Interfering Effects Of Shenyuan Granules On Myocardial Injury In Diabetic Nephropathy Mice And Its Mechanism Based On FGF23/CaMKⅡ

Objective:In this experiment,spontaneous diabetic db/db mice were used as the research model,and the mechanism of myocardial injury in db/db mice and the intervention effect of Shenyuan granule were investigated by observing the endoplasmic reticulum stress mediated by FGF23/CaMKⅡsignal pathway.Method:Experimental part:Forty-five db/db male mice were randomly divided into three groups,the Modle Group（MD）,the Ibesartan Group（IBT）,and the Shenyuan Granules Group（SYKL）;littermate wild-type mice will automatically become the normal control group（Normal Control Group,NC）.The mice in the NC group were fed with normal mice,and the MD,SYKL,and IBT groups were given high-phosphorus diets（phosphate 1.2%,calcium 1.6%）.After one week of adaptive feeding,the mice in each group were given 12 weeks of drug intervention.NC group and MD group were given double steaming water,IBT group were given irbesartan(30mg·kg^-1),and SYKL group were given Shenyuan Granule(6.0g·kg^-1),and the dosage volume of mice in each group was the same(20mL·kg^-1·d^-1).During this period,the general conditions such as the spirit,diet,drinking water,weight and urine output of the mice in each group were monitored.The urine microalbumin content and blood glucose level of the mice in each group were detected.Twelve weeks later,mice of each group were sacrificed,and blood,heart and kidney tissues of the mice were collected.Part of the kidney and heart tissues were fixed with 4%paraformaldehyde,and the rest were frozen in a-80℃refrigerator.The calcium and phosphorus content,serum creatinine,urea nitrogen in the serum of the mice were detected by automatic biochemical analyzer.The pathological changes of mouse heart and kidney were detected by HE staining and Masson staining.The apoptosis of myocardial cells was detected by Tunel staining.The interaction between Klotho protein and FGFR1 protein in kidneys of mice in each group was detected by immunofluorescence（IF）double staining.The IF was used to detect the expression of FGFR4 and PLCγ1 proteins in the heart of the mice.p-CaMKⅡand endoplasmic reticulum stress marker proteins GRP78 and CHOP were detected by immunohistochemical staining in each group.The expression of ANP,BNP,CollagenⅠ,Bax,Bcl-2,GRP78,CHOP,FGFR4,PLCγ1 mRNA in the heart of mice and Klotho mRNA in the kidney of mice were detected by Real-time PCR in each group.The expression of Klotho protein in the kidney of mice and Bax,Bcl-2,GRP78,CHOP,FGFR4,PLCγ1,p-CaMKⅡprotein in the heart of mice were detected by Western Blot in each group of mice.Results:1.General condition of mice in each group:During the feeding period,mice in the NC group had bright and dense fur,normal drinking and eating,good mental status.Compared with the NC group,the MD group had dull fur over time,accompanied with excessive drinking,eating,and urination.The pads was repeatedly drenched,the spirit was sluggish,the action was slow,and the reaction was slow.Compared with the mice in the MD group,the fur of the mice in the SYKL group and the IBT group is slightly shiny,accompanied with more drinking,more food,and more urine.The mouse pads are repeatedly wet,but the spirit,active,response were better than the mice in the MD group.2.The results of biochemical indicators of mice in each group:The biochemical indicators of mice in the NC group were normal,and there were no abnormal fluctuations in blood phosphorus,calcium,Scr,BUN,and mALB.Compared with the mice in NC group,mALB,blood phosphorus,and renal function indicators（Scr and BUN）in MD group were all up-regulated significantly（P<0.01）,while the blood calcium level was down-regulated significantly（P<0.01）.Compared with the mice in the MD group,the levels of mALB,blood phosphorus,and renal function indicators（Scr and BUN）were down-regulated（P<0.05）in the SYKL group and IBT group,while blood calcium levels of mice had been up-regulated significantly（P<0.05）.3.Fasting blood glucose test results in each group:The mice in the NC group had normal blood glucose.Compared with the NC group,the blood glucose of the MD group increased