| Diabetic nephropathy?DN?is one of the most important complications in development of diabetes?DM?,and could eventually lead to End-stage renal disease?ESRD?or even death,however,the treatment of DN is still limited and less effective.The TCM pathogenesis of DN is the asthenia of both spleen and kidney and excess in real evils such as phlegm retention,blood stasis,toxi.Shenyuan Granules is hospital preparations of Hubei Provincial Hospital of TCM,which has the efficacy of tonifying spleen and kidney,and promoting drain real evils in the treatment of DN.Clinical studies have shown that Shenyuan Granules can delay the development of DN,and the preliminary experimental studies also indicate the renal function of DN mouse have significantly improved through its intervention.Modernresearchhasshownthatepithelial-mesenchymal transition?EMT?in renal tubular epithelial cells play an important role in the progress of renal tubule interstitial fibrosis?RIF?in DN,which is closely related to the Klotho/FGFR1/FGF2 signal pathway.Based on previous study,in order to have a further understanding for the mechanisms of action,the present study investigated the effects of Shenyuan granules in regulating the Klotho/FGFR1/FGF2 signal pathway in DN.ObjectiveInvestigate the changes of Klotho/FGFR1/FGF2 signal pathway in the process of EMT in DN mice treated by STZ and in HK-2 cells treated by high glucose.Explore the mechanism of Shenyuan granules to treat RIF by inhibiting EMT in DN.Provide a scientific basis for the treatment of DN by“Tonifying spleen and kidney,and promoting drain turbidity”in TCM.Methods1.In vitro experiment?1?Drug-containing serum preparationThe SPF grade Wistar rats were randomly assigned to the control group?n=10?,Shenyuan granule group?n=10?and irbesartan group?n=10?.The rats of Shenyuan granules group were gavaged with Shenyuan granules solution by 3.085g/Kg/d dosage,and irbesartan group0.015g/Kg/d dosage,meanwhile the control group were gavaged with an equal volume of distilled water.On the seventh day,after 1 hour following the second gavage the blood of rats was extracted from the abdominal aorta,then the serum was separated by centrifugation and stored at-20?.?2?UPLC-MS/MS was adopted to detect the active ingredients in Shenyuan-containing serum of rats.Chromatographic separation was fulfilled on a ACQUITYUPLCRBEHC18 column?1.7?m,50×2.1mm?with the mobile phase consisting of 0.1%formic acid?A?and0.1%acetonitrile?B?.The gradient elution program was set as follows:020min?90%?40%A?,2030min?40%?25%A?.The flow rate was set at 0.5 mL·min-1,column temperature was 30?,detection wavelength was 203 nm,and injection volume was 10?L.The mass spectrometer?MS?condition were set as follows:the ionization mode was ESI?-?,the acquisition was MSE for 20 minutes,and the mass spectra was obtained over the m/e range of 100-1200m/z for 0.5s.?3?CCK-8 method was used to detect the equivalence between rat serum and 10%fetal bovine serum,so as to determine the concentration of HK-2 cells cultured in rat serum,and LDH method was adopted to assess the cytotoxicity of drug-containing serum on HK-2 cells.?4?Experimental groupsDuplicated model through induced HK-2 cells epithelial mesenchymal transdifferentiation by high glucose?30mmol/L?,and divided into normal group,model group,control group,hypertonic control group,Shenyuan granules group,irbesartan group and ERK inhibitor group.After a48-hour incubation,all cell groups were used for further analysis.?5?Index detectionKlotho and FGF2 were detected by immunofluorescence,Western blot and RT-PCR were used to detect expression levels of Klotho,FGF2,E-Cadherin,?-SMA,FN as well as Collgen?protein and its mRNA,ERK and p-ERK levels in each group were analyzed by Western blot,while both Klotho/FGFR1 and FGF23/FGFR1 immune complex were detected by immunoprecipitation?IP?method.2.In vivo experiment?1?A total of 120 male C57BL/6 mice were used in this experiment.20mice were selected randomly as the normal group,then the rest of 100mice were given a single dose of 150?·kg-1streptozotocin?STZ?intraperitonally.Random blood glucose?RBG?was measured for two times continuously in 72 hours after STZ in injection to confirm the developmentofdiabetesaccordingtothecriterionof RBG?16.7mmol·L-1.62 mice conformed RBG?16.7mmol·L-1were divided into the following groups randomly:the model group with 20mice,Shenyuan granules group with 21 mice,irbesartan group with 21mice.The normal group and the model group mice were given normal saline 2 mL/d by gavage,Shenyuan granules group mice were given Shenyuan granules solution 6.17g/?kg·d?by gavage,and irbesartan group mice were given irbesartan solution 0.03g/?kg·d?by gavage.?2?Duringintervention,Fastingbloodglucose?FBG?,24h Microalbuminuria and mALB?24h mALB?were tested according to the pre-set time point.?3?After intervention,all mice were euthanized,blood was used to detectBloodUreaNitrogen?BUN?andSerum Creatinine?SCr?,nephridial tissue sectionwas used for pathology detection by the method of HE,PAS and MASSON staining.The expression of Klotho?FGF2?E-Cad??