| ObjectiveDiabetic Nephropathy (DN) is one of the most common and serious complications of diabetes with its complicated pathogenesis. This experiment was focused on studying the effect of baicalin on the gap junction (GJ) communication medicated by connexin43 phosphorylation (p-Cx43) in DN rats kidneyso that to explore the GJ communication mechanism in the development of DN and find new ways for the prevention and treatment of DN in TCM.Method(1) In vivo experiment,70 male SD rats,8 weeks old,180g-220g, were adapted to the SPF environment for two days before experiment. Rats were randomly divided into normal group (10 rats)and DN model group (60 rats). Rats in normal group were breeded with normal diet while those in model group were fed with high fat food. A month later, all rats were deprived of food for 12h. DN model was induced with a single intraperitoneal injection of STZ (35mg/kg body weight),while rats in normal control received the same volume of 0.lmol/L citrate buffer. After 5-7 days treatment with STZ, random blood glucose was tested in all rats by cutting tail, and the blood sugar level of 16.7 mmol/L or higher was diagnosis as diabetes mellitus. Later on, all rats were fed with normal diet. After 8 weeks, rats with blood glucose levels of 16.7 mmol/L or higher, strongly positive urine glucose levels and 50% urine volume more than normal group in 24h were considered diabetic nephropathy. Rats that developed DN was assigned to model group, baicalin group and enalapril group randomly. Each group was lavaged with corresponding drug. After treatment for 6 weeks, all rats were weighed and sacrificed after blood glucose measured, double kidneys were removed and capsules were excluded. The left kidneys were fixed in 10% buffered formaline solution for HE staining and immunohistochemistry. The right kidneys were rapidly put into liquid nitrogen and stored at -80℃for western blot.(2)In vitro level, HK-2 were co-cultured with low glucose (5.5mmol/L, LG), high glucose (25mmol/L, HG), high glucose+baicalin (100μmol/L), high glucose± enalapril(10μmol/L) for 24h,48h,72h for proliferation. Average proliferation rate of each group was tested by MTT method.Result(1) Blood glucose levelBefore treatment, the blood glucose level of model rats was statistically significant higher than the normal group (P<0.05). After treating, the blood glucose level of baicalin group and enalapril group were lower than the model group, the difference was statistically significant(P<0.05). Antidiabetic effect of baicalin was better than enalapril.(2)Renal indexAfter treatment, the renal index of model group, baicalin group and enalapril group was higher than the normal group, the difference was statistically significant(P<0.05). Compared with model group, renal index of baicalin group and enalapril group was lower than the model group, but there was no statistical significance(P>0.05). And renal index of baicalin group was a little lower than enalapril group.(3)Renal pathology observationAfter treatment, the glomerular volume increased, renal mesangial cell proliferation, renal tubular epithelial cell edema in model group, baicalin group and enalapril group compared with nomal group. At the mean time, nephropathy was relieved in baicalin group compared with model group and the curative effect was better than enalapril group.(4) Renal PKC expressionPKC was positive expression in renal tubular epithelial cell membrane ofmodel group but notinnormal group. Compared with model group, the particle number of PKC in baicalin group and enalaprilat group was decreased significantly with faded colorand considered as moderate positive expression. And the PKC positive expression of baicalin group was lower than enalaprilat group.(5) Renal p-Cx43 expressionThe weakly positive expression of p-Cx43 was in renal tubular epithelial cell membrane of normal group. P-Cx43 brown granules was observed in model group and considered as strong positive expression. Compared with model group, the number of p-Cx43 brown particle in baicalin group and enalapril group was decreased significantlywith faded color, and diagnosed as moderate positive expression. And p-Cx43 positive expression of baicalin group was lower. In addition, the trend of p-Cx43 protein expression of the kidney was consistent with positive expression of p-Cx43 on renal tubular.(6)Average proliferation rateThe proliferation rate of HK-2 was measured in 24 hour,48 hourand 72 hour. The growth of HK-2 appeared double effect in high sugar environment. It was proliferation after high sugar intervention for 24h, while it was significantly suppressed after intervention for 48 to 72h, and the inhibition rate was proportional to the time. Baicalin increased HK-2 cells in a time-dependent manner. It was statistically significant when compared with model group (P<0.05), and the proliferation effect was better than enalaprilat group.ConelusionBaicalin could relieve the diabetic kidney damage in high glucose environment. In vivo,, baicalin could ameliorate the structural and functional abnormalities and the gap junction communication in kidneys in DN rats. Its mechanism may be corrected with the reduction of PKC and p-Cx43 expression in kidney. In vitro, high glucose could suppress the growth of HK-2, baicalin could promote proliferation of HK-2 in high glucose environment on time-dependent. |
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