-treated mice was moderated after cannabidiol treatment.
CONCLUSIONS AND IMPLICATIONS
Cannabidiol restores liver function, normalizes 5-HT levels and improves brain pathology in accordance with normalization of brain function. Therefore, the effects of cannabidiol may result from a combination of its actions in the liver and brain.
Hepatic encephalopathy (HE) is a syndrome observed in patients with end-stage liver disease. It is defined as a spectrum of neuropsychiatric abnormalities in patients with liver dysfunction, after exclusion of other known brain diseases, and is characterized by personality changes, intellectual impairments and a depressed level of consciousness associated with multiple neurotransmitter systems, astrocyte dysfunction and cerebral perfusion (Riggio et al., 2005; Magen et al., 2008; Avraham et al., 2006; 2008a; 2009; Butterworth, 2010). Subtle signs of HE are observed in nearly 70% of patients with cirrhosis and approximately 30% of patients dying of end-stage liver disease experience significant encephalopathy (Ferenci, 1995). HE, accompanying the acute onset of severe hepatic dysfunction, is the hallmark of fulminant hepatic failure (FHF), and patients with HE have been reported to have elevated levels of ammonia in their blood (Stahl, 1963). In addition, the infiltration of tumour necrosis factor-α-secreting monocytes into the brain of bile duct-ligated mice, a model of chronic liver disease, has been found 10 days after the ligation, indicating that neuroinflammation is involved in the pathogenesis of HE. This infiltration was shown to be associated with activation of the cerebral endothelium and an increase in the expression of adhesion molecules (Kerfoot et al., 2006).
Cannabidiol (CBD) is a non-psychoactive ingredient of Cannabis sativa (Izzo et al., 2009). Many mechanisms have been suggested for its action, such as agonism of 5-HT1A receptors (Russo et al., 2005). It also has a very strong anti-inflammatory activity both in vivo, as an anti-arthritic therapeutic (Malfait et al., 2000; Durst et al., 2007), and in vitro, manifested by inhibition of cytokine production in immune cells (Ben-Shabat et al., 2006). The finding that CBD is devoid of any psychotropic effects combined with its anti-inflammatory activity makes it a promising tool for treating HE, which is exacerbated by an inflammatory response (Shawcross et al., 2004). In the present work, we aimed to explore the effects of CBD in the acute model of HE induced by the hepatotoxin thioacetamide (TAA), focusing on brain function, brain pathology and 5-HT levels, liver function and pathology as possible targets for therapeutic effects of CBD.
Female Sabra mice (34–36 g), 8 to 10 weeks old, were assigned at random to different groups of 10 mice per cage and were used in all experiments. All cages contained wood-chip bedding and were placed in a temperature-controlled room at 22°C, on a 12 h light/dark cycle (lights on at 07h00min). The mice had free access to water 24 h a day. The food provided was Purina chow and the animals were maintained in the animal facility (Specific Pathogen Free Organism unit) of the Hebrew University Hadassah Medical School, Jerusalem. Mice were killed after each treatment by decapitation between 10h00min and 12h00min. Animals were kept at the animal facility in accordance with NIH guidelines and all experiments were approved by the institutional animal use and care committee, No. MD-89.52-4.
Induction of hepatic failure
We adapted the rat model of acute liver failure induced by TAA to mice (Zimmermann et al., 1989). The TAA model in mice has been extensively validated previously (Honda et al., 2002; Fernández-Martínez et al., 2004; Schnur et al., 2004). TAA was obtained from Sigma-Aldrich (Rehovot, Israel) in powder form and dissolved in sterile normal saline (NS) solution; it was injected i.p. as a single dose of 200 mg·kg −1 . Vehicle (NS) was also administered in a separate group of animals that served as controls. Twenty-four hours after injection of TAA all animals (including control) were injected s.c. with 0.5 mL of a solution containing 0.45% NaCl, 5% dextrose and 0.2% KCl in order to prevent hypovolaemia, hypokalaemia and hypoglycaemia. The mice were intermittently exposed to infrared light in order to prevent hypothermia.
