小檗碱抑制脓毒症诱导的肠上皮细胞凋亡

目的:观察小檗碱对脓毒症小鼠肠上皮细胞凋亡的影响及其作用机制。方法:8~10周雄性C57BL/6小鼠,随机分为假手术组(sham组)、脓毒症模型组(CLP组)、小檗碱治疗组(CLP+Ber组)、小檗碱对照组(sham+Ber组)。CLP组行盲肠结扎穿孔术复制小鼠脓毒症模型,其余组除不结扎盲肠外,其它操作同CLP组。各组小鼠术后2 h内分别予双蒸水、小檗碱(50 mg/kg)灌胃。术后20 h,小鼠腹腔注射过量的戊巴比妥钠处以安乐死后开腹取回肠组织,HE染色观察各组小鼠肠组织形态学改变;Western blot法观察各组肠组织凋亡相关蛋白caspase-3降解片段、胞浆组织细胞色素C(Cyt C)、线粒体蛋白Bax及细胞总蛋白Bcl-2、Fas、Fas L、Fas相关死亡域结构蛋白(FADD)的含量变化;real-time PCR法检测各组肠组织酪氨酸羟化酶(TH)和多巴胺β-羟化酶(DBH)的mRNA表达水平。结果:小鼠CLP术后20 h,可见脓毒症模型组小鼠肠绒毛坏死并伴有大量炎症细胞浸润;模型组肠组织caspase-3降解片段显著增多,线粒体Bax、胞浆Cyt C及总蛋白Fas、Fas L含量显著增高,而总蛋白Bcl-2含量明显降低,FADD未见明显改变;脓毒症模型组肠组织TH、DBH的mRNA表达明显增高。术后给予小檗碱治疗后可显著减轻肠组织炎症,逆转caspase-3降解片段、Bax、Cyt C、Fas、Fas L、Bcl-2蛋白和TH、DBH mRNA的表达。结论:小檗碱可显著抑制脓毒症小鼠肠上皮细胞凋亡,其机制可能与抑制内、外源性凋亡途径有关。

Berberine inhibits enterocyte apoptosis in septic mice

AIM: To observe the effects of berberine (Ber) on enterocyte apoptosis in septic mice and its possible mechanism. METHODS: Male C57BL/6 mice (8~10 weeks old) were randomly divided into sham group, cecal ligation and puncture (CLP) group, CLP+Ber group and sham+Ber group. The mice in CLP group underwent CLP ope-ration, and the mice in sham groups suffered a similar operation except the ligation and puncture. After the sham or CLP operation, the mice were administered intragastrically with distilled water or berberine (50 mg/kg) within 2 h. After 20 h, the mice were killed with excess pentobarbital sodium and the ileum tissues were removed. The histological changes of the intestine were observed and the enterocyte apoptosis was examined by determining the protein level of cleaved caspase-3. Furthermore, mitochondrial Bax, cytoplasm cytochrome C (Cyt C) and the total proteins of Bcl-2, Fas, FasL and Fas-associated protein with death domain (FADD) were examined by Western blot. The mRNA expression of tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) was measured by real-time PCR. RESULTS: The extensive ileum injuries, including remarkably increased leukocytes and necrosis of intestinal villus were observed 20 h after CLP. In CLP group, the protein levels of cleaved caspase-3, cytoplasm Cyt C, as well as Fas, FasL were significantly increased, but the Bcl-2 level was decreased. Bax translocation into mitochondria was promoted. However, FADD was not changed significantly. The mRNA expression of TH and DBH was also increased sharply in CLP group. On the contrary, treatment with berberine made a considerable alleviating alteration in the ileum of the septic mice.CONCLUSION: Treatment with berberine provides protective effects on intestinal injury in septic mice by reducing enterocyte apoptosis, and its possible mechanism may be involved in the inhibition of the endogenous and exogenous apoptosis pathways.