| Objective To explore the effect mechanism of Linggui Zhugan Decoction(LGZG),a compound Chinese herbal medicine,on improving mitochondrial damage,regulating mitochondrial respiratory chain function,increasing myocardial ATP content,improving oxidative stress injury,and protecting cardiac function through SIRT3-mediated signaling pathway.Method 1.6-week-old male SPF SS-13 BN male rats 6 were assigned as(1)Normal control group(Control),and forty Dahl/SS rats were randomly divided into five groups,(2)model group(Model),(3)Linggui Zhugan Decoction low dose group(LGZG-L),(4)Linggui Zhugan Decoction medium dose group(LGZG-M),(5)Linggui Zhugan Decoction high dose group(LGZG-H),(6)positive control group(Enalapril).The normal control group was fed with 0.3 % Na Cl diet.Dahl/SS male rats were fed with 8 % Na Cl diet for 8 weeks to establish HFp EF model.Five rats were randomly selected from the control group and the model group with small animal echocardiography to determine the HFp EF model establishment.On the next day after 6 weeks of intragastric treatment,5 rats in each group were randomly selected for echocardiography to detect the values of IVSd,LVIDd,LVPWd,EF,FS and E/A.After the body weight was weighed,blood and heart sample was taken,and the heart mass was weighed.Serum BNP and NT-Pro BNP were detected by ELISA.HE staining was used to observe the pathological changes of myocardium in each group.Masson staining was used to observe the changes of fibrosis of rats in each group.Myocardial apoptosis was detected by TUNEL staining.The ultrastructure of mitochondria was observed by electron microscope.The activity of mitochondrial respiratory chain complex I and ATP content were detected with the kit.Serum LDH and LA levels were detected by biochemical analyzer.The contents of SOD and MDA were detected by kit.The expression of SIRT3 in myocardial tissue was detected by immunohistochemistry.The expression of SIRT3,AMPK,PPARα and PGC-1α m RNA in myocardial tissue was detected by RT-PCR.The protein expressions of SIRT3,p-AMPK,AMPK,PPARα and PGC-1α in myocardial tissue were detected by Western blot.2.Different concentrations of ANGII were used to stimulate H9C2 cardiomyocytes.According to the cell viability,the appropriate ANGII concentration was selected for subsequent experiments.SIRT3 gene was transfected with cardiomyocytes,and an optimal sequence by RT-PCR was selected for subsequent experiments.The cultured H9C2 cardiomyocytes were inoculated in the culture plate and divided into 6 groups according to different treatment conditions,(1)Control group,(2)ANGII group(ANGII),(3)ANGII+ LGZG group(ANGII+LGZG),(4)ANGII+NCsi RNA group,(5)ANGII+SIRT3 si RNA group(ANGII+SIRT3 si RNA),and(6)ANGII+SIRT3 si RNA+LGZG group(ANGII+SIRT3si RNA+LGZG).After cultured at 37°C,5 % CO2 and saturated humidity for 24 h,the cells were collected for the following detection.The viability of cardiomyocytes was detected by MTT colorimetry;the gene silencing efficiency of SIRT3 m RNA was detected by RT-PCR.FITC-labeled wheat germ agglutinin was used to label cardiomyocytes,and optical density analysis was used to detect cardiomyocyte hypertrophy.The apoptosis rate was detected by TUNEL staining kit.The activity of mitochondrial respiratory chain complex I and ATP content were detected by kit.LDH level was detected by biochemical analyzer;the levels of ROS,SOD and MDA were detected by kit.The expression of SIRT3 m RNA in cardiomyocytes was detected by RT-PCR.Western blot was used to detect the expression of SIRT3,p-AMPK,AMPK,PPARα and PGC-1α in cardiomyocytes.Results 1.The results of Animal experiments Cardiac ultrasonography: After 8 weeks of high-salt diet feeding,compared with the Control group,the IVSd and LVIDd of the Model group were thickened(p> 0.05),the LVPWd was significantly thickened,and the E/A value decreased(p<0.05). At the end of the 6-week experiment,compared with the Control group,IVSd,LVIDd,and LVPWd in the Model group were significantly increased(p<0.05,p<0.05,p<0.01),and E/A was significantly reduced(p<0.01).Compared with the Model group,IVSd and LVIDd in each treatment group decreased but there was no statistical significance(p>0.05);LVPWd in LGZG-M and LGZG-H groups decreased significantly(p<0.05),EF and FS in each treatment group