Long-term Clinical Observation And Experimental Study On The Effect Of Qihuang Yishen Prescription On Improving Diabetic Microvascular Complication

BackgroundDiabetic kidney disease(DKD)and diabetic retinopathy(DR)are the main microvascular complications of type 2 diabetes mellitus,which are often accompanied by both.They are the leading causes of end-stage renal disease(ESRD)and visual impairment and blindness in adults in developed countries.Renal fibrosis and retinal fibrovascular hyperplasia are the final pathological changes,respectively.Epithelial-mesenchymal transition(EMT)is the early pathogenesis of fibrosis.It has been found that long non-coding RNA(LncRNA)MALAT1 can interact with Wnt/β-catenin signal pathway,and regulate EMT and participate in the pathogenesis of DKD and DR.Therefore,regulating LncRNA MALAT1 and inhibiting EMT in kidney and retina is the key to prevent and control the progress of DKD and DR.Qihuang Yishen formula(QHYS)is an experience prescription created by my mentor in the early years based on "Qi and Yin deficiency and blood stasis",the core pathogenesis of DKD.Previous studies have explained the clinical efficacy and part of the mechanism of QHYS in the treatment of DKD,and found that it can inhibit podocyte EMT in vitro by regulating the LncRNA MALAT1/Wnt/β-catenin pathway.My mentor believes that the core pathogenesis of DR is consistent with that of DKD,and the two diseases had the same pathological mechanism.Therefore,under the guidance of the theory of " treating different diseases with the same therapeutic principle ",the present study would focus on the pathological process of EMT,and explore the mechanism of QHYS to improve DKD and DR.Objective1.To preliminarily explore the effects of long course of treatment with QHYS on renal outcome and all-cause death outcome in DKD patients.2.To explore the effect and mechanism of QHYS regulating LncRNA MALAT1 and Wnt/β-catenin pathway on renal EMT in DKD model rats.3.To explore the effect and mechanism of QHYS regulating LncRNA MALAT1 and Wnt/β-catenin pathway on retinal EMT in DR Model rats.Methods1.Clinical study:Long-term retrospective study on the effect of QHYS on renal outcome and all-cause death endpoint in patients with DKD.This is a retrospective cohort study based the DKD patients.From January 2006 to December 2014,patients with DKD in the clinical research subject of QHYS in Wangjing Hospital of China Academy of Chinese Medical Sciences were screened,and patients with DKD stage Ⅲ to Ⅳ were included according to the inclusion and exclusion criteria.The patients who took QHYS continuously for at least 12 months were selected as the exposure group,and those who were not treated with QHYS were control group.General data including sex,age,height,weight,DM course,DKD course,DKD stage,DR history,diabetes peripheral neuropathy(DPN)history,complications,applied drugs,and HbAlc level were recorded.Patients were followed up for the endpoint outcomes including compound renal outcomes(entry to ESRD,death from nephropathy),compound microvascular outcomes(retinal photocoagulation,vitrectomy,or anti-vascular endothelial growth factor injection for proliferative retinopathy,or retinal detachment,severe vision loss or compound renal outcomes),all-cause death,and compound cardiovascular outcomes(nonfatal myocardial infarction,nonfatal stroke,hospitalization for heart failure,death from cardiovascular causes).The follow-up was until December 31,2021.The annual incidence of 100 patients was calculated,survival analysis was performed by Kaplan-Meier,differences between groups were tested by Log-rank,potential confounding factors were analyzed by univariate analysis,and multivariate COX regression analysis was used to explore the independent influence of QHYS on the end outcomes.2.Experiment study 1:Study based on EMT to explore the preventive and therapeutic effect and mechanism of QHYS on DKD rats.Male SD rats aged 6 to 7 weeks were fed with high-fat and high-sugar diet for 8 weeks combined with STZ intraperitoneal