gradually（>16.7mmol/L）,and the difference between the two groups was significant（P<0.01）.After 12 weeks of intervention with Shenyuan Granules and irbesartan,blood glucose levels did not decrease in SYKL and IBT groups,and there was no statistically difference compared with MD group（P>0.05）,which indicated that Shenyuan Granules and irbesartan did not improve blood glucose in mice.4.The results of pathological staining in the kidneys of mice in each group:The surface of the kidneys of mice in the NC group was smooth,no bumps and breaks were observed,and the size was normal;HE staining showed that the nephron structure of the mice in the NC group was clear and complete,and no mesangial cells and stroma were proliferated.There was no thickening of the basement membrane,no fibrous tissue hyperplasia,and no tubular edema;Masson staining showed that the nephron had no blue collagen fiber deposition in the NC mice.Compared with the NC group,the kidney volume of the MD group mice increased significantly.HE staining suggested that the glomerular volume of mice in MD group increased,cystic cavity narrowed,mesangial cell proliferation,basement membrane and mesangium thickened.Masson staining suggested that blue collagen fiber deposition was seen in MD group（P<0.01）.Compared with the mice in the MD group,the kidney surface of the mice in the SYKL group and the IBT group was smoother,no bulge and damage were seen,and the kidney volume increased slightly.HE staining showed clear nephrons in the kidneys of mice in the SYKL group and IBT group.Glomerular mesangial cells and stromal proliferation were reduced,basement membrane was slightly thickened.Masson staining suggested SYKL The blue collagen fibers of the kidneys of mice in the group and IBT group were lighter,and the percentage of collagen deposition was lower than that of the mice in the MD group（P<0.01）.5.The results of HW/BW index in each group:The HW/BW index of mice in the NC group was normal,and no significant increase was seen.Compared with NC group mice,MD group mice HW/BW index significantly increased（P<0.01）.Compared with MD group mice,SYKL group and IBT group mice HW/BW index significantly decreased（P<0.01）.6.The results of heart pathological staining in each group:The heart size of the NC group was normal,the color was ruddy,and no fatty tissue was attached to the surface of the tissue.HE staining showed non-hypertrophic myocardium in the NC group.Cardiac cells and myocardial fibers are arranged neatly.There was no rupture of myocardial fibers in the heart.Masson staining showed that blue collagen fibers were not found in the hearts of NC group mice.Compared with the NC group,the heart volume of the MD group was enlarged,the color was dark brown,and the fatty tissue was visible on the surface.HE staining showed that the myocardial cells in the MD group were hypertrophic,and the myocardial cells and muscle fibers were disorderly arranged with myocardial interstitial formation.Myocardial fibers were broken.Masson staining showed myocardial collagen deposition increased significant in MD group（P<0.01）.Compared with the mice in the MD group,the heart volume of the SYKL and IBT groups were slightly smaller,and adipose tissue was attached to the heart surface;HE staining showed that the cardiomyocyte hypertrophy of the mice in the SYKL and IBT groups was reduced,and the arrangement of myocardial cells and myocardial fibers was slightly disordered.It can be seen that a small part of myocardial fibers broke.Masson staining showed that cardiac collagen deposition in the SYKL group and IBT group was significantly reduced（P<0.01）.7.Detection of FGF23 in serum of mice in each group:The serum FGF23 content of mice in the NC group was normal,and it is not observed abnormal fluctuation.Compared with the NC group,the serum FGF23 content in the MD group was significantly increased（P<0.01）.Compared with the mice in the MD group,the serum FGF23 levels in the SYKL and IBT groups were down-regulated at different levels（P<0.05,P<0.01）.8.The expression of mRNA with Real-time PCR in the heart and kidney in each group:The expressions of Klotho mRNA in the kidney of the NC group were normal.The expressions