Study On The Effect And Mechanism Of Shenkang Injection On Chronic Kidney Disease And Renal Fibrosis

[Objective]To investigate the protective effect of traditional Chinese medicine compound Shenkang injection(SK)on chronic kidney disease(CKD)and renal fibrosis,and related mechanism,from the animal,cell and molecular level,combined with systemic pharmacological target prediction method,as well as to provide more data support and scientific basis for the clinical application of SK on CKD and renal fibrosis.[Method]1.System pharmacology:SK molecular database was built through data mining and literature review,and active molecules were screened through drug similarity and drug half-life evaluation;WES drug targeting model was used to predict direct drug targeting of biologically active ingredients;the Cytoscape 3.2 software was used to construct a drug-target,target-pathway and target-disease network,and determine the distribution of targets in tissues and organs based on the BioGPS database.2.Animals:Thirty six C57BL/6 mice were randomly divided into sham group,model group,positive drug(ARB)group,SK high-dose group,SK medium-dose group,and SK low-dose group using random control design(n=6).UUO surgery was performed on the mice in the model group,ARB group,and SK groups to establish a renal fibrosis model.In the sham group,the left ureter was separated without ligating the ureter.Continuous administration of drugs began on the first day after surgery,for a total of 14 days.Sham group and model group were given normal saline 0.13 mL/10 g via tail vein injection and normal saline 0.15 mL/10 g via intragastric administration;ARB group was given normal saline 0.13 mL/10 g via tail vein injection and losartan potassium solution 0.15 mL(0.13 g crude drug)/10 g via intragastric administration;SK high-dose group was given SK 0.13 mL(0.08 g crude drug)/10 g via tail vein injection and normal saline 0.15 mL/10 g via intragastric administration;SK medium-dose group was given SK 0.13 mL(0.04 g crude drug)/10 g via tail vein injection and normal saline 0.15 mL/10 g via intragastric administration;SK low-dose group was given SK 0.13 mL(0.02 g crude drug)/10 g via tail vein injection and normal saline 0.15 mL/10 g via intragastric administration.General conditions such as body weight were observed and recorded during the experiment.MRI scan was performed on the 13th day of drug administration and FA value was measured to determine the degree of renal fibrosis in vivo.After 14 days of administration,eyeballs were removed to take blood from the mice.Kidneys were weighed,freezed and fixed.Serum creatinine,blood urea nitrogen,and blood CysC levels were measured by colorimetric method.The pathological morphology and fibrosis of the affected kidney were observed by HE and Sirius red staining.Renal ultrastructure was observed using transmission electron microscope.Western Blot was used to detect the renal myofibroblast marker a-SMA,ECM component Col ? and JAK2/STAT3 pathway molecular protein phosphorylation levels.Real-time PCR was used to detect the renal myofibroblast marker ?-SMA,FSP-1,ECM component Col ?,JAK2,STAT3,TGF-?1 and negative regulatory molecules SOCS1,SOCS3 mRNA levels in sham group,UUO group and SK medium-dose group.3.Cells:NRK-49F cells were resuscitated and subcultured,stimulated with 10 ng/mL TGF-?1 for 48 hours to induce proliferation and activation of NRK-49F cells.After intervention with different concentrations of SK(0,1,2,4 mg/mL),the cell morphology was observed.Western Blot was used to detect myofibroblast marker ?-SMA,ECM component Col ?,JAK2/STAT3 pathway molecular protein phosphorylation level and expression of negative regulatory molecule Prdx5.Real-time PCR was used to detect the levels of a-SMA,JAK 2 and STAT3 mRNA in NRK-49F cells.[Result]1.System pharmacology:DL?0.6 was adopted as the screening criterion and "HL=long"was used to define the appropriate HL range.Finally,88 compounds were selected as active molecules.Using the WES algorithm with CTD and TTD screening,85 potential compound targets related to the treatment of CKD were identified,including STAT3.Key pathways were obtained through KEGG database mapping,including NF-?B,MAPK,TRP ion channels,and VEGF pathways.In combination with advances in CKD pathogenesis knowledge,pathways for CKD treatment modules were constructed,including inflammation,proliferation,differentiation,migration,permeability,degradation and other modules.Analysis of the target-disease interaction network showed that the treatment of CKD by SK mainly interacted with 6 diseases:urogenital diseases,metabolic diseases,endocrine diseases,cardiovascular diseases,pathological processes and immune diseases.Tissue localization results showed that SK functioned through tissue modules including kidney,liver,lung,and heart.2.Animal experiment(1)Renal function detection:Compared with the sham group,the levels of serum CREA,BUN,and Cys-C of mice in the UUO group were significantly increased(p<0.01);compared with the UUO group,serum CREA level in the ARB and high-dose SK group was significantly reduced(p<0.01);compared with the UUO group,BUN level in the ARB and SK groups was significantly reduced(p<0.05 or p<0.01);compared