injection to establish the DKD rat model.A total of 40 DKD model rats were randomly divided into five groups:DKD model group,QHYS low-dose group(QHYS-L),QHYS medium-dose group(QHYS-M),QHYS high-dose group(QHYS-G)and losartan potassium group(LP)with 8 in each group,and 8 normal rats were selected as the normal group(NC).The rats were gavage continuously for 12 weeks.24hUTP,fasting blood glucose,renal function,liver function were measured.Renal tissue were stained with HE and Masson.The glomerular foot process and basement membrane were observed by transmission electron microscopy.The expressions of Nephrin and FSP-1 in podocytes were observed by immunohistochemistry.Western blot was used to detect the expressions of Nephrin,Desmin,FSP-1,Wntl,β-catenin and active-β-catenin.The expression levels of LncRNA MALAT1 were detected by RT-qPCR.3.Experiment study 2:Study based on EMT to explore the preventive and therapeutic effect and mechanism of QHYS on DR rats.The methods of animal feeding and model establishment were same as experiment study 1.The 50 DR rats were randomly divided into 5 groups:model group(DR),QHYS-L group,QHYS-M group,QHYS-G group and Calcium Dobesilate group(CD),and 10 normal rats were selected as normal group.After twelve weeks of gavage,retinal structure was observed by HE staining,and the retinal microvascular morphology was observed by retinal digestion patch PAS staining.The Occludin expression was detected by immunohistochemistry.After the rats were injected with 2%Evans blue solution in the tail vein,the leakage of Evans blue was observed by fluorescence scanning of retinal patch.After 24 weeks of gavage,HbAlc was detected,and the Occludin and α-SMA protein expression were detected by immunohistochemistry.The expression of Occludin,E-cad,RPE65,N-cad,α-SMA,Wnt1,β-catenin and active-β-catenin detected by Western blot.The expression level of LncRNA MALAT1 was detected by RT-qPCR.Results1.Clinical study:A total of 100 patients including 50 patients in the exposed group and 50 patients in the control group were included.The median follow-up time was 8.92 years.(1)Comparison of endpoint outcomes(exposure group vs.control group):20 cases(8 vs 12)had complex renal outcomes,including 17 cases(7 vs 10)of ESRD and 9 cases(1 vs 8)of deaths due to nephropathy.There were 19 patients with all-cause death(2 vs 17),32 patients with complex microvascular outcomes(10 vs 22),and 26 patients with complex cardiovascular outcomes(14 vs 12).(2)The annual incidence of end-point outcome events in 100 patients(exposure group vs.control group)showed statistically significant differences in deaths from renal causes(0.2 vs 1.59),all-cause deaths(0.4 vs 3.38)and complex microvascular outcomes(1.98 vs 4.37)(P<0.05).(3)The independent effect of QHYS on the end point outcome analyzed by multivariate COX regression analysis:Compared with the control group,the risk of death from nephropathy was reduced by 85.6%in the exposure group[HR:0.164,95%CI:(0.020,1.343),P=0.092],and the risk of all-cause death was reduced by 87.8%in the exposed group[HR:0.122,95%CI:(0.027,0.546),P=0.006],the risk of complex microvascular outcomes was reduced by 62.3%in the exposure group[HR:0.377,95%CI:(0.176,0.809),P=0.012].However,there was no significant difference in the deaths from renal causes,but there was significant difference in all-cause death and complex microvascular outcomes.2.Experiment study 1:(1)Drug effect.Compared with NC group,the body weight of DKD group decreased,and renal weight index increased,and 24hUTP significantly increased,and FBG,BUN,ALT,AST increased,and ALB decreased.The glomerular volume of DKD group increased,and positive area of glomerular collagen fiber increased,and foot process was fused,and glomerular basement membrane thickened.Compared with DKD group,the body weight of rats in QHYS-L and QHYS-M groups increased,and the renal weight index,24hUTP,FBG and BUN of QHYS-M and LP groups decreased to varying degrees,and the average glomerular volume of rats in QHYS-M and QHYS-G groups decreased,and the positive area of glomerular collagen fibers decreased,and the morphology of foot process improved.Overall,QHYS-M group had better effect.