Mechanism-based Effects Of Scutellarin On Carbon Tetrachloride-induced Liver Injury Through Gut Microbiota

Purpose:To demonstrate the mechanism-based protective effects of scutellarin（SCU）on carbon tetrachloride（CCl₄）-induced liver injury in mice and screen out the dominant bacteria at genus level that are responsible for the effects of SCU.Furthermore,we used antibiotics cocktails（ampicillin,neomycin sulfate,metronidazole and vancomycin）to interfere and deplete gut microbiota in mice,observing and assessing the alterations of pharmacological effects of SCU and analyzing the change of dominant bacteria at genus level,respectively.Eventually,we used the method of co-cultivating two groups of mice,model group and high concentration of SCU group and observed the change of degree of liver injury in model group,revealing the relationship between gut microbiota and the hepatic effects of SCU,providing experimental basis for its treatment of liver injury.Methods:Preliminary evaluation of SCU in hepatic protection.BALB/c mice were randomly divided into 6 groups 1.Control group（N）;2.Model group（M,1 m L/kg CCl₄）;3.Low dose SCU group（ML,15 mg/kg）;4.Middle dose SCU group（MM,30mg/kg）;5.High dose SCU group（MH,60 mg/kg）;6.Positive control group（BIF,bifendate act as a positive control,200 mg/kg）.Liver injury in mice was induced by intraperitoneal injection of CCl₄ three times per week.SCU and bifendate was given by gavage every day.All animals were treated for an additional five weeks.Mice were euthanized and blood samples,stools and liver tissues were collected 12 h after the final injection of CCl₄.The content of AST、ALT、TBIL、ALB in serum and SOD、MDA in liver tissues were measured by commercial kits as per the manufacturer’s instructions.The liver necrosis and hepatocyte apoptosis were determined by H&E and TUNEL,respectively.The m RNA expression of IL-6、IL-1β、TNF-αand CYP2E1 in liver tissues were determinated by RT-q PCR.The protein expression of CYP2E1 in liver tissues was determinated by immunohistochemistry and western blot.The cytoplasmic and nuclear protein expression of IκBαand NF-κB were assessed by western blot.Upon extracting total DNA of fecal bacteria,gut microbiota structure and composition were analyzed by16S r RNA sequencing.The dominate genera were screened out using Z score and the relationship between genus and liver injury indicators were analyzed by spearman correlation analysis.2.Alterations of hepatoprotective effects of SCU with antibiotics interference.BALB/c mice were randomly divided into 6 groups 1.Control group（NA）;2.Model group（MA,1 m L/kg CCl₄）;3.Low dose SCU group（MLA,15 mg/kg）;4.Middle dose SCU group（MMA,30 mg/kg）;5.High dose SCU group（MHA,60 mg/kg）;The drinking water in the NA,MA,MLA,MMA and MHA group contains a cocktail of antibiotics[ampicillin（1 g/liter）,neomycin sulfate（1 g/liter）,metronidazole（1 g/liter）and vancomycin（0.5 g/liter）].6.High dose SCU group without antibiotics in drinking water（MH）acted as a positive control.Liver injury in mice was induced by intraperitoneal injection of CCl₄ three times per week.SCU was given by gavage every day.All animals were treated for an additional five weeks.Mice were euthanized and blood samples,stools and liver tissues were collected 12 h after the final injection of CCl₄.The content of AST、ALT、TBIL、ALB in serum and SOD、MDA in liver tissues were measured by commercial kits according to the manufacturer’s instructions.The liver necrosis and hepatocyte apoptosis were determined by H&E and TUNEL,respectively.The m RNA expression of IL-6、IL-1β、TNF-αand CYP2E1 in liver tissues were determinated by RT-q PCR.The protein expression of CYP2E1 in liver tissues was determinated by immunohistochemistry and western blot.The cytoplasmic and nuclear protein expression of IκBαand NF-κB were assessed by western blot.Upon extracting total DNA of fecal bacteria,gut microbiota structure and composition were analyzed by16S