with the UUO group,serum Cys-C level in the SK high-dose group was significantly reduced(p<0.05)(2)Renal weight and pathological examination:Compared with the sham group,the affected kidney weight/body weight,affected kidney/contralateral kidney weight,and contralateral kidney weight/weight in the UUO model group significantly increased(p<0.05 or p<0.01).However,each drug had no effect on kidney weight indices of UUO mice Compared with the sham group,the kidney FA value in the UUO group was significantly increased(p<0.01).Compared with the UUO group,the FA value in the ARB-positive drug group and SK high-medium-and low-dose group significantly decreased(p<0.01 or p<0.001),among which the SK medium-dose group was the most significant(p<0.001).Compared with the sham group,renal pathological injury in the UUO group was severe;compared with the UUO group,kidney pathological injury in all treatment groups was alleviated to varying degrees;extracellular matrix accumulation,inflammatory cell infiltration,renal tubular dilatation and/or atrophy were alleviated,and the SK medium-dose group and the high-dose group had better alleviating effects.Compared with the sham group,the Sirius red stain red area in UUO group increased significantly(p<0.001);compared with the UUO group,the Sirius red stain red area of the affected kidney of the mice in the ARB group,SK high-,medium-and low-dose groups was significantly reduced(p<0.05 or p<0.01).SK reduced the proliferation of fibroblasts and accumulation of collagen,and the apoptosis of renal tubules caused by UUO under electron microscope(3)Western blot detection:Compared with the sham group,the ?-SMA protein level in the UUO group was significantly increased(p<0.01);compared with the UUO group,the SK medium-dose group and the low-dose group significantly reduced ?-SMA level(p<0.05)Compared with the sham group,Col ? protein level in the UUO group increased significantly(p<0.01);compared with the UUO group,the SK medium-dose group significantly reduced the elevation of Col ? protein levels(p<0.05).Compared with the sham group,the p-STAT3(Try705 site)/STAT3 ratio in the UUO group increased significantly(p<0.01);compared with the UUO group,SK medium-dose(p<0.01)and low-dose(p<0.01)group significantly reduced the level of STAT3 phosphorylation at the Try705 site.Compared with the sham group,the p-JAK2(Try1007 site)/JAK2 ratio in the UUO group had an upward trend,but there was no statistical difference;compared with the UUO group,SK high-dose group significantly reduced the degree of JAK2 phosphorylation at the Try1007 site(p<0.05).(4)PCR detection:compared with the sham group,ECM components Col ?,Col ?,FN,fibroblast activation markers a-SMA,FSP-1,and JAK2,STAT3,TGF-?,JAK2/STAT3 pathway negative regulators SOCS1,SOCS3 mRNA levels in the UUO group significantly increased(p<0.05 or p<0.01 or p<0.001);compared with the UUO group,the SK medium-dose group significantly reduced Col ?,Col ?,FN,?-SMA,FSP-1,JAK2,TGF-?,SOCS1,SOCS3 mRNA levels(p<0.05 or p<0.01).3.Cell experimentAfter TGF-?1 induction,the cytoskeleton of NRK-49F cells showed internal microfilaments,which gradually thickened and elongated,lossing spindle or stellate fibroblast appearance.Different concentrations of SK reversed the morphological changes and cell proliferation induced by TGF-?1 to a certain extent.Compared with the blank control group,the p-STAT3(Try705 site)/STAT3 and Prdx5 protein levels of the fibroblasts in the model group were significantly increased(p<0.05),and p-JAK2(Try1007 site)/JAK2 increased to a certain extent but not statistically significant(p>0.05);compared with the model group,the phosphorylation level of the STAT3 Try705 site in the 4 mg/mL SK group and the 2 mg/mL SK group were significantly reduced(p<0.05),the phosphorylation level of JAK2 Try 1007 site in the 1 mg/mL SK group and 2 mg/mL SK group was significantly reduced(p<0.05),and Prdx5 protein expression level in 4mg/mL SK group was significantly reduced(p<0.05).Compared with the blank control group,the levels of ?-SMA,JAK2,and STAT3 mRNA of fibroblasts in the model group were significantly increased(p<0.05 or p<0.001);compared with the model group,ARB and different concentrations of SK significantly reduced ?-SMA mRNA levels(p<0.001);compared with the model group,ARB,1 mg/mL,2 mg/mL SK(p<0.05))and 4 mg/mL SK(p<0.01)significantly reduced STAT3 mRNA levels;compared with the model group.ARB and 4 mg/mL SK significantly reduced JAK2 mRNA levels(p<0.05)[Conclusions](1)Systemic pharmacological predictions suggested that multiple compounds in SK might intervene CKD by acting on target molecules of multiple pathway such as NF-?B,MAPK,TRP ion channels,and VEGF,related to inflammation,apoptosis,and proliferation(2)SK could effectively improve renal function index and renal interstitial fibrosis of UUO mice.The mechanism might be achieved by regulating JAK2/STAT3 signaling pathway to inhibit fibroblast activation,verifying part of the results of systemic pharmacology.(3)SK could also inhibit the proliferation and activation of NRK-49F and the production of ECM induced by TGF-?1,and its mechanism of action might be mediated by inhibition of activation of the JAK2/STAT3 pathway.