Administration of CBD
CBD was extracted from cannabis resin (hashish) and purified as previously reported (Gaoni and Mechoulam, 1971) and was dissolved in a vehicle solution consisting of ethanol, emulphor and saline at a ratio of 1:1:18, respectively, and was injected in a single dose of 5 mg·kg −1 i.p, 1 day after either NS or TAA treatment. Similarly, the CBD-related vehicle (the same mixture without CBD) was administered at the same time points following either NS or TAA treatment. A dose of 5 mg·kg −1 CBD was chosen based on the studies done by Magen et al. (2009; 2010;) and on preliminary experiments done in our laboratory, which demonstrated that this does produced a maximal effect compared to 1 and 10 mg·kg −1 . Four groups of animals were studied: control naïve animals treated with either CBD or its vehicle, and corresponding TAA-treated animals.
Assessment of neurological function
Neurological function was assessed by a 10-point scale based on reflexes and task performance (Chen et al., 1996): exit from a 1 m in diameter circle in less than 1 min, seeking, walking a straight line, startle reflex, grasping reflex, righting reflex, placing reflex, corneal reflex, maintaining balance on a beam 3, 2 and 1 cm in width, climbing onto a square and a round pole. For each task failed or abnormal reflex reaction a score of 1 was assigned. Thus, a higher score indicates poorer neurological function. The neurological score was assessed 1 day after induction of hepatic failure by TAA (day 2). The mice were then divided between treatment groups so that all groups had similar baseline neurological scores after TAA induction. The post-treatment neurological score was assessed 1 day after administration of CBD or vehicle (day 3).
Assessment of activity
The activity test was performed 2 days after the induction of hepatic failure. Activity of two mice was measured simultaneously for a 5 min period. Two mice were tested together to lower stress to the minimum, as it has been shown that separation of mice induces stress (van Leeuwen et al., 1997; Hao et al., 2001). Activity was assessed in the open field (20 × 30 cm field divided into 12 squares of equal size) as described previously (Fride and Mechoulam, 1993). Locomotor activity was recorded by counting the number of crossings by the mice at 1 min intervals.
Results are presented as the mean number of crossings·min −1 .
Cognitive function studies were performed 8 days after the induction of hepatic failure. The animals were placed in an eight-arm maze, which is a scaled-down version of that developed for rats (Olton and Samuelson, 1976; Pick and Yanai, 1983). Mice were deprived of water 2 h prior to the test and a reward of 50 µL of water was presented at the end of each arm, in order to motivate them to perform the task. Animals were divided between treatment groups so that all groups had similar baselines neurological scores after TAA induction. The mice were tested (no. of entries) until they made entries into all eight arms or until they completed 24 entries, whichever came first. Hence, the lower the score the better the cognitive function. Food and water were given at the completion of the test. Maze performance was calculated on each day for five consecutive days. Results are presented as area under the curve (AUC) utilizing the formula: (day 2 + day 3 + day 4 + day 5) − 4*(day 1) (Pick and Yanai, 1983).
Brain histopathology and immunohistochemistry
Two days after the induction of hepatic failure, mice were killed by decapitation and brains were excised and fixed in 4% neutral-buffered paraformaldehyde.
