保肾通络方对高糖培养下肾小球系膜细胞外基质增生及外泌体miR-192表达的影响

Effect of Baoshen Tongluo recipe on the proliferation of mesangial extracellular matrix and the expression of miR-192 in exosomes under high glucose culture

目的观察保肾通络方对高糖培养下肾小球系膜细胞外基质增生及外泌体miR-192表达的影响,探讨其防治糖尿病肾病(DN)的作用机制。方法将20只SD大鼠随机分为正常对照组、保肾通络方低剂量组、保肾通络方中剂量组及保肾通络方高剂量组,每组5只。保肾通络方低、中、高剂量组大鼠分别予浓度为0.5、1.0、2.0 g/mL的保肾通络方颗粒剂药液灌胃,正常对照组大鼠予等容积蒸馏水灌胃。各组大鼠均连续灌胃7 d,通过腹主动脉取血并分离出含药血清。将永生化小鼠肾小球系膜细胞分为正常对照组、高糖对照组、保肾通络方低剂量组、保肾通络方中剂量组及保肾通络方高剂量组。正常对照组肾小球系膜细胞予DMEM低糖培养基+正常对照组大鼠血清培养,高糖对照组肾小球系膜细胞予含30 mmol/L葡萄糖的高糖培养基+正常对照组大鼠血清培养,保肾通络方低、中、高剂量组肾小球系膜细胞分别予高糖培养基+10%的保肾通络方低、中、高剂量含药血清培养。培养24 h后,采用CCK-8法检测各组细胞增殖活性情况,免疫荧光法检测细胞外基质蛋白胶原蛋白Ⅳ(CollagenⅣ)及纤维连接蛋白(FN)表达情况,实时荧光定量聚合酶链式反应(Real-time PCR)法检测CollagenⅣ、FN及外泌体miR-192的mRNA表达情况。结果高糖对照组培养后肾小球系膜细胞增殖活性与正常对照组比较明显升高(P<0.05),保肾通络方低、中、高剂量组培养后肾小球系膜细胞增殖活性与高糖对照组比较明显降低(P<0.05),但保肾通络低、中、高剂量组细胞增殖活性组间比较差异均无统计学意义(P>0.05)。高糖对照组培养后CollagenⅣ及FN灰度值与正常对照组比较均升高(P<0.05),保肾通络方低、中、高剂量组培养后CollagenⅣ及FN灰度值与高糖对照组比较均降低(P<0.05),但保肾通络方低、中、高剂量组CollagenⅣ及FN灰度值组间比较差异均无统计学意义(P>0.05)。高糖对照组培养后CollagenⅣ、FN及外泌体miR-192的mRNA表达与正常对照组相比均明显上调(P<0.05),保肾通络方低、中、高剂量组培养后CollagenⅣ、FN及外泌体miR-192的mRNA表达与高糖对照组比较均明显下调(P<0.05),但保肾通络方各剂量组CollagenⅣ、FN及外泌体miR-192的mRNA表达组间比较差异均无统计学意义(P>0.05)。结论保肾通络方可以降低DN肾小球系膜细胞增殖活性,抑制细胞外基质增生,减少CollagenⅣ及FN蛋白的表达,并能够下调CollagenⅣ、FN及外泌体miR-192的mRNA表达,从多途径对DN进行干预治疗。

Objective To observe the effect of Baoshen Tongluo recipe on the proliferation of mesangial extracellular matrix and the expression of miR-192 in exosomes under high glucose culture,and to explore the mechanism of its prevention and treatment of DN.Methods Twenty SD rats were randomly divided into normal control group,low,middle and high dose group of Baoshen Tongluo recipe,with five rats in each group.The rats in the low,middle and high dose groups of Baoshen Tongluo formula were respectively given 0.5 g/mL,1.0 g/mL and 2.0 g/mL Baoshen Tongluo recipe granule solution by gavage,and the rats in the normal control group were given equal volume distilled water by gavage.The rats in each group were given intragastric gavage for 7 days,blood was taken from abdominal aorta and drug serum was separated.The conditioned immortalized rats mesangial cells were divided into normal control group,high glucose control group,low,middle and high dose group of Baoshen Tongluo recipe.The cells in the normal control group were cultured with DMEM low glucose middle and normal control group serum,the cells in the high glucose control group were cultured with 30 mmol/L glucose high glucose middle and normal control group serum,and the cells in the low,middle and high dose groups were cultured with high glucose middle and 10%low,middle and high dose serum respectively.After 24 hours of culture,CCK-8 was used to detect the proliferation activity of each group of cells,the expression of extracellular matrix protein collagenⅣ(CollagenⅣ)and fibronectin(FN)was detected by immunofluorescence method,and the mRNA expression of collagenⅣ,FN and miR-192 in exosomes was detected by real-time fluorescence quantitative polymerase chain reaction(Real-time PCR).Results Compared with the normal control group,the proliferative activity of the cells in the high glucose control group was significantly higher(P<0.05).Compared with the high glucose control group,he proliferative activity of the cells in the low,middle and high dose groups of Baoshen Tongluo formula was significantly lower(P<0.05),but there was no significant difference between the groups of each dose of Baoshen Tongluo recipe (P>0.05).The gray values of collagenⅣand FN in the high glucose control group were higher than those in the normal control group(P<0.05).The gray values of collagenⅣand FN in the low,middle and high dose groups of Baoshen Tongluo recipe were lower than those in the high glucose control group(P<0.05),but there was no significant difference between the groups of each dose of Baoshen Tongluo recipe(P>0.05).The mRNA expression of collagenⅣ,FN and exocrine miR-192 in the high glucose control group was significantly higher than that in the normal control group(P<0.05).The mRNA expression of collagenⅣ,FN and exocrine miR-192 in the low,middle and high dose groups of Baoshen Tongluo recipe was significantly lower than that in the high glucose control group(P<0.05),but there was no significant difference in the mRNA expression of collagenⅣ,FN and exocrine miR-192 between the groups of each dose of Baoshen Tongluo recipe(P>0.05).Conclusion Baoshen Tongluo recipe can also reduce the proliferation of DN mesangial cells,inhibit the proliferation of extracellular matrix,reduce the expression of collagenⅣand FN,and down regulate the expression of miR-192 in exosomes,so as to intervene DN through multiple ways.