increased to varying degrees,but only EF in LGZG-M group and LGZG-H group increased significantly(p<0.05,p<0.01).The E/A of each treatment group was significantly increased(p<0.05).Cardiac index: Compared with the Control group,the body weight of the Model group decreased significantly(p<0.01),and the heart weight and cardiac index increased significantly(p<0.01).Compared with the Model group,the body weight of the LGZG-L,LGZG-M,LGZG-H and Enalapril group was significantly increased(p < 0.01,p<0.05,p <0.01,p<0.01).Compared with the Model group,the heart mass and cardiac index of each group decreased to varying degrees after intervention.The heart mass of the LGZG-L and LGZG-H group decreased significantly(p<0.05,p<0.01,p<0.05).cardiac index of each group decreased significantly(p <0.01).Serum BNP,NT-Pro BNP: Compared with the Control group,the serum BNP and NT-Pro BNP of the Model group were significantly increased(p<0.01).Compared with Model group,the levels of BNP in LGZG-M,LGZG-H and Enalapril groups were significantly decreased(p<0.01).The NT-Pro BNP of each treatment group was significantly reduced(p <0.01).HE staining: The heart tissue of the Model group showed that the myocardial fibers in the middle layer were arranged in a spiral shape and gathered into bundles.The morphological structure of myocardial fiber was blurred,part of the nucleus was split,cytoplasm was broken and dissolved,visible myocardial fiber degeneration and necrosis,interstitial fibrous tissue hyperplasia;myocardial cell thickening,inflammatory cell infiltration,nuclear displacement.LGZG-H group,LGZG-M group and Enalapril group were more obvious.Masson staining: The visual field of the Model group showed that the myocardial cells were hypertrophic,the myocardial fibers were arranged disorderly,and the local fracture of the muscle fibers was observed.Many collagen fibers were distributed in the myocardial interstitium,and many collagen fibers were seen around the blood vessels.The accumulation of collagen fibers increased significantly and the degree of fibrosis increased significantly(p<0.01).The LGZG-H group,LGZG-M group and Enalapri group were significantly improved(p<0.05),and LGZG-H group improved most obviously.TUNEL staining: Compared with the Control group,the number of cardiomyocyte apoptosis in the Model group was significantly increased(p<0.01).Compared with the Model group,the number of apoptotic cells in each intervention group was significantly reduced,among which LGZG-H,LGZG-M and Enalapril were significantly reduced,with statistical significance(p<0.05).Mitochondrial ultrastructure: The Model group showed that the arrangement of mitochondria in myocardial cells was disordered,the mitochondria were swollen,the mitochondrial cristae were blurred or even broken,some mitochondrial cristae and matrix disappeared,showing vacuolar changes,myofibrillar structure was seriously damaged,most of the regional sarcomere had contracted,M line and Z line were blurred,and myofibrils in a small number of parts were dissolved and broken.After 6-week treatment,the degree of mitochondrial damage in each group was reduced.LGZG-H group improved most obviously.Mitochondrial respiratory chain complex I activity and ATP content: Compared with the Control group,the activity of mitochondrial respiratory chain complex I and ATP content in the Model group were significantly decreased(p<0.01).Compared with the Model group,the activity of mitochondrial respiratory chain complex I in LGZG-M group,LGZG-H group and Enalapril group was significantly increased(p<0.05,p < 0.01,p < 0.01,p < 0.01).The content of ATP in LGZG-H group and Enalapril group increased significantly(p<0.01).Serum LDH and LA levels: Compared with Control group,serum LDH and LA in Model group were significantly increased(p<0.01).Compared with the Model group,the serum LDH of LGZG-M group,LGZG-H group and Enalapril group decreased significantly(p<0.05).The serum LA of LGZG-L and LGZG-H group decreased significantly(p <0.05,p<0.01).SOD and MDA content: Compared with the Control group,the total SOD activity in the Model group was significantly decreased and the MDA content was significantly increased(p<0.01).Compared with the Model group,the total SOD activity in LGZG-M,LGZG-H and Enalapril group were