Study On The Mechanism Of Keluoxin Capsule For Diabetic Nephropathy Based On PERK-ATF4-CHOP Signaling Pathway

With the rapid increase of the global prevalence of diabetes mellitus,diabetic nephropathy has become the main cause of end-stage renal disease worldwide,increasing the risk of premature death and bringing a heavy economic and social burden.Apoptosis of renal tubular epithelial cells is an important part of renal tubular injury,and it is also the key link of diabetic nephropathy.Previous studies have found that the accumulation of advanced glycation end products under high glucose conditions is one of the important reasons for the apoptosis of renal tubular epithelial cells in diabetic nephropathy by activating the PERK-ATF4-CHOP signaling pathway and inducing endoplasmic reticulum stress.Therefore,in-depth exploration of the characteristics of this link and the corresponding intervention measures is of great significance for understanding the molecular mechanism of diabetic nephropathy and finding new drug intervention targets.Keluoxin capsule(formerly known as Tangweikang capsule)is the first new Chinese medicine approved for the treatment of diabetic nephropathy developed by Professor Lin Lan,the chief researcher of the Chinese Academy of Chinese Medical Sciences.It has the effects of nourishing the liver and kidney,supplementing qi and Yin,activating blood circulation and removing blood stasis.A large number of studies have been carried out in the early stage,involving pharmacy,pharmacodynamics,clinical efficacy evaluation and mechanism exploration,which fully affirmed the efficacy of Keluoxin capsule in the treatment of diabetic nephropathy.Studies have shown that Keluoxin capsule can not only inhibit the production of advanced glycation end products in patients with diabetic kidney disease,but also inhibit the overexpression of TIMP-1 in renal cortex of diabetic rats and enhance the expression of MMP-9,which is mainly located in renal tubular epithelial cells,promote the degradation of extracellular matrix,protect the renal tubular basement membrane,inhibit the progression of renal interstitial fibrosis,and protect the renal tubules.However,the specific action pathway of keluoxin capsule in protecting renal tubular epithelial cells from injury is still unclear.Based on the previous findings of the research group,this study was funded by the Beijing Natural Science Foundation(7202172)to explore how Keluoxin capsules can further exert renal protection after inhibiting the formation of advanced glycation end products.Objective1.To clarify the mechanism of keluoxin capsule in intervening the apoptosis of DN renal tubular epithelial cells by regulating PERK-ATF4-CHOP signal pathway after inhibiting the formation of advanced glycation end products.2.To confirm that the traditional Chinese medicine pathogenesis of DN,which is characterized by "deficiency of both qi and yin,stasis and blood blockage",is related to the pathological features of "endoplasmic reticulum stress-apoptosis" in modern research.Methods1.In vivoThe db/db mice model of spontaneous diabetic nephropathy was used.After 2 weeks of adaptive feeding,all db/db mice were randomly divided into 4 groups:model group(db/db+Vehicle),Keluoxin low dose group(db/db+KLXL),Keluoxin high dose group(db/db+KLXH),and valsartan group(db/db+Valsartan),with 12 mice in each group.Twelve db/m mice were used as normal control group(db/m+vehicle);The low dose group and the high dose group were given 0.78g.d-1.kg-1、3.12g.d-1.kg-1 Keluoxin suspension,valsartan group was given 10.40mg.d-1.kg-1 suspension,the normal group and the model group were given the same volume of carboxymethylcellulose sodium solution,which was gavaged once a day at a fixed time,with l0ml.kg-1 for 12 weeks.All groups of mice were weighed daily for food intake and water consumption,weighed at a fixed time every week,blood was taken from the tail tip every 4 weeks to measure blood glucose,and 24-hour urine was collected to record urine volume.uALB was detected by ELISA,Ucr was detected by automatic biochemical analyzer,and UAER,ACR and CCR were calculated.After 12 weeks of administration,eyeballs were taken for blood,AGEs was detected by ELISA,and Scr,BUN,TC,TG,LDL-C and HDL-C were detected by automatic biochemical analyzer.Weigh