| Objective:To explore the effect and mechanism of n-acetyl-l-tryptophan(L-NAT)pretreatment on carbon tetrachloride(CC14)induced liver fibrosis in mice.Methods:This study consists of two parts:in vivo study and in vitro study.In vivo study:(1)Establishment of mouse liver fibrosis model:40 male C57BL/6 mice were randomly divided into four groups:blank group(control group),model group(CC14 group),low concentration of n-acetyl-l-tryptophan group(CCL4+L-NAT 2.5)and high concentration of n-acetyl-l-tryptophan group(CCL4+ L-NAT 5).The mice in the blank group were given olive oil(2.5ml/kg)through intraperitoneal injection,the other groups were given 20%CC14 solution(2.5ml/kg)through intraperitoneal injection,twice a week,for 6 weeks.The mice in the low concentration group of n-acetyl-l-tryptophan(CCL4+L-NAT 2.5)and the high concentration group of n-acetyl-l-tryptophan(CCL4+L-NAT 5)were given intraperitoneal injection of n-acetyl-l-tryptophan(2.5mg/kg and 5mg/kg)on the basis of CCL4 twice a week for 6 weeks.After 72 hours the last CC14 was injected,the mice in each group were euthanized,and the liver tissue and peripheral blood were collected;(2)The content of αSMA protein in liver tissue was observed by immunofluorescence;(3)ALT and AST in serum of mice were detected by transaminase kits;(4)The morphological changes of liver were observed by HE staining;(5)The collagen deposition was observed by Masson and Sirius red staining;(6)Western blotting was used to detect the protein expression of Bax,Bcl-2,MMP9 andαSMA,and the protein expression of LATS1/Yap1 and TGF β/Smad pathway in the liver tissues of mice.In vivo study:(1)CCK-8 method was used to detect the effects of different concentrations of n-acetyl-l-tryptophan o on the proliferation of HSC-6 mouse cells in vitro;(2)TGF β was used to stimulate hsc-6 mouse hepatic stellate cells to construct mouse hepatic fibrosis model which were divided into blank group(control),n-acetyl-l-tryptophan(L-NAT 20 group),model group(TGF β 10 group)and n-acetyl-l-tryptophan+model group(TGF β 10+L-NAT 20 group)(3)Western blot was used to detect the expression of Bax,Bcl-2,α-SMA and MMP9 protein,and the expression of TGF β/Smad pathway related proteins.Results:The in vivo results showed that:(1)the levels of ALT and AST in serum of mice in N-acetyl-L-tryptophan pretreatment group were significantly lower than those in CC14 group(2)HE results showed that N-acetyl-Ltryptophan liver tissue morphology change pretreatment group was obviously lighter than CCL4 group;(3)The results showed that the content of collagen fiber and the formation of fiber septum in the N-acetyl-L-tryptophan preconditioning group were significantly lower than those in the CCl4 group.(4)Frozen section immunofluorescence showed that the distribution of α-SMA collagen in N-acetyl-L-tryptophan pretreatment group was less than that in CC14 group;(5)Compared with CC14 group,the protein expressions of Bcl-2 and LATS1 in liver tissues of N-acetyl-L-tryptophan preconditioning group were significantly increased,while the protein contents of Bax,MMP9,α-SMA and YAP 1 were significantly decreased.(6)Compared with CC14 group,n-acetyl-l-tryptophan pretreatment group down regulated the expression of TGFβ/Smad pathway protein in liver tissue.The results in vitro showed that:(1)In different time(12h,24h,48h),20 μmol/L n-acetyl-l-tryptophan had no significant effect on the proliferation of hsc-6 hepatic stellate cells;(2)Compared with TGF β group,the protein contents of Bcl-2 in TGF β 10+L-NAT 20 group were significantly increased,while the protein contents of Bax,α-SMA and MMP9 were significantly decreased;(3)Compared with TGF βgroup,the expression of TGF β/Smad pathway in TGF β 10+L-NAT 20 group was significantly decreased.Conclusion:N-acetyl-L-tryptophan can reduce the liver fibrosis induced by CC14 in mice,and the possible mechanism is the inhibition of apoptosis by N-acetyl-L-tryptophan.Regulation of LATS1/YAP1 is related to the expression of TGFβ/Smad protein pathway. |
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