-SMA?FN?Collgen?in kidney were detected by Western blot or RT-PCR respectively,and the total ERK and phosphorylation ERK level were detected by Western blot,while both Klotho/FGFR1 and FGF2/FGFR1 immune complex were detected by immunoprecipitation?IP?method.Result1.In vitro experiment?1?Compared with the retention time and MS data of control serum,it was detected that the medicated serum of Shenyuan granules contained epimedium glycoside,astragalus glycosides,emodin prototype compounds,however,rhubarb phenol and rhein were not detected.It was proved that this method was simple,high sensitivity,linearity,precision,good repeatability and recovery rate.Adopt linear regression equation to calculate,the result showed that epimedium glycoside,astragalusglycosides,thecontentofemodinin Shenyuan-containing serum were 0.87?g/mL,0.76?g/mL,13.6?g/mL respectively.?2?CCK-8 results demonstrated that there was no statistical significance between 10%,15%,20%blank rat serum and 10%FBS group?P>0.05?.Therefore,a concentration of 20%rat serum was selected for further experiments.LDH results showed that there was no statistical difference between the effects on cell LDH activity by the 10%FBS group and the 20%blank rat serum group or 20%Shenyuan granules-containing serum group?P>0.05?,demonstrating that the drug-containing serum had no cytotoxic properties.?3?Compared with the normal group,the expression of Klotho,E-Cadherin protein and its mRNA in model group were significantly decreased?P<0.05?,while the expression of p-ERK,FGF2,?-SMA,FN,Collgen?protein and its mRNA were significantly increased?P<0.05?.Meanwhile,when the results in the Shenyuan granules group compared with the model group,the expression of Klotho,E-Cadherin protein and its mRNA in model group were significantly increased?P<0.05?,while the expression of p-ERK,FGF2,?-SMA,FN,Collgen?protein and its mRNA were significantly decreased?P<0.05?.Immunoprecipitation verified the high Klotho/FGFR1 immune complex expression and low FGF2/FGFR1 immune complex expression in normal group.Compared to normal group,Klotho/FGFR1 immune complex expression in model group decreased significantly?P<0.05?,while FGF2/FGFR1 immune complex expression increased significantly?P<0.05?.Compared to model group,Klotho/FGFR1 immune complex expression in Shenyuan granules group increased significantly?P<0.05?,while FGF2/FGFR1immune complex expression decreased significantly?P<0.05?.2.In vitro experimentCompared with the normal group,the FBG,24h mALB and Scr in model group mice rose continuously since three days after STZ injected?P<0.05?,while the expression of Klotho?E-Cad and Klotho/FGFR1immune complex were significantly decreased?P<0.05?,with the expression of FGF2?p-ERK??-SMA?FN?Collgen?and FGF2/FGFR1 immune complex significantly increased?P<0.05?.Meanwhile the renal pathology in model group showed glomerular hyperplasiaandhypertrophy,glomerularatrophy,hardening,glomerular mesangial cells hyperplasia,broadening mesangial area,renal tubular structure arrangement was disorder,part of the renal tubular atrophy and lumen collapse,with interstitial had a large number of lymphocyte infiltration.Compared with model group,the FBG in Shenyuan granules group and irbesartan group had no significant difference?P>0.05?,while the 24h mALB significantly decreased?P<0.05?,and the Scr in Shenyuan granules group significantly decreased?P<0.05?.Compared with model group,the expression of Klotho?E-Cad and Klotho/FGFR1 immune complex in Shenyuan granules group and irbesartan group significantly decreased?P<0.05?,while the expression of FGF2?p-ERK??-SMA?FN?Collgen?and FGF2/FGFR1 immune complex significantly increased?P<0.05?.The renal pathology in Shenyuan granules group and irbesartan group showed significantly alleviated.The degree og glomerular atrophy,hardening and mesangial cell proliferation all showed mitigated,and mesangial matrix broadening,renal tubular lesions mitigated too,with a small amount of lymphocytic infiltrates interstitial lymphocytic infiltrates.Conclusion1.Main pharmacological ingredients of drug-containing serum from rats treated with Shenyuan granules are epimedium glycoside,astragalus glycosides and emodin,which provides a reference for further research on pharmacodynamics mechanism of Shenyuan granules.2.Shenyuan-containing serum can inhibit FGF2/FGFR1 signal pathway by up-regulating Klotho expression in HK-2 cells induced by high glucose.Therefore,Shenyuan granules could be able to improve the transdifferentiation of renal tubular epithelial cells,which might be one of the mechanisms that the granules improves diabetic nephropathy.3.Shenyuan granules protected and reversed the EMT of renal tubular epithelial cells in DN C57BL/6 mice,which may be achieved via the regulation of Klotho/FGFR1/FGF2 signal pathway. |
PDF Full Text Request