the weight of both kidneys,calculate the kidney coefficient,and take the right kidney tissue for fixation and freezing.The morphological changes of renal tissue were observed by HE,PAS and Masson staining,the ultrastructure of renal tubular epithelial cells was observed by transmission electron microscope,the apoptosis of renal tubular epithelial cells was observed by TUNEL staining,the localized expression of GRP78 protein was detected by immunohistochemistry,the expression of GRP-78 mRNA was detected by RT-PCR,and PERK,p-PERK,P-eIF2α,eIF2α,ATF4,CHOP,Bcl-2,Bax,cleaved Caspase-3,cleaved caspase-9 protein expression were detected by Western blot.2.In vitro(1)The apoptosis injury model of human renal tubular epithelial cell line(HK-2)induced by glycosylated bovine serum albumin(AGE-BSA)was established.The effect of AGE-BSA on the viability of HK-2 cells was detected by CCK-8 method,and the optimal concentration and time of AGE-BSA induced HK-2 cell modeling were screened.Hoechst 33342 staining was used to observe the nuclear changes of HK-2.Annexin V-FITC/PI double staining flow cytometry was used to detect the effect of AGE-BSA on apoptosis of HK-2 cells.(2)Keluoxin medicated serum was used to intervene AGE-BSA induced HK-2 cells.The relative survival rate of HK-2 cells was detected by CCK-8 method,and the best intervention concentration of medicated serum was selected.Hoechst 33342 staining,fluorescence microscope observation and photography,annexin V-FITC/PI double staining flow cytometry to detect the effect of drug containing serum on AGE-BSA induced apoptosis of HK-2 cells.The effects of Keluoxin containing serum on the expression of GRP78 protein and mRNA in HK-2 cells were detected by Western blot and RT-PCR.Construct PERK-shRNA vector,silence PERK and block PERK-ATF4-CHOP pathway.Western blot was used to detect the effects of Keluoxin medicated serum on the expression of PERK,p-PERK,P-eIF2α,eIF2α,ATF4,CHOP,Bcl-2,Bax,Cleaved Caspase-3 and Cleaved Caspase-9 proteins.Results of the study1.In vivo(1)Effects of Keluoxin capsule on food intake and water consumption of db/db mice:compared with db/m mice,the food intake of db/db mice in the model group was significantly higher(P<0.001).Compared with db/db mice in the model group,the food intake of mice in the Keluoxin low and high dose groups decreased significantly from the 5th to the 9th week of administration(P<0.05).In the later stage of intervention,with the progress of the disease,the food intake of mice in the model group decreased,and there was no significant difference between the model group and each administration group.Compared with db/m mice in the normal group,the water consumption of db/db mice in the model group was significantly higher(P<0.001).Compared with db/db mice in the model group,Keluoxin intervention can reduce the drinking water volume of db/db mice,especially in the high-dose Keluoxin group at the 3rd to 9th weeks,11th and 12th weeks(P<0.05,P<0.01,P<0.001).(2)Effects of Keluoxin capsule on body weight,kidney weight and kidney coefficient of db/db mice:The body weight of db/db mice in the model group was significantly higher than that in the normal group(P<0.001).The highest value was reached in the 10th week of administration,and the body weight showed a decreasing trend since the 11th and 12th weeks.Compared with the model group,valsartan had no significant effect on the body weight of db/db mice.The body weight of low-dose and high-dose Keloxin groups began to decrease at the 5th week,especially in the high-dose Keloxin group at the 8th to 12th week of administration(P<0.05,P<0.01,P<0.001).Compared with db/m mice in the normal group,the weight of bilateral kidneys of db/db mice in the model group increased significantly(P<0.001).Compared with db/db mice in the model group,the weight of bilateral kidneys in the low-dose and high-dose Keloxin groups and valsartan groups decreased significantly(P<0.05,P<0.01).In terms of the ratio of kidney weight to body weight,db/db mice in the model group had an upward trend compared with db/m mice in the normal group.While the ratio of kidney weight to body weight in valsartan group was significantly lower than that in the model group(P<0.01).