(2)Mechanism of action.Compared with the NC group,the proportion of Nephrin-positive glomeruli decreased and the proportion of FSP-1 positive glomeruli increased in the DKD group.Western blot results showed that epithelial marker protein Nephrin was down-regulated,mesenchymal marker proteins Desmin and FSP-1 were up-regulated,and pathway proteins Wntl,β-catenin and active-β-catenin were up-regulated and LncRNA MALAT1 was up-regulated by RT-qPCR in the DKD group.Compared with DKD group,the proportion of Nephrin-positive glomeruli increased in QHYS-M and QHYS-G groups,while the proportion of FSP-1 positive glomeruli decreased.Western blot results showed that Nephrin was up-regulated and Desmin and FSP-1 were down-regulated in QHYS-M and QHYS-G groups to varying degrees.Wntl,β-catenin and active-β-catenin pathway proteins were down-regulated either.RT-qPCR results showed that the expression of LncRNA MALAT1 in QHYS-M group was down-regulated.3.Experiment study 2:(1)Drug effect.Compared with the NC group,the model group showed opacity of lens,decreased body weight,increased HbAlc,retinal tissue structure disorder,unclear layers,thin thickness,loose and irregular arrangement of cells in each layer.Microvascular overlay showed tortuous retinal microvessels and occlusive capillaries.Retinal tissue appeared obvious spots,patches,diffuse leakage area.Compared with DR group,the lens opacity and weight increase of QHYS group and CD group occurred later.The HbAlc decreased,and retinal tissue structure improved,thickness recovered,microvascular flow recovered,and retinal tissue leakage reduced in QHYS-M group.(2)Mechanism of action.Compared with the NC group,immunohistochemistry showed no significant changes in Occludin expression in the DR group at 12 weeks after gavage.However,the expression of Occludin in the DR group significantly decreased,while α-SMA expression significantly increased at 24 weeks after gavage.Western blot showed that the expression of Occludin,E-cad and RPE65 decreased,while the expression of N-cad and α-SMA increased in the DR group.The expressions of pathway proteins Wnt1,β-catenin and active-β-catenin were increased,and the expression of lncRNA MALAT1 was significantly up-regulated in DR group.Compared with DR group,after 24 weeks after gavage,immunohistochemistry showed that the Occludin expressions of RPE cells in QHYS groups and CD group increased,and the α-SMA expression decreased.Western blot showed that the expression of Occludin,E-cad and RPE65 increased,and the expression of N-cad and α-SMA decreased,and the expression of pathway proteins Wnt1,β-catenin and active β-catenin decreased in QHYS groups and CD group.RT-qPCR showed that LncRNA MALAT1 expression was significantly down-regulated in QHYS groups.Overall,the QHYS-M group had the most significant effect.Conclusions1.The long course of intervention of QHYS may reduce the incidence of nephrotic causative death,cumulative all-cause death and complex microvascular outcome in DKD stage Ⅲ to Ⅳ patients,and QHYS may be an independent protective factor for the occurrence of all-cause death and complex microvascular outcome in DKD patients.2.QHYS can down-regulate LncRNA MALAT1 level in renal tissue of DKD model rats,and inhibit Wnt/β-catenin pathway activation,up-regulate podocyte marker proteins Nephrin,down-regulate mesenchymal marker proteins Desmin and FSP-1,inhibit EMT process,and alleviate glomerular hypertrophy and sclerosis.3.QHYS can down-regulate the LncRNA MALAT1 level in retinal tissue of DR Model rats,and inhibit the activation of Wnt/β-catenin pathway,up-regulate the epithelial markers Occludin,E-cad,RPE65,down-regulate the expression of mesenchymal protein N-cad,α-SMA,and inhibit the EMT process,and reduce retinal histopathologic damage,and improve retinal microvascular morphology,and reduce retinal tissue leakage,and protect the blood-retina barrier.