| Objective:Diabetic nephropathy is a common and serious complication of diabetes mellitus. In recent years, studies have shown that apoptosis plays pivotal role in the development of nephropathy.Recent studies have found that apoptosis is correlated with the occurrence as well as the development of DN. Observing the morphological and blood biochemical changes as well as employing various methods such as Western Blot, RT-PCR in detecting the mRNA as well as the proteins of GRP78, ATF4, CHOP and the apoptosis of the kidney, we aim to explore the effect of astragalus injection combined with puerarin injection in treating the kidney injury in KKAy mice with diabetes. Therefore, this would shed light upon the molecular mechanism of astragalus injection and puerarin injection in treating and preventing DN, thus providing scientific evidences of the application of astragalus and puerarin injection, which would also offer guidance to the clinical application by discovering new targets of treating DN.Methods:1. In vivo(1) Diabetic, male KKAy mice (fasting blood glucose≧13.9mmol/L) were randomly divided into a model group and a treatment group (used astragalus injection combined with puerarin injection), while age-matched male C57BL/6J mice were selected as controls. The treatment group received daily intraperitoneal injections of astragalus and puerarin, while the model group received injection of an equivalent volume of saline. Mice were euthanized after16weeks. Serum samples were obtained from animals in each group, and blood glucose, creatinine, urea nitrogen, uric acid, cholesterol, and triacylglycerol levels were measured.(2) Tissue samples from the kidney were used for morphometric studies.(3) Renal apoptosis was detected by TUNEL assay.(4) The expressions of GRP78, ATF4, and CHOP were evaluated using reverse transcription-polymerase chain reaction (RT-PCR), and western blot analysis.(5) The protein expression and location of GRP78, ATF4, and CHOP were observed by immunohistochemical method.2. In vitro(1) HK-2cells were cultured in DMEM media supplemented with10%FBS, and then divided into three groups: The normal group (NG),5.5mmol/L glucose.The model group (MG),25mmol/L glucose.The treatment group (TG),25mmol/L glucose, astragalus injection and puerarin injection.(2) HK-2cells were incubated with different concentrations of glucose for48h. Expressions of GRP78, ATF4, CHOP were examined by Real Time PCR and Western blot.Results:1. In vivo(1) The blood glucose levels in the model group were also increased (P<0.01) compared with blood glucose levels in the normal group. Elevated blood glucose levels were significantly reduced (P﹤0.01) after astragalusinjection combined with puerarin injection treatment. Significant differences (P﹤O.01) were found in plasma concentrations of creatinine, blood urea nitrogen, uric acid, cholesterol, and triacylglycerol between the normal group and the model group, The treatment group showed significantly low levels of plasma creatinine (P<0.05), blood urea nitrogen (P<0.01), uric acid, cholesterol, and triacylglycerol (P<0.01) following administration of astragalus injection combined with puerarin injection when compared to the model group.(2) Compared with the normal group, a variety of DN-induced changes in renal morphology were detected in pathology results from the model group, including thickening of the basal membrane and vacuolar degeneration in the renal tubular epithelial cells in KKAy mice. However, treatment with astragalus and puerarin reversed these changes to some degree.(3) Compared with the model group, the renal cell apoptosis were significantly decreased in the treatment group, which was indicated by the TUNEL assay.(4) The model group displayed significantly higher levels of GRP78, ATF4, and CHOP, when compared to levels measured in the normal group. Treatment with astragalus and puerarin significantly reversed increases in GRP78, ATF4, and CHOP, and the immunohistochemical results were consistent with these.2. In vitro(1) Significant reductions in the expressions of GRP78, ATF4, and CHOP, were apparent in diabetic mice treated with astragalusand puerarin compared to relative levels in the model group.Conclusions (1) Astragalus injection combined with puerarin injection significantly lower blood glucose and lipid levels, improved renal function, ameliorated changes in renal histopathology, and delay the apoptosis process of renal cells.(2) KKAy mice aged16weeks showed apparent kidney injuries and also manifested activation of ERS as well as the ERS-induced apoptosis. Astragalus injection combined with puerarin injection may have a curative effect in treating DN by inhibiting the ERS via modulating GRP78, ATF4and CHOP, factors associated with ERS. |
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