of cardiac ANP,BNP,CollagenⅠ,Bax,GRP78,CHOP,FGFR4,PLCγ1 mRNA were relatively low,and the expression of cardiac Bcl-2 mRNA remained at a high level in the heart of the NC group.Compared with NC group,the expression of Klotho mRNA in the kidney of MD group was significantly reduced（P<0.01）,and the expressions of cardiac ANP,BNP,CollagenⅠ,Bax,GRP78,CHOP,FGFR4,PLCγ1 mRNA increased significantly（P<0.05,P<0.01）,the expression of cardiac Bcl-2 mRNA decreased significantly（P<0.01）.Compared with the MD group,the expressions of Klotho mRNA in the kidney of the SYKL and IBT groups were significantly increased（P<0.01）,and the expressions of cardiac ANP,BNP,CollagenⅠ,Bax,GRP78,CHOP,FGFR4,and PLCγ1 mRNA was reduced（P<0.05,P<0.01）,the expression of cardiac Bcl-2 mRNA increased significantly（P<0.01）.9.The expression of protein with Western Blot in the heart and kidney in each group:The expressions of renal Klotho protein in the NC group were normal.The expression of cardiac ANP,BNP,CollagenⅠ,Bax,GRP78,CHOP,FGFR4,PLCγ1 proteins were relatively low,and the expression of cardiac Bcl-2 protein remained at a high level.Compared with NC group,the expressions of renal Klotho protein in the MD group were significantly reduced（P<0.01）,and the expression of cardiac ANP,BNP,CollagenⅠ,Bax,GRP78,CHOP,FGFR4,PLCγ1proteins increased（P<0.01）,the expression of cardiac Bcl-2 protein decreased significantly（P<0.01）.Compared with the MD group,the expressions of renal Klotho protein in the SYKL and IBT groups were significantly increased（P<0.01）,and the expressions of cardiac ANP,BNP,CollagenⅠ,Bax,GRP78,CHOP,FGFR4,and PLCγ1 proteins were reduced（P<0.01）,the expression of cardiac Bcl-2 protein increased significantly（P<0.01）.10.The expression of protein with IHC staining in the heart of mice in each group:The expressions of p-CaMKⅡ,GRP78 and CHOP proteins with IHC staining in the cardiomyocytes of NC group mice were lower than that of the other three groups.Compared with NC group mice,the expression of cardiac p-CaMKⅡ,GRP78 and CHOP proteins with IHC staining in MD group were increased significantly（P<0.01）.Compared with the MD group,the expressions of cardiac p-CaMKⅡ,GRP78 and CHOP proteins with IHC staining in the SYKL and IBT group were increased significantly（P<0.01）.11.Tunel staining results of mice in each group:It isn’t found the obvious staining in the nucleus of myocardial cells in the NC group.Compared with the NC group mice,the cardiomyocyte nuclear staining of the MD group mice increased,indicating a significant increase in cardiomyocyte apoptosis（P<0.01）.Compared with the MD group mice,the number of cardiomyocyte apoptosis in the SYKL and IBT groups was significantly reduced（P<0.01）.12.The expression levels of cardiac FGFR4 and PLCγ1 proteins with IF staining in each group:Compared with the mice in the NC group,the fluorescence intensity of cardiac FGFR4 and PLCγ1 proteins with IF staining in the MD group mice were increased significantly（P<0.01）.Compared with the mice in the MD group,the fluorescence intensity of cardiac FGFR4 and PLCγ1 proteins with IF staining in the SYKL and IBT groups was significantly increased and decreased significantly（P<0.01）.13.The expression levels of renal Klotho and FGFR1 proteins with IF double-staining in each group:The expression of renal Klotho and FGFR1 proteins with IF double-staining in the kidney of NC group mice were significant high compared with the other three groups.Compared with NC group mice,the expression of Klotho and FGFR1proteins with IF double-staining in MD group were significantly reduced.Compared with mice in MD group,the expression of renal Klotho and FGFR1 protein with IF double-staining in the SYKL group and IBT group mice were significantly reduced.Conclusion:1.Shenyuan Granules can improve calcium and phosphorus metabolism and kidney function,and reduce kidney pathological damage in db/db mice;2.Shenyuan Granules can improve the myocardial injury in db/db mice by inhibiting endoplasmic reticulum stress;3.Shenyuan Granules can up-regulate the expression of Klotho protein,which can regulate the FGF23/CaMKⅡsignaling pathway,and then reduce CaMKⅡphosphorylation to weak ER stress signaling pathway.