(3)Effects of Keluoxin capsule on renal function of db/db mice:compared with db/m mice,24-hour urine volume,UAER,ACR,BUN and Scr of db/db mice in the model group increased significantly(P<0.001),Ucr and Ccr decreased significantly(P<0.001).Compared with db/db mice in the model group,24-hour urine volume in each dose group of Keluoxin decreased,especially in the high dose group of Keluoxin(P<0.01,P<0.001).UAER in each dose group of Keluoxin decreased at 4,8 and 12 weeks,especially at 12 weeks(P<0.05,P<0.001),ACR,BUN and Scr decreased significantly(P<0.05,P<0.01,P<0.001)and Ccr increased significantly(P<0.05,P<0.01).Ucr increased slightly,but the difference was not statistically significant(P>0.05).(4)Effect of Keluoxin capsule on glucolipid metabolism in db/db mice:compared with the normal group of db/m mice,the FBG,TC,TG,and LDL-C levels in the model group of db/db mice were significantly increased,while the levels of HDL-C were significantly decreased(P<0.001).Compared with the model group,FBG in the high and low dose groups of Keluoxin showed a decreasing trend and was positively correlated with the administered dose,but the differences were not statistically significant(P>0.05).The levels of TC,TG and LDL-C in the high-dose Keluoxin group decreased significantly(P<0.05,P<0.001),and the levels of HDL-C increased significantly(P<0.01).The low-dose Keluoxin group showed a decreasing trend,but the difference was not statistically significant(P>0.05).(5)Histopathological effects of Keluoxin capsule on the kidney of db/db mice:HE staining showed that compared with the normal group of db/m mice,local renal tubular degeneration,necrosis and lysis were observed in db/db mice,and detached renal tubular epithelial cells were seen in individual renal tubules.Neutrophils and neutrophil debris were seen in the lumen of most of the tubules and in the renal interstitium,and leukocyte tubular pattern was common.PAS staining showed that db/db mice had thickened glomerular basement membrane,increased thylakoid stroma,and thickened tubular basement membrane and other changes.Masson staining saw a significant increase in tubular basement membrane and interstitial collagen fiber deposition and severe fibrosis.Transmission electron microscope observation of renal tubular epithelial cells in db/db mice in the model group showed morphological features of apoptosis such as nuclear membrane wrinkling,chromatin condensation and nucleus deformation,and TUNEL fluorescence staining showed a significant increase of green fluorescent nuclei in renal tubular epithelial cells,all of which indicated increased apoptosis.After 12 weeks of intervention with Keluoxin capsule and valsartan,renal tubular injury and apoptosis of renal tubular epithelial cells in db/db mice were reduced to different degrees.(6)In vivo mechanism study of the renal protective effect of Keluoxin capsule in db/db mice:compared with the normal group of db/m mice,the serum AGEs level of db/db mice in the model group was significantly increased(P<0.001).The mRNA and protein expression of GRP78 was significantly elevated in renal tissues and was mainly localized in renal tubular epithelial cells.Renal tissue p-PERK,p-eIF2α,ATF4,CHOP,Bax,Cleaved-caspase-3,Cleaved-caspase-9 protein expression was significantly elevated and Bcl-2 protein expression was significantly decreased(P<0.001).After the intervention of Keluoxin and valsartan,serum AGEs level,mRNA of GRP78 and GRP78,p-PERK,p-eIF2α,ATF4,CHOP,Bax,Cleaved-caspase-3,Cleaved-caspase-9 protein expression in kidney tissue were significantly decreased in db/db mice(P<0.05,P<0.01,P<0.001),and Bcl-2 protein expression was significantly increased(P<0.01,P<0.001).2.In vivo(1)Establishment of apoptosis injury model of HK-2 cells induced by AGE-BSA:CCK-8 method showed that after treatment with different concentrations of AGE-BSA for 24 hours,there was no significant change in cell viability in BSA control group compared with normal control group.The viability of HK-2 cells in AGE-BSA group decreased in a dose-dependent manner when the concentrations of AGE-BSA were 100μg/ml,200μg/ml,and 400μg/ml,the viability of HK-2 cells was significantly reduced by 17.89%,27.59%,and 50.13%,respectively,compared with the normal control group(P<0.001).Same concentration(400μg/ml)after 6h,12h and 24h of AGE-BSA intervention,there was no significant change in cell viability