Study On The Effect Of Shenkang Injection On Renal Interstitial Fibrosis And Endoplasmic Reticulum Stress In UUO Model Mice

Object:This study was aimed to explore the effect and mechanism of Shenkang injection on renal interstitial fibrosis in UUO model mice,and to provide scientific basis and guidance for clinical application of traditional Chinese medicine in treating renal interstitial fibrosis.Methods:After one week of adaptive feeding,60 male C57BL/6 mice were randomly divided into two groups according to weight:Sham group(n=10)and operationl group(n=50).The left ureter was free but not ligated in the sham-operated group under anesthesia,while the left ureter was ligated in the operation group under anesthesia.After operation,the mice of operationl group were randomly divided into five groups:UUO group(model group),ARB group(losartan potassium group)and H group(high dose group of Shenkang injection),M group(middle dose group of Shenkang injection)and L group(low dose group of Shenkang injection),with 10 mice in each group.Drug intervention began on the first day after operation:the mice of Sham group and UUO group were given normal saline(0.13ml/10g)by tail vein injection and normal saline(0.15ml/10g)by gavage;the mice of ARB group was given saline(0.13ml/10g)by tail vein injection and losartan potassium solution(0.15ml/10g)by gavage;and the mice of SKI-H group,SKI-M group and SKI-L group were treated with high,middle and low dose of Shenkang injection(0.13ml/10g)by tail vein injection and normal saline(0.15ml/10g)by gavage respectively.The drug was given continuously for 14 days.During the experiment,the general condition,weight and food-intake of the mice were observed and recorded.On the 14th day,urine of the mice was retained to detecte the urine microalbuminuria(UALB).At the end of the medication,the blood was got from the eyeball and the level of BUN,CREA,UA and Cys-c was measured.Then kidney tissue of all mice were sampled and given HE dyeing and Sirius red staining to observe the pathological changes of kidney tissues;and Apoptosis measured by TUNEL.And the expression distribution of p-PERK,p-eIF2α,ATF4,CHOP,GRP78,caspase-12 and caspase-3 in kidney tissue were observed under immunohistochemical staining.Changes of endoplasmic reticulum in renal tubular epithelial cells observed by scanning electron microscopy.Result:1.The Results of Serology and Urine Test:Compared with sham-operated group,the serum creatinine(CREA),urea nitrogen(BUN),urea(UA),cystatin C(Cys-c)and urinary microalbumin(UALB)in the mice of model group did not change significantly(P>0.05);compared with the mice of model group,the above indexes in each treatment group did not change significantly(P>0.05).2.Pathological observation:HE staining and Sirius red staining showed that there was no obvious atrophy of glomeruli,tight and orderly arrangement of renal tubules,no dilation and atrophy of renal tubules,infiltration of inflammatory cells and proliferation of interstitial fibrous tissue in sham-operated mice.In the model group,a large number of tubular epithelial cells necrosis,exfoliation,tubule dilatation,inflammatory cell infiltration and partial glomerulonephritis were observed in the renal tissue of the model group.The renal interstitial area was significantly enlarged and the positive staining degree of collagen was significantly increased.Compared with the sham operation group,the percentage of collagen positive staining area in the model group was significantly higher than that in the sham operation group.Compared with the model group,the renal tubules of each treatment group were dilated,The increasing degree of renal interstitial area and the positive staining degree of interstitial collagen were lower than those in the model group,especially in the high dose group of Shenkang injection.3.Electron microscopic observation:In the sham-operated group,the endoplasmic reticulum of renal tubular epithelial cells presented a layered tubular structure,with a small number of ribosomes attached to the endoplasmic omentum,and normal structures such as nuclei and mitochondria.In the model group,endoplasmic reticulum of renal tubular epithelial cells showed swelling in varying degrees,tubular structure changed and even disappeared,endoplasmic reticulum cavity expanded and expanded,some of them showed vacuolar structure,and ribosomes attached to membrane increased.Nuclear swelling,partial rupture;proliferation of renal interstitial fibroblasts.The above-mentioned situation in each treatment group was alleviated to some extent.4.Immunohistochemical staining detection:1)There was only a small amount of GRP78 expression in kidney tissue of mice in sham-operated group;compared with sham-operated group,the expression of GRP78 in model group was significantly increased(P<0.05),mainly in tubular area.Compared with the model group,GRP78 in each treatment group decreased in varying degrees,and there was significant difference between the high and medium dose groups of Shenkang injection(P<0.05).2)In the sham-operated group,the expression of P-PERK,p-eIF2α,ATF4 protein and CHOP protein were small.Compared with the sham-operated group,the expression of the above-mentioned proteins in the model group increased significantly(P<0.05),mainly in the renal tubules and their surrounding areas.Compared with the model group,the expression of P-PERK,p-eIF2a and CHOP proteins in the kidney tissue of mice in the high,middle,low dose Shenkang injection group and ARB group decreased,with statistical significance(P<0.05);ATF4 proteins in each group also decreased,but the difference was not Significant(P>0.05).In each treatment group,the change of Shenkang injection high dose group was more significant.3)The expression of caspase 12 and caspase 3 protein in sham-operated mice was slightly higher than that in model mice(P<0.05).Compared with model mice,the expression of caspase 12 and caspase 3 in each treatment group decreased in varying degrees,especially in high dose Shenkang injection group(P<0.05).5.TUNEL detection:under fluorescence microscope,the number of apoptotic cells in the normal group was very small;the number of apoptotic cells in the model group was large and mainly located in the renal tubule area;the number of apoptotic cells in the high,middle and low dose Shenkang injection group was less than that in the model group,and was more obvious in the high and middle dose group.Conclusion:1.Shenkang injection was able to improve the Renal Interstitial Fibrosis in UUO model micee,and the effect is more obvious at high dose of Shenkang injection;2.Apoptosis mediated by endoplasmic reticulum stress PERK-eIF2α-ATF4-CHOP pathway was occurred in the post operative kidney of UUO model mice;The downstream cytokine may be related to the activation of caspase12 and caspase3.3.Shenkang injection was able to improve the Renal Interstitial Fibrosis in UUO model mice.The mechanism may be related to decrease the levels of cytokines in PERK-eIF2α-ATF4-CHOP pathway in endoplasmic reticulum stress and other related proteins and inhibit cell apoptosis.