r RNA sequencing.The change of dominate genera were analyzed using Z score and the relationship between genus and liver injury indicators were analyzed by spearman correlation analysis.3.Effects of co-cultured on liver injury.BALB/c mice were randomly divided into 2 groups 1.Model group（M,1 m L/kg CCl₄）and 2.High dose SCU group（MH,60 mg/kg）.Upon the first administration of CCl₄ and SCU,an equal number of mice in M and MH group were selected and fed in one cage.Liver injury in mice was induced by intraperitoneal injection of CCl₄ three times per week.SCU was given by gavage every day.All animals were treated for an additional five weeks.The content of AST、ALT in serum were determinated by commercial kits.The liver necrosis was assessed by H&E.Results:1.The serum AST,ALT and TBIL levels were significantly elevated in the M group relative to the N group（P<0.01）,which was dose-dependently reduced by SCU treatment（P<0.05,P<0.01）.Serum ALB levels were no significant difference among 6 groups（P>0.05）.Next,we performed histopathology assay to further evaluate the protective effect of SCU.There was a large amount of hepatocyte necrosis and inflammatory cell infiltration in the M group relative to the N group（P<0.01）.SCU treatment significantly reduced hepatocyte necrosis and inflammatory cell infiltration relative to the M group（P<0.05,P<0.01）.Finally,we performed TUNEL assay to evaluate the protective effect of SCU.The hepatocyte apoptosis was significantly increased in the M group relative to the N group（P<0.01）,which was significantly ameliorated by SCU treatment（P<0.05,P<0.01）.CCl₄ poisoning induced the m RNA and protein expression of CYP2E1（P<0.01）,which was dose-dependently inhibited by SCU treatment（P<0.05,P<0.01）.CCl₄ challenge leads to an increase of hepatic MDA（P<0.01）and a decrease of hepatic SOD（P<0.01）.In contrast,SCU treatment dose-dependently blunted the MDA content（P<0.05,P<0.01）and elevated SOD activity（P<0.05,P<0.01）.Compared with the N group,hepatic m RNA levels of IL-6,IL-1βand TNF-αwere remarkably increased in the M group（P<0.01）.However,these incasements were significantly reduced by SCU treatment（P<0.05,P<0.01）.CCl₄ stimulated significantly down-regulated the cytoplasmic expression of IκBαand NF-κB（P<0.01）,up-regulated the nuclear expression of NF-κB（P<0.01）,which was reversed by SCU treatment（P<0.01）.Theαdiversity was significantly increased in the M group relative to the N group（P<0.05,P<0.01）.Theαdiversity was significantly decreased in the MH group compared to the M group（P<0.05,P<0.01）.We observed a distinct clustering of microbiota composition for all groups byβdiversity and the structure of microbes in the MH group was more similar to that in the N group.Bacteroidetes and Firmicutes were the dominate phylum in all groups.Compared to the N group,the Bacteroidetes/Firmicutes ratio was significantly elevated in the M group（P<0.01）.Compared to the M group,the Bacteroidetes/Firmicutes ratio was significantly elevated in the M group（P<0.01）.The result of Z score demonstrated that Lactobacillus,Bifidobacterium and Akkermansia were potential dominate genus due to their high abundance and significant difference.Compared to the N group,the relative abundance of Lactobacillus and Bifidobacterium were significantly decreased in the M group（P<0.05,P<0.01）.Compared to the M group,the relative abundance of Lactobacillus and Bifidobacterium were significantly increased in the ML,MM and groups（P<0.05,P<0.01）.The relative abundance of Akkermansia was no significant difference between N and M group.Compared to the M group,the relative abundance of Akkermansia was significantly increased in the ML,MM and groups（P<0.05,P<0.01）.The result of spearman correlation analysis demonstrated that Lactobacillus was negatively correlated with AST、IL-6、IL-1β、TNF-αand CYP2E1（P<0.05,P<0.01）and positively correlated with SOD（P<0.05）.Bifidobacterium and Akkermansia were no significantly