in BSA control group compared with normal control group.The viability of HK-2 cells in AGE-BSA group decreased in a time-dependent manner.After 12h and 24h of AGE-BSA intervention,the viability of HK-2 cells decreased significantly(P<0.01,P<0.001).Annexin V-FITC/PI double staining flow cytometry showed that there was no significant difference in the apoptosis rate of HK-2 cells in BSA control group compared with normal control group(P>0.05).The apoptosis rate of HK-2 cells increased in a dose-dependent manner when the concentration of AGE-BSA was 100μg/ml,200μg/ml,and 400μg/ml,the apoptosis rate of HK-2 cells increased significantly(P<0.05,P<0.001).Hoechst 33342 staining and fluorescence microscopy showed that the nuclei of HK-2 cells in normal control group or BSA control group were normal blue and plump.The nuclei of HK-2 cells in AGE-BSA treatment group were dense and heavily stained,with some white color,accompanied by apoptotic morphological changes such as nuclear pyknosis and nuclear fragmentation,and the apoptosis increased with the increase of intervention concentration.(2)In vitro study on the mechanism of Keluoxin medicated serum intervention in AGE-BSA induced apoptosis of HK-2 cells:CCK-8 method showed that the activity of HK-2 cells in AGE-BSA model group decreased significantly compared with BSA control group(P<0.001).Compared with the model group,10%and 20%Keluoxin containing serum significantly increased the activity of HK-2 cells(P<0.001).Annexin V-FITC/PI double staining flow cytometry showed that the apoptosis rate of HK-2 cells in AGE-BSA model group was significantly higher than that in BSA control group(P<0.001).Compared with the model group,the apoptosis rate of HK-2 cells in the low and high dose group of Keluoxin containing serum decreased significantly(P<0.001).Hoechst 33342 staining showed that the nuclei of HK-2 cells in BSA control group were intact.The nuclei of HK-2 cells in AGE-BSA treatment group were pyknosis and fragmentation,showing bright fluorescence.Different concentrations of medicated serum could inhibit the morphological changes of HK-2 cells and reduce the apoptosis rate(P<0.001).Using 4-PBA as the positive control drug,the mRNA and protein expression of GRP78 were detected by Western blot and RT-PCR.The results showed that the mRNA and protein expression of GRP78 in Keluoxin containing serum high and low dose group and 4-PBA group were significantly lower than that in AGE-BSA group(P<0.05,P<0.01,P<0.001).PERK-shRNA3 vector with the highest inhibition rate was selected,and PERK was silenced to block PERK-ATF4-CHOP pathway.Western Blot detected the expression of p-PERK,PERK,P-eIF2α,eIF2α,ATF4,CHOP,Bcl-2,Bax,Cleaved Caspase-3 and Cleaved Caspase-9 proteins.The results showed that the expression levels of p-PERK/PERK,p-eIF2α/eIF2α,ATF4,CHOP,Bax,Cleaved-caspase-3 and Cleaved-caspase-9 in HK-2 cells were significantly reduced in different dose groups of Keluoxin and PERK-RNAi group(P<0.05,P<0.01,P<0.001)and elevated Bcl-2 protein expression levels in HK-2 cells(P<0.05,P<0.001),which were particularly significant in the Keluoxin medicated serum high dose and PERK-RNAi groups.Conclusion(1)Keluoxin capsule significantly reduced body weight,decreased proteinuria,improved lipid metabolism,protected renal function,and reduced histopathological damage to the kidney in db/db mice,but had no significant effect on fasting glucose.The nephroprotective effects of Keluoxin on db/db mice may be related to reducing the production of AGEs,inhibiting the apoptosis of renal tubular epithelial cells and endoplasmic reticulum stress mediated by the activation of PERK-ATF4-CHOP pathway.(2)AGE-BSA inhibited the viability of HK-2 cells and promoted the apoptosis of HK-2 cells in a time and dose-dependent manner.The concentration of AGE-BSA was 400μg/ml for 24 hours is the best intervention concentration and time for inducing apoptosis injury of HK-2 cells.(3)Keluoxin capsule medicated serum significantly increased the viability of HK-2 cells induced by AGE-BSA and reduced HK-2 cell apoptosis.After silencing the key gene PERK,it was further clarified that the mechanism of keluoxin capsule reducing HK-2 cell apoptosis was related to inhibiting the activation of PERK-ATF4-CHOP signal pathway and reducing endoplasmic reticulum stress.