The brain was cut along the midline and separated into two pieces containing brain and cerebellum hemispheres. Both sections were embedded en block in paraffin and 6 µm sagittal sections were adhered to slides. Serial sections were taken in 15 groups of slides (10 slides each, three sections per slide) at 100 µm intervals. These slides were used for glial fibrillary acidic protein (GFAP) immunohistochemistry (a total of 90 sections), according to standard protocol. Briefly, paraffin sections were deparaffinized and hydrated in xylene and alcohol solutions, rinsed with tris buffer saline. Citrate buffer (pH 6) was used for antigen retrieval. The endogenous peroxidase was blocked with H2O2 (0.3% in phosphate buffer saline). Sections were then incubated in blocking buffer for 1 h. A series of reselected sections were then treated with primary antibody against GFAP (1:2500, DakoCytomation, Denmark), overnight at 4°C, and then with goat anti-rabbit (1:200, Vector Burligame, CA, USA) as secondary antibody. Immunoreactions were visualized with the avidin–biotin complex (Vectastain) and the peroxidase reaction was visualized with diaminobenzidine (DAB) (Vector), as chromogen. Sections were finally counterstained with haematoxylin and examined under light microscope (Zeiss Axioplan 2). Images were captured with a digital camera (NIKON DS-5Mc-L1) mounted on microscope. Astrocytes were evaluated at the hippocampal area of both hemispheres. A total of five to seven randomly selected visual fields per hemisphere section were evaluated. A square with 100 square subdivisions each of 3721 µm 2 as defined by an ocular morphometric grid adjusted at the prefrontal lens, was centred at each visual field. The number of GFAP-positive astrocytes·mm −2 was evaluated. Only those cells with an identifiable nucleus were counted. In addition, in an attempt to evaluate the level of activation of the astrocytes (cell size, extension of cell processes) the number of small square subdivisions with a positive GFAP signal and their % of the total number of square subdivisions counted, were calculated.
Two independent observers who were blinded to sample identity performed all quantitative assessments. In cases where significant discrepancies were obvious between the two observers, the evaluation was repeated by a third one.
Two days after the induction of hepatic failure, mice were killed by decapitation and their livers were excised and fixed in 4% neutral-buffered paraformaldehyde.
Liver histopathological analysis and scoring of necrosis (coagulative, centrilobular) were performed as described previously (Avraham et al., 2008a).
Serum ammonia, liver enzymes and bilirubin levels
Serum for alanine transaminase (ALT), aspartate transaminase (AST), bilirubin and ammonia measurements was obtained on day 3 in glass tubes, centrifuged, and analysed on the day of sampling using a Kone Progress Selective Chemistry Analyzer (Kone Instruments, Espoo, Finland). All serum samples were processed in the same laboratory using the same methods and the same reference values.
On day 12, mice killed by decapitation and their brains were dissected out for determination of 5-HT levels. The assays for 5-HT were performed by standard alumina extraction, and HPLC with electrochemical detection using dehydroxybenzylamine (DHBA) as an internal standard (Avraham et al., 2006).
On the first day of this experiment, 20 mice were administered with TAA (200 mg·kg −1 ) and 20 saline. The following day, neurological evaluation was performed and saline-treated and TAA-treated mice were each assigned to two different subgroups with approximately equal neurological score, which were administered either saline or CBD (5 mg·kg −1 ). On the third day, mice were evaluated for neurological and locomotor function, after which they were killed and their brains and livers were dissected out and fixed with 4% formaldehyde. Blood was drawn and separated for plasma, in which liver enzymes were quantified.
This was identical to experiment 1 on days 1–3, only the mice were not killed on day 3 but were evaluated for cognitive function using the eight-arm maze test, on days 8–12. On day 12, the mice were killed and their livers and brains were dissected out for determination of 5-HT levels.
All data are expressed as mean ± SEM. Statistical analysis was performed using one-way anova followed by Bonferroni’s post hoc test.
Neurological function, evaluated 2 days after induction of hepatic failure, was impaired in thioacetamide (TAA) mice and was restored by cannabidiol (CBD). **P < 0.01 versus control, #P < 0.01 versus TAA.
Locomotor function, evaluated 3 days after induction of hepatic failure, was decreased in thioacetamide (TAA) mice and was restored by cannabidiol (CBD). **P < 0.01 versus control, #P < 0.01 versus TAA.
Cognitive function, tested 8 days after induction of hepatic failure, was impaired following thioacetamide (TAA) and was improved by cannabidiol (CBD). *P < 0.05 versus control, #P < 0.01 versus TAA. AUC, area under the curve.
