| Diabetic nephropathy(DN)is one of the common and serious complications of diabetes mellitus(diabetes mellitus,DM),and also an important cause of end stage renal disease(ESRD).Due to the increase of the incidence of DM,the incidence of DN is also increasing rapidly.However,there is no specific method to prevent the progress of DN.Autophagy stress means that the clearance rate of autophagy vesicles is lower than the generation rate of autophagy vesicles.Cordyceps sobolifera is one of the precious traditional Chinese medicine.It can relieve spasm,course wind and dissipate heat,and deeply search for the function of clearing collaterals,phlegm,heat and blood stasis.It coincides with the theory of miniature mass of renal collateral put foward by professor WANG about the core pathogenesis of DN.Modern researches have demonstrated that cordyceps sobolifera plays a pivotal role in the therapeutic strategies for DN,and has a significan effect in renal protection by inhibiting inflammation,delaying renal fibrosis and reducing oxidative damagethe.Vitamin E(VE),a kind of natural antioxidant,can not only attenuate DN by regulating oxidative stress and inflammation,but also affect DN progress by regulating autophagy stress.However,taking high-dose supplements of VE is not always effective for prevention of disease,and it can even be harmful to the health.On the basis of our previous research findings that cordyceps sobolifera alleviates autophagy stress,and high dose VE effectively protect renal function of type 1 DM rats via alleviation of autophagy stress induced by lysosomal membrane permeabilization as well,we further studied whether the combination of cordyceps sobolifera and medium-dose VE have the function of delaying the development of type 1 DN via alleviation of autophagy stress under the guidance of the theory of miniature mass of renal collateral through in vivo experiments,so as to prove the therapeutic value and significance of culture of Chinese medicine through modern scientific research.OBJECTIVE:On the basis of previous research finding that cordyceps sobolifera plays a specific role in a therapeutic strategy for DN as well as high-dose VE attenuates diabetic nephropathy via alleviation of autophagy stress,we further explored whether cordyceps sobolifera could effectively reduce the dosage of VE under the guidance of the theory of miniature mass of renal collateral.That is,we observed the effect of cordyceps sobolifera combined with medium-dose VE on type 1 DN rats,and studied whether the renal protective function of cordyceps sobolifera combined with medium-dose vitamin E delaying the progression of type 1 DN were related to via alleviation of autophagy stress.METHOD:The renal tissue samples of 17 clinical patients were taken as the research object.Through the identification of their clinical data,the 9 renal tissue samples from patients with diabetic nephropathy confirmed by renal biopsy were classified into diabetic renal group(DN),and the 8 renal tissue samples from patients with albuminuria and hematuria but with small renal histopathological changes were classified into control group.The IF were used to detect the expression of LC3? and SQSTM1/p62 in renal tissues of 17 patients.In addition,all the rats were randomly divided into shan operation group(SO),sham operation+cordyceps sobolifera combined with vitamin E group(SO+CVE),model group(DN),model group+cordyceps sobolifera combined with vitamin E group(DN+CVE).The DN rats were made by unilateral nephrectomy and STZ injection.SO group and DN group were given normal saline for 12 weeks,SO+CVE group and DN+CVE group were given 1 mg/kg cordyceps sobolifera+300 mg/kg VE for 13 weeks.The changes of blood glucose and body weight were monitored and recorded every week during gavage.After the blood and urine were collected at the end of the 13th week,all the animals were sacrificed to collect kidney tissue samples.The renal injury indexes such as Scr,24h urinary microalbumin and NGAL were detected by ELISA.The histological changes of kidney were observed by Masson staining and PAS staining.The western blot(WB)was used to detect the level of Beclinl,LC3?,SQSTM1/p62,LAMP2,COX-?,NF-?B,?-SMA and TGF-?1 in the kidney,the expression of LC3? and SQSTM1/p62 by immunofluorescence(IF)as well.Immunohistochemical(IHC)was used to detect the level of ?-SMA and Col-? in the kidney.The datum were analyzed by SAS 9.2.RESULTS:The expression of LC3? and SQSTM1/p62 of renal tubular in DN group was significantly higher than that in control group,suggesting the occurrence of autophagy stress in patients with DN(P<0.01).What's more,compared with the SO group,the body weight of DN group decreased,and the level of the ration of kidney weight and body weight,blood glucose,Scr,24-hour urine albumin and NGAL were increased(P<0.01);compared with DN group,the level of the body weight and the blood glucose of DN+CVE did not change significantly(P>0.05),whereas,the ration of kidney weight and body weight,the Scr,24-hour urine albumin and the NGAL level of DN+CVE decreased(P<0.01).PAS and Masson staining showed that the the masangial area of DN group was significantly widened,the mesangial matrix was aggregated,glomerular basement membrane was thickened,and renal interstitial fibrosis was obvious.After 13 weeks of intervention with cordyceps sobolifera and VE,the glomerular mesangial matrix dilation and renal tubulointerstitial damage were improved.WB showed that,compare with SO group,the expression of Beclinl,AGT7 and LAMP2 in DN group decreased,and the LC3?,SQSTM1/p62,COX-?,NF-?B,and ?-SMA level in DN group increased(P<0.05);compared with DN group,the expression of Beclinl,AGT7 and LAMP2 in DN group up-regulated,and the LC3?,SQSTM1/p62,COX-?,NF-?B,and?-SMA level in DN group down-regulated(P<0.05).IF showed that the level of LC3? and SQSTM1/p62 in DN group was higher than that in SO group(P<0.05);the level of LC3? and SQSTM1/p62 of DN+CVE group was lower than that in SO group(P<0.05).IHC showed that,compare with SO group,the expression level of ?-SMA and Col-? in DN group improved;compared with DN group,the expression level of ?-SMA and Col-? in DN+CVE group declined.CONCLUSIONS:1.Cordyceps sobolifera can effectively reduce the dosage of high-dose vitamin E,to say,cordyceps sobolifera combined with medium-dose vitamin E plays a pivotal role in protecting the renal function of type 1 DN rats,but it has no effect on the level of blood sugar;2.Cordyceps sobolifera combined combined with medium-dose vitamin E contribute to the development of therapeutic strategies for type 1 DN via alleviation of autophagy stress,and the mechanism of autophagy stress may be related to the disfunction of the lysosomal-mitochondrial axis;3.Cordyceps sobolifera combined combined with medium-dose vitamin E plays a key role in inhibiting inflammation and delaying the progression of renal fibrosis. |
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