Astragaloside ? Attenuates Chronic Intermittent Hypoxia-induced Myocardial Injury By Modulating Ca²⁺ Homeostasis

Objective:Obstructive sleep apnea(OSA)or obstructive sleep apnea syndrome(OSAHS)is a common disease which affects the health of Chinese and world people.The incidence of cardiovascular diseases in these patients is significantly increased,one of the reasons for the close relationship between them is that obstructive sleep apnea hypopnea syndrome can cause chronic intermittent hypoxia(CIH)in the body for a long time.At present,the pathogenesis of myocardial injury caused by CIH and the drug treatment are still limited.Astragaloside(As-IV)obtained by separation and purification is the most important component of Chinese herbal medicine in Astragalus.It has many pharmacological activities and is widely used in the treatment of various diseases.However,the application and mechanism of As-IV in the anti myocardial injury are not clear.In this study,we treated the rats with myocardial injury induced by exposure to CIH and given different doses of As-IV(40mg/kg/day or 80mg/kg/day)by regular gavage for 4 weeks;Meanwhile,we used H9C2 cardiomyocytes to detect the damage of CIH to cardiomyocytes by CIH experiment,and 100?M As-IV.The mechanism of astragaloside in reducing myocardial cell injury was studied by As-IV treatment;Then,by observing the changes of cardiac function,degree of myocardial fibrosis,apoptosis and the evaluation of Ca²⁺homeostasis in the cells,we can evaluate whether As-IV can alleviate the myocardial damage caused by CIH and play a pharmacological role in protecting the heart,so as to provide a new treatment method and basis for the treatment of cardiovascular complications in OSAHS patients.Methods:1.Male SD rats,weighing 200-250g,were fed adaptively for one week.12h white,12h black,temperature 22?±1?,humidity 45-55%,free water.They were randomly divided into normoxia group,intermittent hypoxia group,intermittent hypoxia+astragaloside IV(low)group and intermittent hypoxia+astragaloside IV(high)group(n=12).2.The rats in intermittent hypoxia group and intermittent hypoxia+astragaloside IV group(low and high)were fed in intermittent hypoxia environment,the oxygen concentration changed by 21%-5%,every 3 minutes a cycle:5%O₂ for 90 s,20 cycles/h,8 h/day,lasting for 4 weeks.The rats in normoxia group were fed in normoxia condition.The rats in intermittent hypoxia+astragaloside IV group(low and high)were given 40 mg/kg/day and 80 mg/kg/day by gavage respectively,and the other groups were given 0.5%sodium carboxymethyl cellulose with the same volume of solvent.It lasted for 4 weeks.3.After 4 weeks,the myocardial function was detected.The left ventricular end diastolic diameter(LVIDD),left ventricular end systolic diameter(LVISD),left ventricular end diastolic volume(LVEDV),left ventricular end systolic volume(LVESV)and left ventricular ejection fraction(LVEF)were measured by color Doppler ultrasound.4.All rats were killed by weighing and intraperitoneal injection of200 mg/kg pentobarbital sodium.The heart tissue was weighed,part of which was frozen,part of which was fixed,and part of which was fresh for isolation of myocardial cells.5.HE staining was used to evaluate the morphological changes of myocardial cells,Masson staining was used to qualitatively evaluate the level of myocardial fibrosis,TUNEL staining was used to observe the number of apoptotic myocardial cells in myocardial tissue sections,and the effects of chronic intermittent hypoxia and astragaloside IV on the level of myocardial cell apoptosis were evaluated.Western blot was used to detect the expression levels of cleaved caspase-3 and cleaved caspase-9,so as to further evaluate the level of cardiomyocyte apoptosis.6.Ca²⁺immunofluorescence was used to detect the intracellular Ca²⁺.The activity of SERCA2a was quantitatively analyzed by Ca²⁺-ATPase enzyme assay kit.7.H9C2 cardiomyocytes maintained 37°C When the cells reached 90%confluence,they were pretreated with As-IV and then treated with CIH.Cell grouping:(1)normal culture group(normoxic group):incubated in normal cell environment,the level of apoptosis and calcium related protein were detected48 hours later(2)Intermittent hypoxia group(CIH):the cells were incubated under chronic intermittent hypoxia with repeated circulation of 21%oxygen for 25 minutes/5%oxygen for 35 minutes,and the levels of apoptosis and calcium related protein were detected 48 hours later(3)Astragaloside IV treatment group(CIH+100?M As-IV):add100?M Astragaloside IV was incubated under chronic intermittent hypoxia with repeated circulation of 21%oxygen for 25 minutes/5%oxygen for 35 minutes.After 48hours,the levels of apoptosis and calcium related protein were detected.8.Western blot was used to detect the expression of Ca²⁺transport regulatory proteins:SERCA2a,RYR2,p-Ca MKII/Ca MKII,NCX1.9.Annexin V/PI staining and flow cytometry were used to detect the apoptosis of H9C2 cardiomyocytes.Western blot was used to detect the expression of cleaved caspase-3 and cleaved caspase-9.10.Ca²⁺immunofluorescence was used to detect the Ca²⁺level of H9C2 cells.The activity of SERCA2a was quantitatively analyzed by Ca²⁺-ATPase enzyme assay kit.11.Western blot was used to detect the expression of Ca²⁺transport regulatory proteins in H9C2 cells:SERCA2a,RYR2,p-Ca MKII/Ca MKII,NCX1.Results:1.Compared with the normal control group,the values of LVIDD,LVISD,LVEDV and