| objective:1.To investigate the effect of Cordyceps Sinensis on the renal tubular epithelial Cell apoptosis following renal ischemia and reperfusion in rats;2.To study the mechanisms of the endoplasmic reticulum stress pathway associated renal tubular epithelial cell apoptosis following renal ischemia and reperfusion in Rat;3. To investigate the effect of Cordyceps Sinensis on the endoplasmic reticulum stress pathway associated renal tubular epithelial cell apoptosis following renal ischemia and reperfusion in Rat.Methods:The 40 healthy male Sprague-dawley rats were randomly divided into four groups:normal group(n=5),shame operation group(n=15),renal ischemia and reperfusion model group(n=15),and treatment group with Cordyceps Sinensis.the rat models were established by following methods:Firstly,right kidney was excised.Then,left renal pedicle was clamped for 60 min of ischemia.Finally,the reperfusion was accomplished by releasing the clamps.The rats in the normal group was not treated,and the rats in the shame operation group was only subjected to excise right kidney without clamping left renal pedicle.The rats in the renal ischemia and reperfusion model group and treatment group with Cordyceps Sinensis were sacrificed at 24h,48h,72h after reperfusion respectively,the urine volume were observed,renal function in rats was detected,and the pathology changes were observed by hematoxylin and eosin(HE) stainning,as well as analyzing the condition of pathology damage by tubulointerstitial semi-quantitative scoring method.and the renal tubular epithelial cells apoptosis was detected with TUNEL.Meanwhile,the expression development in the renal tissue of elF2αphosphorylation,caspase-12,bcl-2,bax,caspase-3mRNA and/or proteins were measured with methods of reverse transcription-Polymerase chain reaction(RT-PCR),inununohistochemistry and Western blot.Finally, the effect of Cordyceps Sinensis on the expression of these genes and proteins were investigated.Results:1.The pathology changes following renal ischemia and reperfusion in rats include tubule cell necrosis,focal areas of proximal tubular dilation and distal tubular casts,effacement and loss of proximal tubule brush border, interstitial inflammatory cell infiltration, etc. Increase of BUN and Scr level,reduction of urine output,and increase of renal tubular epithelial cells apoptosis were found, which reached a peak 24h after reperfusion. Compared with the rats in the shame operation group, the differences were statistically significant (P<0.01).Correlation analysis among renal tubular epithelial cells apoptosis, tubulointerstitial injury, BUN level and Scr level showed an positively correlation (r2=0.891,0.952,0.945). Cordyceps Sinensis could increase the urine volume,lower the level of BUN and Scr,improve the pathology changes,reduce the renal tubular epithelial cells apoptosis. Compared with the rats in the I/R group at 24h,48h,72h after reperfusion respectively, the differences were statistically significant (P<0.05 or P<0.01)2.The expression of elF2αphosphorylation,caspase-12,bcl-2,bax and caspase-3mRNA and proteins in the renal tissue after reperfusion enhance. However,the ratio of bcl-2 and bax mRNA and/or proteins lower. The expression of elF2αphosphorylation, caspase-12,bax and caspase-3 mRNA and/or proteins reached a peak 24h after reperfusion,but the expression of bcl-2 mRNA and/or proteins reached a peak 72h after reperfusion.The changes in the parameters keep in parallel with the pathology and tubular epithelial cells apoptosis in the kidney tissue.3.Compared with I/R group at 24h,48h and 72h after reperfusion respectively, treatment with Cordyceps Sinensis could decrease elF2αphosphorylation protein expression,there were statistical signifcance in the difference(P<0.01).In this study,we have observed that Cordyceps Sinensis treatment significantly down-regulated caspase-12mRNA and protein expression at 24h,48h and 72h after reperfusion (P<0.01).Furthermore,Cordyceps Sinensis treatment could decrease bax mRNA expression(P<0.01) and Protein expression(P<0.01)at24h,48h and 72h after reperfusion,compared with I/R group respectively. Meanwhile,Cordyceps Sinensis treatment could also significantly down-regulated caspase-3mRNA and protein expression at 24h,48h and 72h after reperfusion (P<0.01).However,we have observed that Cordyceps Sinensis treatment significantly up-regulated bcl-2 mRNA and protein expression at 24h,48h and 72h after reperfusion (P<0.05 or P <0.01), compared with I/R group respectively.Conclusion:1.We have observed that the rats with ischemia and reperfusion injury presented pathology changses including tubule cell necrosis, focal areas of proximal tubular dilation and distal tubular casts,effacement and loss of proximal tubule brush border, interstitial inflammatory cell infiltration, etc,increase of BUN level and Scr level,decrease of urine output,which suggests successful I/R rat models.2.Apoptosis play an active role in the renal I/R injury,which may suggest that it is an important lose pathway of renal tubule cells. Our investigation demonstrated that Cordyceps Sinensis treatment could protect against renal tubule cells apoptosis.3.Renal isehemia and reperfusion may induce ER, which initiated PERK mediated URP signal pathway. Subsequently,caspae-12 was activated.we observed URP signal pathway paralleled with the changes of the apoptosis cell counting after renal ischemia/reperfusion in rats,which suggested that ER maybe play an important role in renal tubule cells apoptosis. Meanwhile,the across-talking of ERS pathways and chondriosome pathways plays an important role in renal tubule cells apoptosis.4. Cordyceps Sinensis treatment could provide a protection against renal tubule cells apoptosis,which acts on by influencing the over-activated PERK/elF2a signaling,reducing the activation of caspase-12,and working on the across-talking of ERS pathway and chondriosome pathway.which suggested that ERS pathway could become to be the target of Cordyceps Sinensis. |
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