| BackgroundLiver fibrosis is a repair response to damage caused by multiple etiologies and is a common pathway for chronic liver disease,which can progress to cirrhosis,liver cancer,and even death once the damaging factors persist.Early-stage liver fibrosis can be reversed,but the exact mechanism is not fully understood.Therefore,it is important to investigate the mechanisms of liver fibrosis and thus identify new potential therapeutic targets.NOX2 and NLRP3 inflammasome play a key role in liver fibrosis,but the mechanism of their mutual regulation in liver fibrosis remains unclear.The intestinal tract contains a large and diverse microbiota that is involved in a variety of activities in the body and is also closely associated with disease development.It has been shown that NOX2 and NLRP3 inflammasome regulate disease progression through the intestinal microbiota.Ursolic acid（UA）is a monomeric compound found in herbal plants,and early studies have shown that it has anti-inflammatory and anti-fibrotic effects,but the exact mechanism is still not fully elucidated.Using NOX2 and NLRP3 inflammasome inhibitors,our team previously found that UA could improve liver fibrosis in CCl4-modeled mice by inhibiting the NOX2/NLRP3 inflammasome signaling pathway.This study further enriched the modeling methods of liver fibrosis mice and used NOX2 and NLRP3gene knockout mice to deeply explore the specific molecular mechanism of UA inhibiting liver fibrosis and its impact on intestinal flora through in vivo experiments,providing a more favorable basis for UA treating liver fibrosis.Objective1.To elucidate the relationship between NOX2 and NLRP3 inflammasome in liver fibrosis and whether UA exerts anti-fibrotic effects by inhibiting the NOX2/NLRP3 inflammasome signaling pathway.2.To observe the changes of intestinal microbiota in mice with liver fibrosis and to investigate whether UA reverses liver fibrosis by inhibiting the NOX2/NLRP3 inflammasome signaling pathway and thereby improving intestinal microbiota.Method1.Wild-type C57BL/6 male mice,NOX2 gene knockout(NOX2-/-)mice and NLRP3 gene knockout(NLRP3-/-)mice were used to construct liver fibrosis models in two ways,respectively.In the MCD-induced liver fibrosis model,the control group was given MCS diet for 8 weeks;the MCD group was given MCD diet for 8weeks;the UA group was given MCD diet for 8 weeks,of which the latter 4 weeks were treated with 50 mg/kg UA by gavage 7 times a week.In the CCl4-induced liver fibrosis model:the control group was given an intraperitoneal injection of olive oil for 8 weeks at 1 ml/kg twice a week;the CCl4 group was given an intraperitoneal injection of 20%CCl4（olive oil dilution）for 8 weeks at 1 ml/kg twice a week;the UA group was given an intraperitoneal injection of 20%CCl4（olive oil dilution）for8 weeks at 1 ml/kg twice a week,of which the latter 4 weeks were gavage with 50mg/kg UA for 7 times a week.2.Observation of pathological structure of liver tissue by HE,Sirius red staining,and Masson staining3.ELISA kit was used to detect ALT,AST,and hydroxyproline in mice.4.Western blot was used to detect the protein content ofα-SMA,Collagen I,TIMP1,NOX2,and NLRP3 inflammasome in liver tissues.5.Real-time quantitative PCR was performed to detect the m RNA expression ofα-SMA,Collagen I,TIMP1,NOX2,and NLRP3 inflammasome in liver tissues.6.16S rRNA sequencing to detect intestinal flora.Results1.Expression of NOX2 and NLRP3 inflammasome in the liver of MCD-induced mice and the therapeutic effect of UA1.1 UA attenuates liver injury and fibrosis in MCD-induced miceLiver in the MCD group exhibited more pronounced structural destruction of the liver,lipid accumulation,increased inflammatory cell infiltration and collagen deposition,and higher liver fibrosis scores than the control group（MCS group）;compared with the MCD group,liver in the UA group had reduced lipid accumulation,decreased inflammatory cell infiltration,less collagen deposition,and lower liver fibrosis scores.1.2 UA treatment attenuates liver function impairment and hydroxyproline levels in MCD-induced liver fibrosis miceThe AST,ALT,and hydroxyproline levels were increased in the MCD group of mice compared with the MCS group;the AST,ALT,and hydroxyproline in the UA group were significantly lower than those in the MCD group.1.3 UA inhibits the expression of NOX,NLRP3 inflammasome,and liver fibrosis-related indexes in MCD-induced miceThe protein expressions ofα-SMA,TIMP1,collagen I,NOX2,and NLRP3inflammasome in the livers of mice in the MCD group were higher than those in the MCS group;the protein expressions ofα-SMA,TIMP1,collagen I,NOX2,and NLRP3 inflammasome in the livers of mice in the UA group were significantly lower than those in the MCD group.q PCR experiments to detect the m RNA levels of the same indicators showed similar results to WB