significantly increased(p<0.01).The contents of MDA in each group were significantly decreased.Expression of SIRT3 in myocardium detected by immunohistochemistry: Compared with the Control group,the SIRT3 positive cells in the Model group were significantly reduced,and the distribution density was also significantly reduced(p < 0.01).Compared with Model group,the distribution density of SIRT3 positive cells in LGZG-H group increased significantly(p<0.01).RT-PCR detection: Compared with Control group,the expression of SIRT3,PPARα and PGC-1α m RNA in Model group was significantly decreased(p<0.01),and no significant change in AMPK m RNA expression was observed(p>0.05).Compared with Model group,the expression of SIRT3 m RNA in LGZG-M,LGZG-H and Enalapril groups increased significantly(p<0.01).The expression of PPARα m RNA in LGZG-H was significantly increased(p<0.05).The expression of PGC-1α m RNA was significantly increased in LGZG-M,LGZG-H and Enalapril groups(p<0.05,p<0.01,p<0.01).Western blot: Compared with Control group,the protein expression of SIRT3,p-AMPK,PPARα and PGC-1α in Model group were significantly decreased(p< 0.01).Compared with the Model group,SIRT3 was significantly increased in the LGZG-H group and the Enalapril group(p<0.05,p<0.01).The protein expression of p-AMPK and PPARα were significantly increased in LGZG-M,LGZG-H and Enalapril groups(p<0.01).The protein expression of PGC-1α was significantly increased in LGZG-H group and Enalapril group(p< 0.01).2.The results of cellular experiments Cardiocyte viability: Compared with 0 μM ANGII,the cell viability decreased gradually with the increase of ANGII concentration.In the subsequent experiments,0.1μM ANGII was selected as the concentration to induce cardiomyocyte injury.Gene silencing efficiency of SIRT3 m RNA: RT-PCR was used to detect the expression level of SIRT3 m RNA in each group.The results showed that the three SIRT3 si RNA(A,B,C)m RNA decreased.Compared with NC si RNA,SIRT3 si RNA(A)decreased significantly(p < 0.01).Cardiomyocyte hypertrophy: Compared with ANGII group,there was no significant change in the optical density value of ANGII+NC si RNA group(p>0.05),the average optical density of ANGII+SIRT3 si RNA group increased significantly(p<0.01),and the values of ANGII+LGZG group decreased significantly(p<0.01).Compared with ANGII +SIRT3 si RNA group,the average optical density of ANGII+SIRT3 si RNA + LGZG group decreased significantly(p<0.01).There was no significant difference between ANGII+SIRT3si RNA+LGZG group and ANGII+LGZG group.Cell apoptosis rate: Compared with the Control group,the apoptosis rate of the ANGII group increased(p<0.01).Compared with ANGII group,the apoptosis rate of ANGII + SIRT3 si RNA group increased(p<0.01),and the apoptosis rate of ANGII + LGZG group decreased(p<0.01).Compared with ANGII + SIRT3 si RNA group,the apoptosis rate of ANGII+SIRT3si RNA+LGZG group increased(p<0.01).Compared with ANGII+LGZG group,the apoptosis rate of cardiomyocytes in ANGII+SIRT3 si RNA+LGZG group was increased(p<0.05).Activity of mitochondrial respiratory chain complex I,ATP content and LDH level: Compared with the control group,the activity of mitochondrial respiratory chain complex I and ATP content in the ANGII group were significantly decreased(p<0.01),and the LDH content was significantly increased(p<0.01).Compared with the ANGII group,the mitochondrial respiratory chain complex I activity and ATP content in the ANGII + SIRT3 si RNA group were further decreased(p<0.05),and the LDH content continued to increase(p<0.01).The mitochondrial respiratory chain complex I activity and ATP content in the ANGII+LGZG group were significantly increased(p<0.01,p<0.05),and the LDH content was significantly decreased(p<0.01).Compared with ANGII+SIRT3 si RNA group,the activity of respiratory chain complex I and ATP content in ANGII+SIRT3 si RNA+LGZG group were significantly increased(p<0.01,p<0.05),and LDH content was significantly decreased(p<0.01).Compared with ANGII+LGZG group,there was no significant difference in ATP content in ANGII+SIRT3 si RNA+LGZG group(p>0.05),the activity of granular respiratory chain complex I decreased and LDH content increased(p<0.05,p <0.01).ROS,SOD and MDA content: Compared with the Control group,the optical density