摘要目的 探讨组蛋白去乙酰化酶抑制剂———曲古抑菌素 A(trichostatin A,TSA)对s-100诱导的小鼠自身免疫性肝炎的影响.方法 将26只6周龄C57BL/6小鼠随机分为对照组、模型组和TSA组,每组6只,另外8只小鼠用于提取肝组织s-100蛋白.模型组和TSA组小鼠腹腔注射s-100和完全弗氏佐剂,建立小鼠自身免疫性肝炎模型,第21天,TSA组小鼠连续7 d腹腔注射 TSA 2 mg/(kg ·d),对照组和模型组小鼠腹腔注射等量含1% 二甲基亚砜的0.9% 氯化钠溶液.检测各组小鼠血清ALT和AST水平,观察肝组织病理学.蛋白质免疫法检测肝组织内核因子-κB和乙酰化组蛋白 H3;实时定量PCR检测肝组织内核因子-κB、HDAC3、TLR4及TNF-α mRNA水平;ELISA法检测血清 TNF-α.各组间比较采用 t检验.结果 对照组、模型组和TSA组小鼠血清AL T 分别为(122.00 ± 45.29)、(459.33 ± 167.58)和(217.33 ± 49.25)U/L,对照组、TSA组与模型组比较,差异均有统计学意义(t值分别为4.76、3.41,均 P<0.05);AST 分别为(127.83 ± 18.55)、(389.67 ± 87.14)和(249.50 ± 71.72) U/L,与模型组比较,差异均有统计学意义(t值分别为7.20、3.04,均 P<0.05).TSA减轻了s-100诱导的肝组织病理炎症;模型组小鼠肝组织中核因子-κB蛋白、核因子-κB mRNA、TNF-α mRNA、HDAC3 mRNA和TLR4 mRNA的相对表达量分别为2.43 ± 0.42、9.51 ± 0.36、10.53 ± 0.74、2.90 ± 0.22和4.50 ± 0.73,较对照组1.28 ± 0.49、1.28 ± 0.49、1.06 ± 0.14、1.72 ± 0.73和1.01 ± 0.31明显升高(t值分别为4.68、37.14、30.69、4.33和10.85,均 P< 0.05),TSA组核因子-κB蛋白、核因子-κB mRNA、TNF-α mRNA、HDAC3 mRNA及TLR4 mRNA相对表达量分别为1.30 ± 0.36、1.30 ± 0.36、2.38 ± 0.36、2.13 ± 0.32和2.40 ± 0.51,也明显低于模型组(t值分别为4.58、30.62、24.12、2.81、5.81,均 P<0.05).对照组、模型组和 TSA 组小鼠血清 T NF-α分别为(122.37 ± 68.12)、(1361.44 ± 207.13)和(691.64 ± 162.12)ng/L,与模型组比较,差异均有统计学意义(t值分别为13.92、6.24,均 P<0.05).模型组乙酰化组蛋白 H3蛋白相对表达量为1.10 ± 0.08,高于对照组的0.96 ± 0.17(t= 2.27,P<0.05),TSA组为1.30 ± 0.04较模型组表达更高(t= -0.30,P<0.05).结论 TSA可能通过增强组蛋白乙酰化、抑制核因子-κB通路及炎性因子,缓解自身免疫性肝炎.

abstractsObjective To investigate the effect of trichostatin A(TSA),a histone deacetylase inhibitor, on s-100-induced autoimmune hepatitis in mice.Methods A total of 26 six-week-old male C57BL/6 mice were randomly divided into control group,model group and TSA group(six in each group),and the rest 8 mice were used to extract the s-100 protein from liver tissue.Mice of model group and TSA group were injected intraperitoneally with s-100 with complete Freund's adjuvant to induce autoimmune hepatitis model.At day 21, TSA group mice were injected intraperitoneally with TSA 2 mg/(kg·d)for 7 days,and 0.9% sodium chloride solution containing 1% dimethyl sulfoxide was injected into the control and model group mice.Alanine transaminase(ALT)and aspartate aminotransferase(AST)in serum were measured and liver histopathology was observed.The protein levels of nuclear factor(NF)-κB and acetylated histone H3 in liver tissue were detected by Western Blot.The hepatic mRNA levels of NF-κB,HDAC3,toll-like receptor 4(TLR4)and TNF-α were measured by real-time PCR.ELISA was used to determine the TNF-α in serum.The results were analyzed with t test.Results The serum levels of ALT in control group,model group and TSA group were(122.00 ± 45.29),(459.33 ± 167.58)and(217.33 ± 49.25)U/L,respectively.The differences between model group and control group or TSA group were significant(t=4.76 and 3.41,respectively,both P<0.05).The serum levels of AST in control group,model group and TSA group were(127.83 ± 18.55),(389.67 ± 87.14)and (249.50 ± 71.72)U/L,respectively.The differences between model group and control group or TSA group were also significant(t= 7.20 and 3.04,respectively,both P< 0.05).The inflammation of the liver histopathology induced by s100 was alleviated by TSA.The relative expressions of NF-κB protein,NF-κB mRNA,TNF-α mRNA,HDAC3 mRNA and TLR4 mRNA in the liver tissue of model group mice were 2.43 ± 0.42,9.51 ± 0.36,10.53 ± 0.74,2.90 ± 0.22,and 4.50 ± 0.73,respectively,which were significantly higher than those of the control group(1.28 ± 0.49,1.28 ± 0.49,1.06 ± 0.14,1.72 ± 0.73,and 1.01 ± 0.31, respectively)(t=4.68,37.14,30.69,4.33 and 10.85,respectively,all P <0.05).In TSA group,the relative expressions of NF-κB protein,NF-κB mRNA,TNF-α mRNA,HDAC3 mRNA and TLR4 mRNA were decreased(1.30 ± 0.36,1.30 ± 0.36,2.38 ± 0.36,2.13 ± 0.32 and 2.40 ± 0.51,respectively),which were statistically lower than those in model group(t=4.58,30.62,24.12,2.81 and 5.81,respectively,all P<0.05).The serum TNF-α levels in control group,model group and TSA group were(122.37 ± 68.12), (1361.44 207.13)and(691.64 ± 162.12)ng/L,respectively.Compared with model group,the differences were statistically significant(t=13.92 and 6.24,respectively,both P<0.05).The relative expression of ac-H3 protein in the model group was 1.10 ± 0.08,which was higher than that in the control group 0.96 ± 0.17(t=2.27,P<0.05).That in TSA group was 1.30 ± 0.04,which was higher than the model group(t=-0.30, P <0.05).Conclusion Histone deacetylase inhibitor TSA alleviates autoimmune hepatitis by enhancing histone acetylation and inhibiting NF-κB and inflammatory factors.