Brain and liver histopathology
Figure 4 shows images of slices from brains of animals from the control group (A), control + CBD group (B), TAA group (C) and TAA + CBD group (D), immunostained for the detection of astrogliosis. Astrogliosis was observed in visual fields studied in TAA animals, as evident both by the increase in the number of GFAP-positive cells·mm −2 ( Figure 4E ; anova : F3,298= 26.8, P < 0.001; Bonferroni: P < 0.001) and by the increased % of GFAP-positive surface (Figure 4F ; anova : F3,298= 19.21, P < 0.001; Bonferroni: P < 0.001). Both parameters were unaffected in CBD-treated controls (Figure 4E and F ). However, the number of GFAP-positive cells·mm −2 in TAA + 5 mg·kg −1 CBD-treated animals was reduced compared to TAA-treated animals ( Figure 4E ; Bonferroni: P= 0.002). In contrast, CBD had no effect on the % of GFAP-positive surface in TAA animals ( Figure 4F ). Overall, it seems that TAA administration increased the number of activated astrocytes and CBD significantly reduced this effect. However, astrocytes in both CBD- and vehicle-treated TAA animals did not differ as regards their cellular size or extension of processes.
Glial fibrillary acidic protein (GFAP) immunohistochemistry indicating the astrocytic reaction throughout the parahippocampal area in naïve controls (A, B) and thioacetamide (TAA)-treated animals (C,D) following treatment with vehicle (A,C) or cannabidiol (CBD) (B,D). CBD treatment had no effect on the astrocytic activation of naïve animals. However, in the case of animals with hepatic encephalopathy, CBD treatment induced significant reduction in the total number of activated astrocytes, although the level of individual cell activation was not impaired. E. Quantification of GFAP-positive cells·mm −2 ; the number was reduced in TAA mice treated with 5 mg·kg −1 CBD compared to TAA mice treated with vehicle. ***P < 0.001 versus control, #P < 0.01 versus TAA. F. Quantification of GFAP-positive surface in µm 2 ; 5 mg·kg −1 CBD had no effect on the GFAP-positive surface in the brains of TAA-treated mice. ***P < 0.001 versus control. Scale bars: 100 µm.
TAA-treated animals showed the typical TAA-induced liver necrosis lesions that have been described in detail previously (Avraham et al., 2008a). The statistical analysis of liver histopathology scores did not reveal significant differences in the extent and severity of necrotic lesions between CBD-treated and untreated mice (data not shown).
Brain 5-HT levels, measured 12 days after induction of hepatic failure, were increased in the brains of thioacetamide (TAA) mice and were restored by cannabidiol (CBD).
Indices of liver function. The levels of ammonia (A), bilirubin (B), aspartate transaminase (AST) (C) and alanine transaminase (ALT) (D) were all increased in the plasma of thioacetamide (TAA) mice and were all reversed by cannabidiol (CBD). **P < 0.01 versus control, #P < 0.01 versus TAA.
TAA administration induces acute liver failure which leads to CNS changes related to those seen in HE (Zimmermann et al., 1989; Magen et al., 2008; Avraham et al., 2006; 2008a; 2009;). The hepatotoxicity of TAA is due to the generation of free radicals and oxidative stress (Zimmermann et al., 1989). However, it is not clear whether TAA affects the brain directly or the liver (Albrecht et al., 1996). In previous studies both a CB1 antagonist and a CB2 or TRPV1 agonist have been shown to ameliorate the brain and liver damage that occurs in liver disease and HE (Avraham et al., 2006; 2008a,b; 2009; Mallat and Lotersztajn, 2008). Also CBD, an agonist of the 5-HT1A receptor, was found to ameliorate brain damage in a chronic model of HE induced by bile duct ligation. Hence, we investigated the potential of CBD as a treatment for HE induced by FHF. Our results indicated that it has a neuroprotective role in HE induced by FHF; CBD was found to restore liver function, normalize 5-HT levels and improve the brain pathology in accordance with normalization of brain function. We also showed that CBD affects both central functions: neurological score, motor and cognitive functions, brain 5-HT levels as well as astrogliosis and peripheral functions: reduced liver enzymes, ammonia and bilirubin. Therefore, we conclude that it acts both centrally and peripherally. In addition, it has been shown that CBD can cross the blood – brain barrier and act centrally (for review see Pertwee, 2009). Therefore, its effect may result from a combination of its actions in the liver and the brain. However, to elucidate its mechanism of action future experiments are needed to determine the effects of central administration of CBD.