experiments.2.Expression of NOX2 and NLRP3 inflammasome in the liver of CCl4-induced mice and the therapeutic effect of UA2.1 UA attenuates liver injury and fibrosis in CCl4-induced miceMice in the CCl4 group exhibited more hepatic structural disorders,fibrous spacing,and collagen fiber deposition and increased liver fibrosis scores than the Control group;mice in the UA group exhibited improved liver fibrosis compared to the CCl4 group.2.2 UA treatment attenuated liver function impairment and hydroxyproline levels in CCl4-induced liver fibrosis miceThe levels of AST,ALT,and hydroxyproline in the serum of mice in the CCl4group were higher than those in the Control group;the levels of AST,ALT,and hydroxyproline in the serum of mice in the UA group were lower compared with the CCl4 group.2.3 UA inhibits the expression of NOX,NLRP3 inflammasome,and liver fibrosis-related indicators in CCl4-induced miceThe protein expression ofα-SMA,TIMP1,Collagen I,NOX2,and NLRP3inflammasome proteins was higher in the CCl4 group than in the Control group.The protein expression ofα-SMA,Collagen I,and TIMP1 was significantly lower in the UA group than in the CCl4 group,and NOX2 and NLRP3 inflammasome protein expression was also lower.Meanwhile,the m RNA expression ofα-SMA,TIMP1,Collagen I,NOX2,and NLRP3 inflammasome was upregulated in the CCl4 group of mice compared with the control group.After UA treatment the expression levels of these indicators decreased.3.Ursolic acid ameliorates liver fibrosis by regulating the NOX2/NLRP3inflammasome signaling pathway3.1 UA alleviates liver fibrosis in MCD-induced mice by regulating NOX23.1.1 Effect of UA on liver injury and fibrosis in NOX2-/-MCD-induced miceLipid accumulation,liver structural destruction,collagen deposition,and inflammatory cell infiltration in the liver of mice in the NOX2-/-+MCD group were significantly reduced compared with the MCD group.There was no significant difference between the NOX2-/-+MCD group and the NOX2-/-+MCD+UA group.3.1.2 Effect of UA on hepatic impairment and hydroxyproline levels in NOX2-/-MCD-induced miceThe levels of AST,ALT,and hydroxyproline in the serum of mice in the NOX2-/-+MCD group were significantly decreased compared with the MCD group.The differences between the NOX2-/-+MCD and NOX2-/-+MCD+UA groups were not statistically significant.3.1.3 Effect of UA on liver fibrosis-related indexes and NLRP3 inflammasome in NOX2-/-MCD-induced miceCompared with the MCD group,the protein and m RNA expression ofα-SMA,Collagen I,TIMP1,and NOX2 were downregulated in the NOX2-/-+MCD groups of mice,while NOX2-/-+MCD groups also had lower protein and m RNA expression of NLRP3 inflammasome.The protein and m RNA levels of each index were downregulated in the NOX2-/-+MCD+UA group compared with the NOX2-/-+MCD group,but there was no statistical difference.3.2 UA alleviates liver fibrosis in CCl4-induced mice by regulating NOX23.2.1 Effect of UA on liver injury and fibrosis in NOX2-/-CCl4-induced miceCompared with the CCl4 group,mice in the NOX2-/-+CCl4 group showed a significant reduction in liver structural disorder,reduced fibrous septum formation,and collagen fiber deposition.The differences between the two groups of NOX2knockout mice were not statistically significant.3.2.2 Effects of UA on liver function and hydroxyproline levels in NOX2-/-CCl4-induced miceThe serum levels of AST,ALT,and hydroxyproline were lower in NOX2-/-+CCl4 group than in the CCl4 group.The levels of AST,ALT,and hydroxyproline in serum were not significantly different between NOX2-/-+CCl4group and NOX2-/-+CCl4+UA group.3.2.3 Effects of UA on liver fibrosis-related indicators and NLRP3inflammasome in NOX2-/-CCl4-induced miceCompared with the CCl4 group,the protein and m RNA expression ofα-SMA,Collagen I,TIMP1,and NOX2 were downregulated in the NOX2-/-+CCl4 group of mice;meanwhile,NOX2-/-+CCl4 group also had lower protein and m RNA expression of NLRP3 inflammasome.The protein and m RNA levels of each index were downregulated in the NOX2-/-+CCl4+UA group compared with the NOX2-/-+CCl4 group,but there was no statistical difference.3.3 UA alleviates liver fibrosis in MCD-induced mice by regulating NLRP3inflammasome3.3.1 Effect of UA on liver injury and fibrosis in NLRP3-/-MCD-induced miceLipid accumulation,liver structural destruction,collagen deposition,and inflammatory cell infiltration in the liver of mice in the NLRP3-/-+MCD group were significantly reduced compared with the MCD group.The differences between the two groups of NLRP3 knockout mice were not statistically significant.3.3.2 Effect of UA on liver function and hydroxyproline levels in NLRP3-/-MCD-induced miceThe levels of AST,ALT,and hydroxyproline in the serum of mice in the NLRP3-/-+MCD group was significantly decreased compared with the MCD group.The differences between the NLRP3-/-+MCD