| In a variety of mechanisms of diabetic nephropathy, endoplasmic reticulum stress (ERS) was confirmed that has close relationship with diabetic nephropathy, ERS is a common mechanism of early injury response in diabetic organ parenchymal cells, got widespread attention.In the three pathways of ERS, UPR is the most in-depth study, is also considered a sign of occurrence of endoplasmic reticulum stress. On the endoplasmic reticulum membrane, there exists important protein:IRE1,PERK,ATF4.The three across membrane proteins can efficiently amplify stress signals, and start ERS.Generally, PERK、 ATF6and IRE1forming a non-active compound with grp78(glucose regulating protein78, GRP78), but GRP78will dissociate from PERK and combine with the unfolded protein when There are a lot of free unfolded proteins in cells. PERK is exposed and phosphorylated, the activated PERK make eif2a phosphorylated too. All these response will reduce the transport and synthesis of new proteins, effectively alleviate the protein overload in endoplasmic reticulum lumen.Meanwhile, phosphorylation eif2α can specifically increase the expression of transcriptional activity factor-4(ATF4). It can induce expression of apoptosis related factor CHOP, make excessive injuried cells start the apoptotic program, thereby reducing the protein load of cells; so activation of PERK and phosphorylation eif2a can be considered to be the sign of endoplasmic reticulum stress.the characteristics of Traditional Chinese medicine in the treatment of diabetic nephropathy is multi-channel, multiple targets, multiple links, which advantage have got more and more recognition. This experiment put a perspective on the ERS, discusses the treatment mechanism of Huangqi (Radix Astragali seu Hedysari) injection combined Puerarin injection on DN, look forward to provide basis for traditional Chinese medicine on DN.Objective:Observe the effects of Astragalus and Puerarin mongholicus Injection on endoplasmic reticulum stress-related factors such as GRP78PERK and eif2a in KKAy diabetic mice and human kidney tubular epithelial cells, at the same time, Observe morphological and biochemical changes.And to further explore effects of Astragalus and Puerarin mongholicus Injection in prevention and treatment of DN, reveal the mechanism of Astragalus and Puerarin mongholicus Injection in adjusting the endoplasmic reticulum stress reaction, provide a experimental basis for Astragalus and Puerarin mongholicus Injection in the treatment of DN, which will be a new targets for clinical medicine.Methods:1、in vivo: (1) After random blood glucose detection, KKAy mice which random blood glucose higher than13.9mmol/L would be the subjects. KKAy mice fed fat diet were randomly divided into model group and treatment group, treatment group were intervented by puerarin and astragalus injection, the c57BL/J mice given normal diet were used to be control group. treatment group were given Puerarin injection combine Astragalus mongholicus injection by intraperitoneal injection from twelve weeks old. These mice were sacrificed respectively at16weeks old, take blood about1ml, Mice serum biochemical tests GLU、UREA、CREA、TG、 CHOL were measured.(2) After the mice were sacrificed in16weeks old, cut the kidney tissue to make HE staining tissue sections,observe pathological change of renal tissue under light microscope. Product electron microscopy specimens, observe ultrastructure by TEM.(3)Immunohistochemistry was used to detect expression of GRP78protein in kidney tissue of mice.(4)detect expression of GRP78, eif2α and PERK mRNA in kidney tissue of mice by Real-time PCR(5)detect expression of GRP78、eif2α and PERK protein in kidney tissue of mice by western-blot.2、in vitro:(1) Cultured human renal tubular epithelial cells (HK-2), grouped according to the following requirements:model group:stimulated by high glucose; treatment groups:joint stimulation by high glucose, astragalus and puerarin injection; control group:normal cultured human kidney tubule epithelial cells(2)Detect expression of GRP78、eif2α and PERK mRNA in human kidney tubule epithelial cells by Real-time PCR(3)Detect expression of GRP78、eif2α and PERK protein in human kidney tubule epithelial cells by western-blot.Result:1、in vivo(1) Blood biochemical tests in mice showed that, compared with normal mice, the blood glucose, creatinine, urea, cholesterol and triglycerides of model group were higher, glucose and urea of treatment group mice was significantly lower than model group.(p<0.05)(2) Through HE staining, we can observed that glomerular structure is complete and clear, there is no interstitial fibrosis in control group kidney tissue; at the same time, model group could be found early pathological changes of diabetic nephropathy:mesangial matrix increase, a small vacuoles appeared in the cytoplasm of renal tubular epithelial cells, slight tubular atrophy. in treatment group, structure of glomerular and tubular were integrated and clear, small vacuoles were visible, the pathological injury is not significantly.TEM observation shows that in control group mice kidney,structure of glomerular and mitochondrial is intact, foot processes arrange orderly. The model group are mild glomerular basement membrane thickening, swelling of endoplasmic reticulum and the nucleus, mitochondrial swelling, foot process disorganized.Compared with the same weeks-old model mice, the treatment group mice are mild glomerular basement membrane thickening, increased glomerular mesangial matrix mild; tubular epithelial cell mitochondria, endoplasmic reticulum mild swelling.(3)Immunohistochemistry showed that expression of GRP78proteins in model group mice kidney was significantly higher than control group;compared with the control group,proteins expression in treatment group slightly increased, but significantly lower than model group.(4) The result of Real-time PCR shows that the expression of PERK. eif2α and GRP78mRNA in model group was higher than control group,and the treatment group was obviously reduced compared with model group.(5) The result of Western blot showed that the expression of PERK. eif2a and GRP78proteins in model group were higher than control group and treatment group,meanwhile,there was no significant differences.2、in vivo(1) the expression of PERK mRNA, eif2a mRNA, GRP78mRNA of human renal tubular epithelial cells in model group was significantly higher than control group, the treatment group significantly decreased compared with the model group.(2) Expression of eif2a, GRP78protein in Model group is higher than control group and the treatment group, no significant difference between the treatment group and the control group; expression of PERK protein has no significant difference between the three groups.Conclusion:(1) Astragalus injection combined with puerarin injection can decrease blood sugar and blood lipids, protect renal function.(2) In16weeks old, KKAy mice appear significant renal dysfunction,high expression of endoplasmic reticulum stress-related mRNA and protein proved ERS is activated, and the endoplasmic reticulum stress is an important part of the progression of diabetic nephropathy.Astragalus injection combined with puerarin injection plays a role in the treatment of diabetic nephropathy by adjusting the expression of mRNA and protein of endoplasmic reticulum stress-related factor such as PERK, eif2a and GRP78. (3) High glucose can induce renal tubular epithelial cells occur endoplasmic reticulum stress response, Astragalus and puerarin injection can effectively mitigate symptom of treatment group, and probably treat diabetes kidney injury by affecting the unfolded protein response to protect renal tubular epithelial cells. |
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