基于生信技术从AGE/RAGE通路探讨参七糖络丸“清中通络”改善2型糖尿病认知功能障碍的分子机制

Molecular mechanism of Shenqi Tangluo Pills on improving type 2 diabetes cognitive impairment from AGE/RAGE signal pathway based on bioinformatics technology

目的:基于网络药理学和分子对接技术预测具有“清中通络”作用的参七糖络丸改善2型糖尿病认知功能障碍(T2DM-CI)的分子机制并进行实验验证。方法:选取60只SPF级7周龄db/db小鼠纳入实验,随机分为模型对照组,阳性对照组和参七糖络丸高、中、低剂量组,另选12只同龄db/m小鼠作为空白对照组。阳性对照组每日给予二甲双胍(0.26 g/kg)灌胃,参七糖络丸高、中、低剂量组每日分别给予76、38、19 g/kg参七糖络丸灌胃,空白对照组和模型对照组每日给予等量蒸馏水灌胃,持续干预8周。首先测定小鼠体质量和空腹血糖,采用Morris水迷宫测定小鼠学习记忆功能,采用HE染色法观察脑组织病理形态变化;采用网络药理学和分子对接技术预测参七糖络丸干预T2DM-CI的可能靶点,并采用分子生物学技术验证相关靶点。结果:与正常对照组比较,模型对照组小鼠空腹血糖和体质量显著升高(P<0.05),逃避潜伏期显著增加(P<0.05),穿越平台次数显著减少(P<0.05),海马不同分区神经元排列紊乱,胞膜模糊,胞核固缩;与模型对照组比较,阳性对照组和参七糖络丸各剂量组均显著降低小鼠体质量(除参七糖络丸低剂量组外)和空腹血糖水平(P<0.05),阳性对照组和参七糖络丸高、中剂量组小鼠逃避潜伏期显著增加(P<0.05),穿越平台次数显著减少(P<0.05),各给药组小鼠海马组织病理损伤均有改善;与参七糖络丸低剂量组比较,参七糖络丸高剂量组表现出更加显著的改善作用(P<0.05),参七糖络丸的干预呈现显著的剂量依赖。网络药理学预测结果表明槲皮素、汉黄芩素、山柰酚是与疾病靶点连接最为广泛的活性化合物,而PPI网络互作发现JUN、MAPK1、RELA是“药物-疾病”交集靶点中最为关键的靶点,分子对接结果表明槲皮素、汉黄芩素、山柰酚和JUN、MAPK1、RELA靶点均能较好结合,KEGG富集发现AGE/RAGE信号通路的异常响应可能是参七糖络丸干预T2DM-CI的核心机制,分子生物学检测结果表明与空白对照组比较,模型对照组小鼠脑组织和血清中AGEs含量显著升高(P<0.05),脑组织中RAGE蛋白表达显著升高(P<0.05),JUN、ERK、NF-κB p65蛋白磷酸化水平显著升高(P<0.05);与模型对照组比较,参七糖络丸高剂量组小鼠脑组织和血清中AGEs含量显著降低(P<0.05),脑组织中RAGE蛋白表达显著降低(P<0.05),JUN、ERK、NF-κB p65蛋白磷酸化水平显著降低(P<0.05)。结论:参七糖络丸显著改善T2DM-CI小鼠学习记忆功能的作用与有效抑制AGE/RAGE信号通路及下游信号分子在脑组织中的异常响应有关。

Objective:Based on network pharmacology and molecular docking technology,predict the molecular mechanism of Shenqi Tangluo Pills with the function of Qingzhong Tongluo'to improve cognitive impairment in type 2 diabetes(T2DM-CI)and carry out experimental verification.Methods:A total of 50 SPF 6-week-old db/db mice were selected,and then randomly divided into model control group,positive control group and high,medium and low dose Shenqi Tangluo Pills groups.Another 10 db/m mice of the same age were selected as the blank control group.The positive control group was given metformin(0.26 g/kg)by gavage every day,the high,middle and low dose groups of Shenqi Tangluo Pills were given different concentrations of Shenqi Tangluo Pills(76,38,19 g/kg)by gavage every day,the blank control group and model control group were given the same amount of distilled water by gavage every day for 8 weeks.Firstly,the body mass and fasting blood glucose of mice were measured.Morris water maze was used to measure the learning and memory function of mice.HE staining was used to observe the pathological changes of brain tissue;Subsequently,network pharmacology and molecular docking techniques were used to predict the possible targets of Shenqi Tangluo Pills intervention in T2DM-CI,and molecular biology techniques were used to verify the prediction results.Results:Compared with the normal control group,the fasting blood glucose and body mass of rats in the model control group were significantly increased(P<0.05),the escape latency of mice in the model control group was significantly increased(P<0.05),and the number of times they crossed the platform was significantly reduced(P<0.05).The neurons in different areas of hippocampus in the model control group were disordered,the membrane was vague,and the nucleus was pyknotic.Compared with the model control group,both the positive control group and the high,medium and low dose groups of Shenqi Tangluo Pills significantly reduced the body mass(except for the low dose group of Shenqi Tangluo Pills)and fasting blood glucose level of mice(P<0.05).The escape latency of the positive control group and the high and medium dose groups of Shenqi Tangluo Pills significantly increased(P<0.05),and the number of times of crossing the plaform significantly decreased(P<0.05),and the pathological changes in hippocampus of mice in each treatment group were alleviated.Compared with the low dose group of Shenqi Tangluo Pills,the mice in the high dose group of Shenqi Tangluo Pills showed more significant intervention effect(P<0.05),and the intervention of Shenqi Tangluo Pills showed significant dose dependence.The prediction results of network pharmacology showed that quercetin,wogonin and kaempferol were the most widely connected active compounds with disease targets,while the PPI network interaction found that JUN,MAPK1 and RELA were the most critical targets among the'drug disease'intersection targets.The results of molecular docking showed that quercetin,scutellarin,kaempferol and JUN,MAPK1,RELA targets could bind well,KEGG enrichment found that AGE/RAGE signal pathway may be the core signal pathway of Shenqi Tangluo Pills intervention in T2DM-CI,The results of molecular biological verification showed that compared with the blank control group,the content of AGEs in the brain tissue and serum of the model control group was significantly increased(P<0.05),the expression of RAGE protein in the brain tissue was significantly increased(P<0.05),and the phosphorylation levels of JUN,ERK,NF-kB p65 protein in the brain tissue were significantly increased(P<0.05).Compared with the model control group,the content of AGEs in brain tissue and serum of mice in the high-dose Shenqitangluo pill group decreased significantly(P<0.05),the expression of RAGE protein in brain tissue decreased significantly(P<0.05),and the phosphorylation levels of JUN,ERK,NF-kB p65 protein in brain tissue decreased significantly(P<0.05).Conclusion:SQTLW can significantly improve the learning and memory function of T2DM-CI mice,which is related to the effective inhibition of AGE/RAGE signal pathway and the abnormal response of downstream signal molecules in brain tissue.