correlation with liver injury indicator,oxidative stress indicator and inflammation indicator（P>0.05）.2.Serum ALT,AST and TBIL levels were markedly elevated in the MA group with respect to the NA group（P<0.01）.Interestingly,serum ALT,AST and TBIL levels of the SCU plus antibiotics groups were significantly higher than those of the MA group（P<0.05）.ALB levels were no significant difference among all groups（P>0.05）.Histological observation and TUNEL assay showed that SCU plus antibiotics treatment had higher histological score and hepatocyte apoptosis areas（P<0.05）,which was consistent with the results of serum parameters.CCl₄ challenge dramatically increased the both m RNA and protein expression of CYP2E1 in the liver（P<0.01）.Treatment of SCU plus antibiotics did not inhibit but activate the m RNA and protein expression of CYP2E1（P<0.05）.Compared with the NA group,SOD activity was decreased significantly（P<0.01）,and MDA level was increased significantly in the MA group（P<0.01）.SCU plus antibiotics treatment showed severer lipid peroxidation and oxidative stress,as proved by higher MDA level and lower SOD activity（P<0.05）.CCl₄ challenge dramatically increased the IL-6,IL-1βand TNF-αm RNA levels in the liver tissues（P<0.01）.However,the IL-6,IL-1βand TNF-αm RNA levels were even higher in the SCU plus antibiotics groups（P<0.05）.The expression of IκBαin the MA group was decreased relative to the NA group（P<0.01）,but this trend was exacerbated by SCU plus antibiotics co-treatment（P<0.05）.In the MA group,we detected reduced cytoplasmic NF-κB and increased nuclear NF-κB expression relative to the NA group（P<0.05,P<0.01）.However,SCU plus antibiotics co-treatment aggravated these changes in NF-κB levels caused by repeated injections of CCl₄（P<0.05,P<0.01）.αdiversity was no significant difference among five groups（P>0.05）and the structure of gut microbiota in the MA group was more similar to that of the NA group.Bacteroidetes and Firmicutes were the dominate phylum in all groups.The Bacteroidetes/Firmicutes radio in the MA group was significantly increased relative to the NA group（P<0.05）.However,the Bacteroidetes/Firmicutes radio was no significant difference between MA group and SCU plus antibiotics groups（P>0.05）.We performed Z score to assess the changes of 15 genera that showed high abundance.The relative abundance of Bifidobacterium and Lactobacillus were significantly increased and the relative abundance of Enterococcus was significantly decreased in the MA group relative to the SCU plus antibiotics groups（P<0.01）.The relative abundance of Akkermansia was significantly decreased in the MA group than that in the NA group（P<0.01）.Spearman correlation analysis demonstrated that Lactobacillus was positively correlated with SOD and negatively correlated with AST（P<0.05）.Bifidobacterium,Akkermansia and Enterococcus were no significantly correlation with liver injury indicator,oxidative stress indicator and inflammation indicator（P>0.05）.3.Compared to the M group,serum AST and ALT was significantly decreased in the M+MH group（P<0.05）.The result of H&E showed that histological score was significantly decreased in the M+MH group relative to the m group（P<0.05）.Conclusions:1.SCU ameliorated CCl₄-induced liver injury in mice in a dose-depend manner and its mechanism may related to suppress CYP2E1-mediated oxidative stress,inhibit IκBα/NF-κB signal pathway-mediated inflammatory response and modulate gut microbiota,especially enhancing the relative abundance of Lactobacillus.2.The blunted relative abundance of Lactobacillus may be responsible for the hepatoprotective,anti-oxidant and anti-inflammation effects of SCU were reversed with antibiotics interference,which indicated that Lactobacillus may mediate the hepatoprotective effects of SCU.3.Mice co-culture ameliorated liver injury in model group,which indicated that gut microbiota may mediate the hepatoprotective effects of SCU.