七味白术散对糖尿病大鼠肠道屏障和TLR4/NF-κB信号通路的影响

Effects of Qiwei Baizhu San on intestinal barrier and TLR4/NF-κB signaling pathway in diabetic rats

摘要:

【背景】七味白术散可用于治疗气阴两虚所致的消渴病，尤其近些年来在改善糖尿病胰岛素抵抗方面得到广泛使用。【目的】观察七味白术散对糖尿病大鼠肠道屏障和炎症因子的影响，并探讨其作用机制。【方法】60只无特定病原体(specific pathogen free,SPF)级SD雄性大鼠随机分为空白组、模型组、二甲双胍组和七味白术散高、中、低剂量组，每组10只。腹腔注射链脲佐菌素(streptozotocin,STZ)的方法建立糖尿病大鼠模型，空白组大鼠给予正常饮食，模型组、二甲双胍组和七味白术散高、中、低剂量组给予高糖高脂饮食。造模成功后分别用二甲双胍和七味白术散灌胃处理，空白组和模型组生理盐水灌胃。观察治疗前后大鼠的一般生存状况；药物干预4周后，全自动生化分析仪检测空腹血糖(fasting blood glucose,FBG)、空腹胰岛素(fasting insulin,FINS)、脂多糖(lipopolysaccharide,LPS)；酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)测定血清白细胞介素-6(interleukin-6,IL-6)、肿瘤坏死因子-α (tumor necrosis factor-α,TNF-α)水平；采用苏木精-伊红(hematoxylin-eosin,HE)染色法观察各组大鼠结肠组织病理改变，免疫组化法观察结肠组织occludin蛋白的表达位置及表达水平，Western blotting法检测回肠组织中zonula occludens-1(ZO-1)、occludin、toll-like receptor 4(TLR4)、核因子kappa B(nuclear factor kappa B,NF-κB)蛋白的表达。【结果】与空白组比较，造模后的各组大鼠空腹血糖值≥11.1mmol/L，差异有统计学意义(P<0.01)。与模型组比较，七味白术散低、中剂量组大鼠FBG有所下降，差异无统计学意义(P>0.05)；七味白术散高剂量组和二甲双胍组大鼠FBG均明显下降，与模型组比较差异有统计学意义(P<0.05)。与模型组比较，七味白术散低、中、高剂量组和二甲双胍组大鼠FINS、LPS、IL-6、TNF-α水平均显著降低，差异有统计学意义(P<0.01)。与空白组比较，模型组大鼠肠道细胞排列紊乱，肠绒毛断裂不全且间隔稀疏，杯状细胞明显减少且分布不均，同时伴有中性粒细胞渗出，occludin蛋白平均光密度值显著降低(P<0.01)。与模型组比较，七味白术散高剂量组及二甲双胍组大鼠肠绒毛排列较为整齐，完整性较好，结肠杯状细胞数量均有不同程度的恢复，中性粒细胞渗出减少，occludin蛋白平均光密度值均有不同程度升高(P<0.01)。Western blotting显示，与模型组比较，七味白术散高剂量组及二甲双胍组大鼠回肠组织ZO-1、occludin蛋白表达量均升高，差异有统计学意义(P<0.01)，TLR4、NF-κB蛋白表达量均降低，与模型组比较差异有统计学意义(P<0.01)。【结论】七味白术散可以通过上调肠道紧密连接蛋白的表达促进肠黏膜机械屏障修复，维持紧密连接的完整性，降低血清LPS水平，抑制TLR4/NF-κB通路，减少IL-6、TNF-α等炎症因子的释放，从而减轻机体炎症反应，改善胰岛素抵抗。

Abstract:

[Background] Qiwei Baizhu San can be used to treat diabetes caused by deficiency of both qi and yin. Especially in recent years, it has been widely used in improving insulin resistance of diabetes mellitus. [Objective] To observe the effects of Qiwei Baizhu San on intestinal barrier and inflammatory factors in diabetic rats and to explore its mechanism. [Methods] Sixty SD male rats in specific pathogen free (SPF) grade were randomly divided into a normal control group, a model group, a metformin group, and high, medium, and low-dose Qiwei Baizhu San groups, with 10 rats in each group. The diabetic rat model was established by intraperitoneal injection of streptozotocin (STZ). Rats in the normal control group were given a normal diet, and rats in the model group, the metformin group, and the high, medium, and low-dose Qiwei Baizhu San groups were all given high-sugar and high-fat diets. After successful modeling, the metformin group and the Qiwei Baizhu San groups were treated with metformin and Qiwei Baizhu San by gavage, respectively. Normal saline was given by gavage in the normal control group and the model group. The general living status of rats before and after treatment was observed. After 4 weeks of drug intervention, fasting blood glucose (FBG), fasting insulin (FINS), and lipopolysaccharide (LPS) were detected by an automatic biochemical analyzer. Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in rat serum were measured by enzyme-linked immunosorbent assay (ELISA). Hematoxylin-eosin (HE) staining was used to observe the pathological changes in colon tissues of rats in each group, and immunohistochemistry was used to observe the expression position and level of occludin protein in colon tissues. Western blotting assay was used to determine the protein expressions of zonula occludens-1 (ZO-1), occludin, toll-like receptor 4 (TLR4), and nuclear factor kappa B (NF-κ B). [Results] As compared with the normal control group, the FBG of rats after modeling in each group was ≥11.1 mmol/L (P <0.01). As compared with the model group, the FBG of rats in the low and medium-dose Qiwei Baizhu San groups decreased, but the difference was statistically insignificant (P >0.05). The FBG of rats in the high-dose Qiwei Baizhu San group and the metformin group decreased as compared with the model group (P <0.05). As compared with the model group, the levels of FINS, LPS, IL-6, and TNF-α in the metformin group and high, medium, and low-dose Qiwei Baizhu San groups decreased (P <0.01). As compared with the blank group, the intestinal cells in the model group were disorderly arranged, the intestinal villi were incompletely broken and sparsely spaced, the goblet cells were significantly reduced and unevenly distributed, accompanied by neutrophil exudation, and the average optical density of occludin protein was reduced (P <0.01). As compared with the model group, the intestinal villi of the rats in the high-dose Qiwei Baizhu San group and the metformin group were more arranged neatly and completely, the number of colonic goblet cells was restored to varying degrees, the neutrophil exudation was reduced, and the average optical density of occludin protein was increased to varying degrees (P <0.01). The results of Western blot showed that as compared with the model group, the protein expressions of ZO-1 and occludin in the ileum tissues of rats in the high-dose Qiwei Baizhu San group and the metformin group increased (P <0.01), and the protein expressions of toll-like receptor 4 (TLR4) and nuclear factor kappa B (NF-κ B) decreased (P <0.01). [Conclusion] Qiwei Baizhu San can up-regulate the expression of tight junction protein in the intestine, promote the repair of intestinal mucosal mechanical barrier, maintain the integrity of tight junctions, reduce the level of serum LPS, inhibit the TLR4/NF-κ B pathway, and reduce the release of IL-6, TNF-α, and other inflammatory factors, thus reducing the inflammatory reaction and improving insulin resistance of the body.