LVESV in echocardiography of rats after long-term intermittent hypoxia treatment were significantly increased,the EF value of cardiac function was significantly decreased,and the cardiac function was significantly damaged.After long-term intermittent hypoxia treatment,echocardiography showed that the values of LVIDD,LVISD,LVEDV and LVESV were significantly decreased,and the EF value of cardiac function caused by chronic intermittent hypoxia was improved.2.Chronic intermittent hypoxia can significantly increase the ratio of HW/BW,and 80 mg/kg As-IV treatment can effectively reduce the ratio(P<0.05).3.HE staining showed that As-IV(40mg/kg and 80mg/kg)could reduce the disorder and swelling of cardiomyocytes induced by CIH.4.Masson staining showed that the myocardial fibrosis induced by CIH increased sharply after As-IV treatment(40mg/kg and 80mg/kg),and showed a significant decreasing trend.5.TUNEL staining showed that chronic intermittent hypoxia(CIH)could significantly increase the number of apoptotic cells in myocardial tissue,while As-IV(40mg/kg and 80mg/kg)could significantly reduce the number of apoptotic cells(brown).6.Western blot results showed that CIH could significantly increase the expression of cleaved caspase-3 and cleaved caspase-9 in heart tissue,while As-IV at the dose of 80 mg/kg could effectively reduce the expression of both(P<0.01).However,As-IV at the dose of 40 mg/kg could also reduce the expression of cleaved caspase-9 to a certain extent(P<0.05),However,the treatment dose could not reduce the expression level of cleaved caspase-3.7.Typical immunofluorescence micrographs of Ca²⁺related tissue sections showed that As-IV treatment(40 mg/kg and 80 mg/kg)significantly inhibited the increase of intracellular Ca²⁺level induced by CIH.Quantitative analysis of SERCA2a activity showed that As-IV at 80 mg/kg dose could effectively reverse the decrease of SERCA2a activity induced by CIH(P<0.05),while As-IV at 40 mg/kg dose could not reverse the change of SERCA2a activity.8.Western blot results showed that CIH could significantly reduce the expression of SERCA2a and Ry R2 in rat cardiomyocytes,while As-IV(40mg/kg and 80mg/kg)could reverse the trend and up regulate the expression of SERCA2a and RYR2.Compared with CIH group,SERCA2a expression was up-regulated in As-IV 40mg/kg group(P<0.05),and up-regulated in As-IV 80mg/kg group(P<0.01).Compared with CIH group,the expression of RYR2in As-IV 40 mg/kg group was up-regulated(P<0.01),and that in As-IV 80 mg/kg group was up-regulated(P<0.01).Western blot results showed that CIH could significantly increase the ratio of p-Ca MKII/Ca MKII and the expression level of NCX1,while As-IV(40mg/kg and 80mg/kg)could significantly reduce the ratio of p-Ca MKII/Ca MKII and the expression level of NCX1.Compared with CIH group,p-Ca MKII/Ca MKII ratio decreased in As-IV 40mg/kg group(P<0.01),and p-Ca MKII/Ca MKII ratio decreased in As-IV 80mg/kg group(P<0.01).Compared with CIH group,the expression of NCX1 in As-IV 40 mg/kg group was lower(P<0.01),and that in As-IV 80 mg/kg group was lower(P<0.01).9.Flow cytometry with annexin V/PI staining showed that CIH could significantly promote the apoptosis of H9C2 cells,and As-IV treatment could effectively inhibit this pro apoptotic effect(P<0.01).Western blot results showed that CIH significantly up-regulated the expression of cleaved caspase-3and cleaved caspase-9 in H9C2 cells,and As-IV could effectively down regulate their expression.Application 100?M Compared with CIH group,the expression of cleaved caspase-3 was decreased in As-IV group(P<0.05)Compared with CIH group,the expression of cleaved caspase-9 decreased in As-IV group(P<0.05).10.Typical micrographs of Ca²⁺immunofluorescence showed that As-IV treatment significantly reduced the increase of intracellular calcium level induced by CIH in H9C2 cells.Quantitative analysis of SERCA2a activity showed that As-IV treatment could improve the decrease of SERCA2a activity in H9C2 cells induced by CIH(P<0.05).11.Western blot results showed that CIH could significantly down regulate the expression of SERCA2a and Ry R2 in H9C2 cells.As-IV treatment could reverse and increase the expression of SERCA2a and Ry R2.Application 100?M Compared with CIH group,the expression of SERCA2a was up-regulated in As-IV group(P<0.01).Application 100?Compared with CIH group,the expression of RYR2 was up-regulated in As-IV group(P<0.01).Western blot results showed that CIH significantly increased the ratio of p-Ca MKII/Ca MKII and the expression of NCX1,while As-IV treatment decreased the ratio of p-Ca MKII/Ca MKII and the expression of NCX1.Application 100?M The p-Ca MKII/Ca MKII ratio in As-IV group was lower than that in CIH group(P<0.05).Application 100?M Compared with CIH group,the expression of NCX1 decreased in As-IV group(P<0.05).Conclusions:Astragaloside IV can effectively reduce the cardiac structural changes and cardiac function loss caused by chronic intermittent hypoxia,and can play a protective role in myocardial injury caused by chronic intermittent hypoxia,and this protective effect is positively correlated with the concentration of astragaloside IV.Astragaloside IV reduced the phosphorylation level of Ca²⁺/calmodulin dependent protein kinase II(Ca MKII),increase the activity of SERCA2a in sarcoplasmic reticulum,up regulate the expression of RYR2 and down regulate the expression of NCX1,the injury plays a protective role.