Previous work from our laboratory has demonstrated an impaired neurological and motor function 3 days, and impaired cognition 12 days after TAA injection to mice (Avraham et al., 2006; 2008a; 2009;). These results were reproduced in the present study ( Figures 1–3 ). In a more recent study from our laboratory, cognitive and motor deficits were observed 21 days after bile duct ligation, a chronic model of liver disease (Magen et al., 2009). The different durations of the development of HE symptoms in the two models apparently result from their different characteristics – an acute versus a chronic model of HE. In the latter model, CBD was found to improve cognition and locomotor activity, in accordance with our present data (Magen et al., 2009). However, in sharp contrast to the findings reported here no evidence for astrogliosis was found in that study (data not reported); in our acute model induced by TAA we observed astrogliosis after 3 days ( Figure 4C ). Those mice with histopathological alterations displayed an increased neurological score and decreased activity level, and 5 mg·kg −1 CBD reversed both the increase in the number of GFAP(+) cells, an index of neuroinflammation ( Figure 4E ), and the neurological and locomotor impairments ( Figures 1 and and2), 2 ), suggesting a link between neuroinflammation and motor and neurological deficits. Similar results were reported by Jover et al. (2006) who demonstrated a decrease in motor activity in bile duct-ligated rats on a high-protein diet, in association with astrogliosis, and by Cauli et al. (2009), who reported that treatment with an anti-inflammatory restored the motor activity in HE. In the work of Jover et al. (2006), astrogliosis was found only in the bile duct-ligated rats on a high protein diet, but not in the bile duct-ligated rats on a regular diet, similar to our previous findings (Avraham et al., 2009) This suggests that TAA causes more severe damage to the brain than bile duct ligation, and that hyperammonaemia is required to worsen the damage in bile duct-ligated rats to an extent that is equivalent to that observed in TAA mice. The reason for this may be that in chronic liver disease induced by bile duct ligation, compensation mechanisms are activated, which moderate the brain damage, while in the acute model induced by TAA, no such mechanisms can come into action because of the severity of the liver insult and the short interval of time between the induction of liver damage and the histopathological examination.
Kerfoot et al. (2006) showed the infiltration of peripheral monocytes into the brain of bile duct-ligated mice 10 days after the ligation and suggested that this infiltration may cause the activation of inflammatory cells in the brain. Therefore, it is conceivable that such a mechanism was responsible for the astrogliosis observed in our study, since we found evidence of liver inflammation (data not shown). As evident from the histopathology results, CBD did not appear to affect the development of TAA-induced necrotic lesions in the liver of mice. However, the levels of liver transaminases in the serum of CBD-treated mice were significantly reduced compared to their untreated counterparts, indicating that this substance contributed to a partial restoration of liver function. Recent evidence elucidating the complicated mechanisms involved in the release of hepatocyte cytosolic enzymes such as ALT and AST in the blood may explain the discrepancy between histopathology and serum biochemistry data observed in the present study. Indeed, it is now generally accepted that the release of cytosolic enzymes during both the reversible and irreversible phases of hepatocyte injury and therefore their appearance in blood does not necessarily indicate cell death and also that enzyme release during reversible cell damage occurs with an apparent lack of histological evidence of necrosis (Solter, 2005). Following this reasoning, it could be hypothesized that although CBD did not reduce the levels of histologically detectable necrosis, it may have ameliorated the minute reversible hepatocyte damage that causes the so-called ‘leakage’ of cytoplasmic ALT and AST in blood. The interaction between hyperammonaemia and inflammation as a precipitating factor for HE has been discussed in two recent reviews (Shawcross and Jalan, 2005; Wright and Jalan, 2007). Further work is required to reveal the exact mechanism/s of the manner by which liver damage is related to dysfunction/damage in the brain, and studies using antagonists of the A2A adenosine receptors, which are potential targets of CBD that may mediate its anti-inflammatory effect (Carrier et al., 2006), need to be carried out in order to elucidate the receptors involved in this effect.
