摘要
目的探讨通络益肾方治疗糖尿病肾病的可能作用机制。方法 35只SD大鼠随机分为空白组、西药组及中药大、中、小剂量组,每组7只。中药大、中、小剂量组小鼠每日分别给予6.20、3.10、1.55 g/ml的通络益肾方2 ml灌胃;西药组小鼠每日灌胃洛汀新(0.09 mg/ml)、糖适平(0.27 mg/ml)混合液2ml。每日1次,连续给药10天。于末次灌胃2 h后制备含药血清。大鼠肾小球系膜细胞株HBZY-1细胞分正常组、模型组、西药组及中药大、中、小剂量组,同步化24 h后,正常组加5.6 mmol/L低糖+10%的胎牛血清100μl,模型组加30 mmol/L高糖+10%的空白血清100μl,中药大、中、小剂量组加30 mmol/L高糖及10%的中药大、中、小剂量含药血清100μl,西药组加30 mmol/L高糖+10%的西药血清100μl。分别培养12 h、24 h、48 h和72 h后,采用流式细胞术检测系膜细胞凋亡情况,RT-PCR检测各组系膜细胞葡萄糖调节蛋白78(GRP78)、c-Jun氨基末端激酶(JNK)mRNA的表达水平。结果与正常组同时间点比较,模型组系膜细胞在24 h、48 h和72 h凋亡率持续增加(P<0.01)。与模型组同时间点比较,各给药组24 h、48 h和72 h时系膜细胞凋亡率均明显下降(P<0.01);中药大、中、小剂量组在各时间点系膜细胞凋亡率随着药物剂量升高而降低,呈剂量依赖性(P<0.01)。与正常组同时间点比较,模型组GRP78 mRNA在24 h、48 h持续升高,而72 h明显下降,JNK mRNA表达在48、72 h时明显增加(P<0.05或P<0.01)。与模型组同时间点比较,各给药组系膜细胞GRP78 mRNA、JNK mRNA在48 h、72 h时明显减低,且以中药大剂量组最低(P<0.05或P<0.01)。结论通络益肾方可抑制肾小球系膜细胞凋亡,其作用机制可能与延缓GRP78 mRNA增幅、下调JNK mRNA表达有关。
Objective To investigate the possible mechanism of Tongluo Yishen Formula（ 通络益肾方） in the treatment of diabetic nephropathy. Methods Totally 35 SD rats were randomly divided into blank group,western medicine group and Chinese medicine group with large,medium and small dose,7 rats in each group. The Chinese medicine group with large,medium and small dose were gavaged with 6. 20,3. 10,1. 55 g / ml Tongluo Yishen Formula respectively daily with the amount of 2 ml; The western medicine group were gavaged with 2 ml mixed liquor of Lotensin（ 0. 09 mg / ml） and Gliquidone（ 0. 27 mg / ml）,once a day,continuous administration for 10 days. Two hours after the last time of intragastric administration,medicated serum was prepared. Rat mesangial cell line HBZY-1 cells were divided into normal group,model group,western medicine group and Chinese medicine groups with large,medium and small dose. After synchronization of 24 h,the normal group was added with 5. 6 mmol / L low sugar and 10%fetal calf serum 100 μl,the model group was added with 30 mmol / L high glucose and 10% blank serum 100 μl,the Chinese medicine groups with large,medium and small dose were added with 30 mmol / L high glucose and 10%large,medium and small dose of medicated serum 100 μl respectively,the western medicine group was added with30 mmol / L high glucose and 10% western medicine serum 100 μL. After culture of 12,24,48 and 72 h respectively,the apoptosis of mesangial cells was detected by flow cytometry,RT-PCR detection of the expression level of glucose regulated protein 78（ GRP78） and c-Jun amino terminal kinase（ JNK） mRNA in the mesangial cells was used in all the groups. Results Compared with the normal group at the same time point,the apoptosis rates of mesangial cell in the model group were continuously increased at 24,48 and 72 h（ P 〈 0. 01）. Compared with the model group at the same time point,the apoptosis rate of mesangial cells was significantly decreased at 24,48 and 72 h of each group（ P 〈 0. 01）; In the large,medium and small dose of Chinese medicine group,the apoptosis rates of the mesangial cells at different time points were decreased with the increase of the drug dose,which was dose dependent（ P 〈0. 01）. Compared with the normal group at the same time point,GRP78 mRNA levels in the model group at 24,48 h continued to rise,while at 72 h it was significantly decreased,mRNA JNK expression significantly increased at 48 h and 72 h（ P 〈 0. 05 or P 〈 0. 01）. Compared with the model group at the same time point,mesangial cells of GRP78 mRNA and JNK mRNA of each group decreased significantly at 48 and 72 h,and the large-dose group was the lowest（ P 〈 0. 05 or P 〈 0. 01）. Conclusion Tongluo Yishen Formula can inhibit the apoptosis of glomerular mesangial cells,and its mechanism may be related to the delay of GRP78 mRNA increase and down regulation of mRNA JNK expression.
出处
《中医杂志》
CSCD
北大核心
2016年第7期605-609,共5页
Journal of Traditional Chinese Medicine
基金
陕西省科技厅2013年度科学技术研究发展计划项目(2013JM4034)
陕西省教育厅2011年度科学研究计划项目(11JK0684)
关键词
通络益肾方
糖尿病肾病
系膜细胞
内质网应激
葡萄糖调节蛋白78
C-JUN氨基末端激酶
Tongluo Yishen Formula（通络益肾方）
diabetic nephropathy
mesangial cell
endoplasmic reticulum stress
glucose regulated protein 78
c-jun N-terminal kinase
