Effect Of Shexiang Baoxin Pill On Ventricular Remodeling In Spontaneously Hypertensive Rats By Inhibiting Endoplasmic Reticulum Stress

Background and objective:It is clear that ventricular remodeling in hypertension is related to endoplasmic reticulum stress(ERS),and inhibition of ERS mediated apoptosis can improve ventricular remodeling in hypertension.Meanwhile,as a Chinese patent medicine for the treatment of angina pectoris,the effect of Shexiang Baoxin Pill(SBP),on improving ventricular remodeling in hypertensive rats,has been confirmed,but its mechanism remains unclear,especially whether SBP can inhibit endoplasmic reticulum stress,and whether the improvement of ventricular remodeling is related to its inhibition of endoplasmic reticulum stress needs to be further confirmed.The purpose of this study was to investigate whether SBP could improve ventricular remodeling in hypertension by inhibiting endoplasmic reticulum stress,so as to provide new evidence and theoretical basis for the application of the drug in treatment of ventricular remodeling in hypertension.Methods:Thirty-two 7-weeks-old male SHR were randomly divided into four groups: model group(SHR,n=8),low-dose Shexiang Baoxin pill group(SBP-L,n=8),high-dose Shexiang Baoxin pill group(SBP-H,n=8),Shexiang Baoxin pill + Tunicamycin group(SBP+TM,n=8),and eight 7-weeks-old WKY as blank control group(WKY,n=8).After adaptive feeding for 1 week,SBP-L,SBP-H,SBP+TM were given 45mg/kg/d,112.5mg/kg/d and 112.5mg/kg/d,of Shexiang Baoxin Pills respectively,Shexiang Baoxin Pills were dissolved in 2ml of distilled water,and administered by gavage for 10 weeks.WKY and SHR were given the same amount of distilled water by gavage.Meanwhile,SBP+TM group was given tunicamycin(0.08 mg/kg,dissolved in 0.4 ml of normal saline)by intraperitoneal injection every Monday and Thursday.The other four groups were injected with the same amount of normal saline.The caudal artery systolic blood pressure of rats was measured before administration and 10 weeks of administration.After 10 weeks of administration,the rats were anesthetized with 3% sodium pentobarbital,and then cardiac ultrasound was performed,and the left ventricle of the heart was taken out to calculate the left ventricular weight index.Myocardial tissue section HE staining was used to observe the myocardial cell morphology changes size,to calculate the cross-sectional area of myocardial cells.Masson staining was used to observe the collagen fibers and muscle fibers in myocardial tissue,and the collagen volume fraction was calculated.Sirius red staining was used to observe the myocardial tissue collagen I and III collagen fibers,to calculate the ratio of type collagen I/III.TUNEL staining was used to observe the apoptosis of myocardial cells and calculate the apoptosis index.The endoplasmic reticulum stress-related proteins GRP78,ATF-6,IRE1 and PERK in left ventricular myocardium were detected by Western blot.Apoptosis-related protein CHOP,Bcl-2,Bax and autophagy-related proteins LC3 II,LC3I,Beclin1 in left ventricular myocardium were detected by Western blot.Results:Compared with WKY group,in the SHR group,LVEDd of the rats in the SHR group was significantly increased(p<0.05)and LVEF was obviously decreased(p<0.05);the left ventricular weight index was significantly increased(p<0.05);the cardiomyocytes were hypertrophied,the cytoplasm was significantly dissolved and necrotic,and the crosssectional area of the cardiomyocytes was significantly increased(p<0.05),myocardial collagen fiber deposition increased,myocardial collagen volume fraction increased significantly(p < 0.05),and collagen I/III ratio increased significantly(p < 0.05).All the above indicators indicated that SHR group had obvious ventricular remodeling.The expression levels of endoplasmic reticulum stress-related proteins GRP78,PERK,IRE1,and ATF-6 were significantly higher(p < 0.05),in SHR group than those in WKY group,indicating that endoplasmic reticulum stress was significantly enhanced in the SHR group.The ratio of autophagy-related protein LC3II/LC3 I and the expression of Beclin1 protein were significantly increased(p < 0.05)in SHR group,indicating that the autophagy of cardiomyocytes in the SHR group was significantly enhanced.In addition apoptotic cells increased,the apoptosis index increased significantly(p <0.05),the expression of apoptosis-related proteins CHOP and Bax protein increased significantly(p < 0.05),the expression of Bcl-2 protein and the ratio of Bcl-2/Bax decreased significantly(p < 0.05),indicating that the apoptosis of cardiomyocytes in the SHR group was significantly increased.Compared with SHR group,there was no significant difference in systolic blood pressure in the SBP-L and SBP-H group(p>0.05);the LVEDd was significantly reduced(p < 0.05),the left ventricular weight index was significantly decreased(p < 0.05),cardiomyocyte hypertrophy was reduced,cytoplasmic necrosis was improved,and the cross-sectional area of cardiomyocytes was significantly reduced(p < 0.05),the myocardial collagen fibers were reduced,the myocardial collagen volume fraction was significantly reduced(p < 0.05),and the ratio of type collagen I/III was significantly reduced(p < 0.05)in SBP-L and SBP-H group,indicating that Shexiang Baoxin Pill can significantly improve the ventricular remodeling in SHR independently of its antihypertensive effect.The expression levels of endoplasmic reticulum stress-related proteins GRP78,PERK,IRE1,and ATF-6 were significantly decreased(p<0.05)in SBP-L and SBP-H group,,indicating that Shexiang Baoxin Pills could inhibit endoplasmic reticulum stress.The ratio of autophagy-related protein LC3Ⅱ/LC3Ⅰ and the expression of Beclin1 protein were significantly increased(p<0.05)in SBPL and SBP-H group,indicating that Shexiang Baoxin Pill could enhance autophagy in cardiomyocytes.Apoptotic cells decreased,the apoptosis index decreased significantly(p < 0.05),the protein expressions of apoptosis-related proteins CHOP and Bax were significantly decreased(p <0.05),and the expression of Bcl-2 protein was significantly increased(p <0.05);the ratio of Bcl-2/Bax was significantly increased(p<0.05),in SBP-L and SBP-H group compared those in SHR group.It indicated that Shexiang Baoxin Pill could inhibit the apoptosis of cardiomyocytes.Compared with SHR-L group,SBP-H group had more significant effect on improvement of ventricular remodeling,inhibition of endoplasmic reticulum stress,enhancement of autophagy of cardiomyocytes,inhibition of apoptosis of cardiomyocytes in spontaneously hypertensive rats.Compared with SBP-H group,in the SBP+TM group,systolic blood pressure of the rats was significantly lower(p < 0.05),the LVEDd increased significantly(p <0.05),the left ventricular weight index increased(p < 0.05),the hypertrophic cardiomyocytes increased,the cytoplasmic lysis and necrosis were obvious,and the cardiomyocytes cross-sectional area was increased significantly(p < 0.05),the deposition of myocardial collagen fibers increased,the myocardial collagen volume fraction increased significantly(p < 0.05),the ratio of type collagen I/III increased significantly(p < 0.05);Expressions of endoplasmic reticulum stress-related proteins GRP78,PERK,IRE1 and ATF-6 were significantly increased(p < 0.05),suggesting that tunicamycin could enhance endoplasmic reticulum stress and attenuate the effect of Shexiang Baoxin Pill on ventricular remodeling of SHR.Accordingly,the ratio of autophagy-related protein LC3II/LC3 I and the expression of Beclin1 protein were significantly decreased(p <0.05);Apoptotic cells increased,and the apoptosis index increased significantly(p < 0.05);The expression of apoptosis-related proteins CHOP and Bax increased significantly(p < 0.05),Bcl-2 protein expression and the ratio of Bcl-2/Bax decreased significantly(p < 0.05).These results indicated that after enhancing endoplasmic reticulum stress was enhanced,the enhanced effect of Shexiang Baoxin Pill on enhancing autophagy and inhibiting apoptosis of cardiomyocytes were also obviously weakened.Conclusion:Shexiang Baoxin Pill can improve ventricular remodeling in spontaneously hypertensive rats by inhibiting endoplasmic reticulum stress,enhancing autophagy of cardiomyocytes and inhibiting apoptosis of cardiomyocytes...