Astrogliosis has also been shown to be involved in learning and memory deficits in a mouse model of Alzheimer’s disease. In this study, astrogliosis was reduced by caloric restriction, which also reversed the cognitive deficits and increased the expression of neurogenesis in related genes (Wu et al., 2008). Further studies, such as expression analysis of such genes using DNA microarray and evaluation of neurogenesis using BrdU staining, needs to be performed in order to explore the mechanisms through which TAA-induced astrogliosis impairs cognition, and through which CBD acts to improve it.
Even though astrogliosis was found a week before cognitive function was observed, and it is not definite whether it was long-lasting, this mechanism seems, in our eyes, to account for the cognitive dysfunction, rather than the increase in 5-HT level ( Figure 5 ). The latter mechanism does not seem to be related to the cognitive dysfunction, even though this increase in 5-HT was reversed by CBD ( Figure 5 ), as 5-HT depletion, not increase, has been shown to cause memory deficits in the eight arm maze (Mazer et al., 1997). On the other hand, there is much evidence that ammonia induces astrocyte swelling which via a number of mechanisms leads to impaired astrocyte/neuronal communication and synaptic plasticity, thereby resulting in a disturbance of oscillatory networks. The latter accounts for the symptoms of HE (for review see Häussinger and Görg, 2010), among them presumably the cognitive dysfunction.
An increased level of 5-HT in the brain of rats after TAA administration was reported by Yurdaydin et al. (1990). In addition, there is indirect evidence that this increase is related to decreased motor activity, as the nonselective 5-HT receptor antagonist methysergide increased motor activity in TAA-injected rats, while the selective 5-HT2 receptor antagonist seganserin did not (Yurdaydin et al., 1996). Likewise, we found that the level of 5-HT was increased following TAA administration and this was restored after CBD treatment ( Figure 5 ). In parallel, motor activity was decreased following TAA injection and increased after CBD treatment, indicating a link between the increase in 5-HT and decrease in motor activity. Hence, it seems that CBD reversed the increased 5-HT level in the brains of TAA mice and thus reversed the decrease in their motor activity. A possible mechanism can be activation of 5-HT1A receptors by CBD (Russo et al., 2005; receptor nomencalture follows Alexander et al., 2008), as these receptors have been reported to inhibit 5-HT synthesis (Invernizzi et al., 1991). We have shown that the effects of CBD in a chronic model of HE, bile duct ligation, are mediated via the 5-HT1A receptors (Magen et al., 2010), and in an earlier study with the same model we demonstrated that the effects of CBD can be also mediated via A2A adenosine receptors (Magen et al., 2009). Thus, the effects of CBD can be mediated by 5-HT1A or/and A2A adenosine receptors. We think that in the current study the effects of CBD were mediated by the 5-HT1A receptor since activation of the receptor by CBD caused depletion of 5-HT ( Figure 5 ). In our previous studies we showed that cognition is multifactorial and not dependent only on 5-HT levels, and therefore there is no direct correlation between cognition and 5-HT levels.
The reversal of astrogliosis was probably related to reduced hepatic toxin formation. Indeed, there is much evidence that ammonia induces astrocyte swelling, which via a number of mechanisms leads to impaired astrocyte/neuronal communication and synaptic plasticity, thereby resulting in a disturbance of oscillatory networks. The latter accounts for the symptoms of HE (for review see Häussinger and Görg, 2010), among them presumably the cognitive dysfunction. Neurological and motor functions were improved 2 and 3 days, respectively, after induction of hepatic failure at the same time as a partial reversal of the astrogliosis and reduced levels of ammonia, bilirubin and liver enzymes were noticed. It seems that the behavioural effects of CBD are dramatic and occur within 3 days.
In summary, the present study demonstrates the therapeutic effects of CBD in an acute model of HE. It appears that this effect of CBD is multifactorial and involves cannabinoid (Avraham et al., 2006), vanilloid (Avraham et al., 2008a; 2009;) and 5-HT1A receptors (Magen et al., 2010). CBD improves the symptoms of FHF by affecting both brain histopathology and liver function, and thus may serve as therapeutic agent for treating human HE.