of ROS in the ANGII group was significantly increased(p<0.01),and the increase of ROS level in the ANGII + SIRT3 si RNA group was more significant(p< 0.01).Compared with ANGII+SIRT3 si RNA group,the optical density of ROS in ANGII + SIRT3 si RNA+LGZG group was significantly decreased(p<0.01).There was no significant difference between ANGII+SIRT3 si RNA+LGZG group and ANGII + LGZG group(p>0.05).Compared with control group,the SOD content in H9C2 cardiomyocytes of ANGII group was significantly decreased(p<0.01),and MDA was significantly increased(p<0.01).Compared with ANGII group,there was no significant change in SOD and MDA after NC si RNA transfection of H9C2 cardiomyocytes(p>0.05).The level of SOD in ANGII + SIRT3 si RNA group was further decreased(p < 0.01),and MDA was further increased(p<0.01).The level of SOD in ANGII+LGZG group was significantly increased(p<0.01),and MDA was significantly decreased(p<0.01).Compared with ANGII + SIRT3 si RNA group,the level of SOD in ANGII +SIRT3 si RNA+LGZG group was significantly increased(p<0.01),and the level of MDA was significantly decreased(p<0.01).Compared with ANGII + LGZG group,the level of SOD in ANGII+SIRT3 si RNA+LGZG group was significantly decreased(p<0.01),and MDA was significantly increased(p<0.05).RT-PCR detection: Compared with Cotrol group,SIRT3 m RNA and PGC-1α m RNA in ANGII group were significantly decreased(p<0.01).Compared with ANGII group,SIRT3 m RNA and PGC-1α m RNA in ANGII+NCsi RNA group had no significant change(p>0.05).SIRT3 m RNA and PGC-1αm RNA were significantly increased in ANGII+LGZG group(p<0.05).Compared with ANGII+NC si RNA group,SIRT3 m RNA level in ANGII+SIRT3 si RNA group was further decreased(p<0.01),PGC-1α m RNA level was further decreased(p<0.05).Compared with ANGII+SIRT3 si RNA group,SIRT3 m RNA and PGC-1α m RNA in ANGII+SIRT3si RNA+LGZG group were significantly increased(p<0.01),and there was a significant difference between ANGII+SIRT3 si RNA+ LGZG and ANGII+LGZG group(p<0.01).Western blot: Compared with control group,the expression of SIRT3,PGC-1α,PPARa and p-AMPK in ANGII group were significantly decreased(p<0.01).Compared with ANGII group,the expression of SIRT3,PGC-1α,PPARa and p-AMPK in ANGII+LGZG group were significantly increased(p<0.01,p<0.05,p<0.01,p<0.05).The expression of SIRT3,PGC-1α and PPARa in ANGII +SIRT3 si RNA group were further significantly decreased(p<0.05,p<0.01,p<0.01).Compared with ANGII+SIRT3 si RNA group,The expression of SIRT3 and PPARa in ANGII+SIRT3 si RNA+LGZG group were significantly increased(p<0.05),PGC1 a and p-AMPK were significantly increased(p<0.05).Compared with ANGII+LGZG group,the expression of SIRT3,PPARa and p-AMPK in ANGII+SIRT3 si RNA+LGZG group were significantly decreased(p<0.01,p<0.05,p<0.01).Conclusion 1.Linggui Zhugan Decoction improved cardiac function such as echocardiography,BNP and NT-Pro BNP in HFp EF model rats,and inhibited morphological changes such as cardiomyocyte hypertrophy,myocardial fibrosis and cardiomyocyte apoptosis.2.Linggui Zhugan Decoction improved myocardial mitochondrial damage in HFp EF model rats,regulated mitochondrial respiratory chain function,increased myocardial ATP content,and inhibited oxidative stress injury.Linggui Zhugan Decoction can also up-regulate the expression of SIRT3,PGC-1α,PPARa and p-AMPK in myocardial tissue of HFp EF model.3.Linggui Zhugan Decoction-containing serum can inhibit ANGII-induced cardiomyocyte injury and apoptosis,improve the mitochondrial respiratory chain activity of cardiomyocytes,increase ATP production,reduce oxidative stress,and improve ANGII-induced cardiomyocyte SIRT3,PGC-1α,PPARa and p-AMPK expression.The cell damage was aggravated after SIRT3 si RNA transfection of H9C2 cardiomyocytes,and the protective effect of Linggui Zhugan Decoction was also partially reduced.SIRT3 may be a key signal molecule for Linggui Zhugan Decoction to improve mitochondrial damage.4.The myocardial protective effect of Linggui Zhugan Decoction may improve the mitochondrial respiratory chain activity of cardiomyocytes by activating the up-regulation of SIRT3-related signal molecule expression pathway,increase ATP production and reduce oxidative stress dysfunction,thereby improving cardiomyocyte hypertrophy and apoptosis. |
PDF Full Text Request