group and NLRP3-/-+MCD+UA group were not statistically significant.3.3.3 Effect of UA on liver fibrosis-related indexes and NOX2 in NLRP3-/-MCD-induced miceThe protein expression ofα-SMA,TIMP1,Collagen I,and NLRP3inflammasome was lower in NLRP3-/-+MCD group mice than in MCD group,however,the protein expression of NOX2 was not significantly different.The protein expression of NOX2 was down-regulated in liver tissues of NLRP3-/-+MCD+UA group mice compared with NLRP3-/-+MCD group.q PCR experiments to detect the m RNA levels of the same indicators showed similar results to WB experiments.3.4 UA alleviates liver fibrosis in CCl4-induced mice by regulating NLRP3inflammasome3.4.1 Effect of UA on liver injury and fibrosis in NLRP3-/-CCl4-induced miceCompared with the CCl4 group,the NLRP3-/-+CCl4 group mice showed reduced liver structural disorders and fibrous septum formation,and significantly reduced collagen fiber deposition.The differences between the two groups of NLRP3knockout mice were not statistically significant.3.4.2 Effects of UA on liver function and hydroxyproline levels in NLRP3-/-CCl4-induced miceThe levels of AST,ALT,and hydroxyproline in the serum of mice in the NLRP3-/-+CCl4 group was significantly decreased compared with the CCl4 group.The differences between the NLRP3-/-+CCl4 group and NLRP3-/-+CCl4+UA group were not statistically significant.3.4.3 Effects of UA on liver fibrosis-related indicators and NOX2 in NLRP3-/-CCl4-induced miceThe protein expression ofα-SMA,TIMP1,Collagen I,and NLRP3inflammasome was lower in NLRP3-/-+CCl4 group mice than in CCl4 group,however,the protein expression of NOX2 was not significantly different.Compared with the NLRP3-/-+CCl4 group,the NLRP3-/-+CCl4+UA group had down-regulated NOX2 protein expression.q PCR results were similar to the WB results.4.UA could regulate intestinal microbiota during liver fibrosis and the effect may be related to the NOX2/NLRP3 inflammasome signaling pathway4.1 Effect of UA on the diversity and composition of intestinal microbiota in MCD-induced miceCompared with the MCS group,theαdiversity of the flora was reduced in the MCD group;compared with the MCD group,theαdiversity of the UA group was significantly restored.βdiversity analysis showed that the samples within each group were close to each other and grouped well in the MCS,MCD,and UA groups.The three groups had differences in microbial community composition.At the phylum level:compared with the MCS group,there was an increase in Firmicutes and a decrease in Bacteroidota in the intestine of mice in the MCD group;compared with the MCD group,there was a decrease in Firmicutes and a rebound in Bacteroidota in the intestine of mice after UA treatment.At the genus level:Akkermansia was increased in the intestine of mice in the MCD group compared with the MCS group;Akkermansia abundance decreased after UA treatment.4.2 Effect of NOX2-/-on the diversity and composition of intestinal flora in MCD-induced miceCompared with the MCD group,theαdiversity of the flora was higher in the NOX2-/-+MCD group;there was no statistical difference inαdiversity between the NOX2-/-+MCD and NOX2-/-+MCD+UA groups.β-diversity analysis showed that the intra-group samples were close and the grouping effect was good.The three groups had differences in microbial community composition.At the phylum level:there were fewer Firmicutes and more Bacteroidota in the intestine of mice in the NOX2-/-+MCD+UA group compared to the MCD group.At the genus level:Helicobacter was lower in NOX2-/-+MCD group than in MCD hepatic fibrotic mice.Helicobacter was also lower in the intestine of the NOX2-/-+MCD+UA group of hepatic fibrotic mice than in the MCD group.4.3 Effect of NLRP3-/-on the diversity and composition of intestinal flora in MCD-induced miceCompared with the MCD group,theαdiversity of the flora was higher in the NLRP3-/-+MCD group;there was no statistical difference inαdiversity between the NLRP3-/-+MCD and NLRP3-/-+MCD+UA groups.β-diversity analysis showed that the intra-group samples were close and the grouping effect was good.The three groups had differences in microbial community composition.At the phylum level:fewer Firmicutes and more Bacteroidota in the intestine of mice in the NLRP3-/-+MCD+UA group compared to the MCD group.At the genus level:Helicobacter was lower in the NLRP3-/-+MCD group mice than in the MCD group;Helicobacter was also lower in the intestine of the NLRP3-/-+MCD+UA group liver fibrosis mice than in the MCD group.Conclusion1.In the development of liver fibrosis,NOX2 acts as an upstream regulator of NLRP3 inflammasome,and UA exerts anti-fibrotic effects by inhibiting the NOX2/NLRP3 inflammasome signaling pathway.2.UA may reverse liver fibrosis by interfering with the NOX2/NLRP3inflammasome signaling pathway and thereby improving intestinal flora. |
