基于cGAS-STING通路探讨增液通腑逐瘀方治疗脓毒症大鼠肠屏障功能障碍的机制

Exploring the mechanism of intestinal barrier dysfunction in septic rats treated with Zengye Tongfu Zhuyu Formula based on cGAS-STING pathway

关键词:  脓毒症  增液通腑逐瘀方  cGAS-STING信号通路  凋亡  坏死性凋亡

Key words:  sepsis  Zengye Tongfu Zhuyu Formula  cGAS-STING signaling pathway  apoptosis  necroptosis

摘要:

[目的] 探讨增液通腑逐瘀方基于cGAS-STING信号通路治疗脓毒症大鼠肠屏障功能障碍的效应机制。[方法] 采用盲肠结扎穿孔术（CLP）建立脓毒症大鼠模型，将36只健康SPF级雄性SD大鼠随机分为假手术组，模型组，增液通腑逐瘀方低、高剂量组（以下分别简称低剂量组、高剂量组），每组各9只。连续给药3 d后取回肠组织，制备病理学切片，采用Chiu’s评分测定肠道损伤程度，电镜下观察肠道细胞超微结构。通过蛋白质免疫印迹法（Western blot）检测肠道环鸟苷酸腺苷酸合酶（cGAS）、干扰素基因的刺激因子（STING）、TANK结合激酶1（TBK1），磷酸化干扰素调节因子3（pIRF3）及磷酸化混合谱系激酶结构域样蛋白（pMLKL）表达。通过原位缺口末端标记（TUNEL）法检测肠道细胞凋亡率。[结果] 与假手术组相比，模型组Chiu’s评分、cGAS、STING、TBK1、pIRF3及pMLKL相对表达量和肠道细胞凋亡率均升高，差异有统计学意义（P<0.01）。电镜下发现模型组微绒毛受损严重，细胞间隙增宽，细胞间连接装置损坏；与模型组相比，低剂量组Chiu’s评分、TBK1及pIRF3相对表达量和肠道细胞凋亡率均降低，差异有统计学意义（P<0.05或P<0.01）；高剂量组Chiu’s评分、cGAS、STING、TBK1、pIRF3及pMLKL相对表达量和肠道细胞凋亡率均降低，差异有统计学意义（P<0.01）；电镜下观察低、高剂量组肠道结构受损情况均有不同程度改善。[结论] 增液通腑逐瘀方可以抑制脓毒症时cGAS-STING信号通路被激活，并通过抑制下游通路关键蛋白和调节因子的合成和磷酸化过程进而抑制细胞凋亡和坏死性凋亡的发生，最终维持肠黏膜屏障完整性，治疗脓毒症肠屏障功能障碍。

Abstract:

[Objective] To explore the effect mechanism of the Zengye Tongfu Zhuyu Formula based on the cGAS-STING signaling pathway in treating intestinal barrier dysfunction in rats with sepsis. [Methods] A rat model of sepsis was established by cecum ligation and perforation（CLP），and 36 healthy SPF-grade male SD rats were randomly divided into the sham-operated group，the model group，and the low- and high-dose groups of Zengye Tongfu Zhuyu Formula，with 9 rats in each group. The intestinal tissues were retrieved after 3 consecutive days of drug administration，and pathological sections were prepared to determine the degree of intestinal damage by Chiu’s score，and the ultrastructure of intestinal cells was observed under an electron microscope. The expression of intestinal cyclic guanosine adenylate synthase（cGAS），stimulator of interferon genes（STING），TANK-binding kinase 1（TBK1），phosphorylated interferon regulatory factor 3（pIRF3），and phosphorylated mixed-spectrum kinase structural domain-like protein（pMLKL） was detected by protein immunoblotting（Western blot）. The apoptosis rate of intestinal cells was detected by the in situ nick end labeling（TUNEL） method. [Results] Compared with the sham-operated group，the relative expression of Chiu’s score，cGAS，STING，TBK1，pIRF3，and pMLKL and the apoptosis rate of intestinal cells were elevated in the model group，and the differences were statistically significant（P<0.01）. Electron microscopy revealed that microvilli in the model group were severely damaged，cell gaps were widened，and intercellular junctions were damaged；compared with the model group，Chiu’s score，relative expression of TBK1 and pIRF3，and apoptosis rate of intestinal cells were reduced in the Zengye Tongfu Zhuyu Formula low-dose group，and the differences were statistically significant（P<0.05 or P<0.01）；in the Zengye Tongfu Zhuyu Formula high-dose group，Chiu’s score，cGAS，STING，TBK1，pIRF3 and pMLKL relative expression and intestinal cell apoptosis rate were all reduced，and the difference was statistically significant（P<0.01）；electron microscopic observation of intestinal structural damage in the Zengye Tongfu Zhuyu Formula low and high dose groups were improved to different degrees. [Conclusion] Zengye Tongfu Zhuyu Formula can inhibit the activation of a cGAS-STING signaling pathway in sepsis，and inhibit apoptosis and necrotic apoptosis by inhibiting the synthesis and phosphorylation of the key proteins and regulatory factors of the downstream pathway，and ultimately maintain the integrity of the intestinal mucosal barrier，and treat the intestinal barrier dysfunction in sepsis.