ObjectiveTo investigate the mechanism by which Kangxian Yixin prescription regulates the circular RNA 0006719 （circ0006719）/microRNA 6852-5p （miR-6852-5p）/transforming growth factor-β₁ （TGF-β₁）/Smad axis to ameliorate myocardial fibrosis in rats with dilated cardiomyopathy （DCM） and angiotensin Ⅱ （Ang Ⅱ）-induced activation of cardiac fibroblasts （CFs）. MethodsA DCM rat model was established by administration of furazolidone. The rats were divided into normal group， model group， captopril （CAP， 10.1 mg·kg^-1·d^-1） group， and low-， medium-， and high-dose Kangxian Yixin prescription groups （0.9， 1.8， 3.6 g·kg^-1·d^-1）， with 15 rats in each group. After modeling， the treatment groups received corresponding drug solutions by gavage， whereas the normal and model groups received an equal volume of normal saline for 60 consecutive days. Echocardiography was used to measure left ventricular end-systolic diameter （LVESD）， left ventricular end-diastolic diameter （LVEDD）， left ventricular ejection fraction （LVEF）， and left ventricular fractional shortening （LVFS）. Transmission electron microscopy （TEM）， hematoxylin-eosin （HE） staining， and Masson staining were used to observe myocardial ultrastructure， pathological changes， and collagen deposition. Immunohistochemistry （IHC） was used to detect the protein expression of collagen type Ⅰ alpha 1 （Col1A1） and collagen type Ⅲ（Col Ⅲ） in myocardial tissue. Real-time quantitative polymerase chain reaction （Real-time PCR）， Western blot， and fluorescence in situ hybridization （FISH） were used to detect the expression and localization of circ0006719， miR-6852-5p， fibrosis-related molecules， and TGF-β₁/Smad pathway-related molecules in myocardial tissue and CFs. CFs from neonatal rats were isolated and cultured in vitro. Cell Counting Kit-8 （CCK-8） assay was used to determine the optimal concentration of Kangxian Yixin prescription for subsequent intervention. An Ang Ⅱ-induced CF activation model was established， and the cells were divided into normal group， Ang Ⅱ group， Ang Ⅱ+CAP group， Ang Ⅱ+Kangxian Yixin prescription group， and Ang Ⅱ+Kangxian Yixin prescription+miR-6852-5p inhibitor group. CF viability and fibrosis-related molecular expression were detected. RNA immunoprecipitation （RNA IP） and dual-luciferase reporter assays were used to verify the targeting relationship between circ0006719 and miR-6852-5p. ResultsCompared with the normal group， the model group showed significantly increased LVESD and LVEDD， significantly decreased LVEF and LVFS， aggravated myocardial ultrastructural and histopathological injury， and significantly increased collagen volume fraction （CVF） in myocardial tissue （P<0.01）. The expression of circ0006719， Col1A1 mRNA， Col ⅢmRNA， and Col1A1， Col Ⅲ， TGF-β₁， Smad3， and Smad4 proteins in myocardial tissue was significantly upregulated， whereas miR-6852-5p expression and Smad7 protein expression were significantly downregulated （P<0.01）. Compared with the model group， the CAP group and the high- and medium-dose Kangxian Yixin prescription groups showed varying degrees of improvement in cardiac function， myocardial fibrosis， circ0006719/miR-6852-5p expression， and TGF-β₁/Smad pathway-related indicators， with more pronounced improvements in the CAP group and the high-dose Kangxian Yixin prescription group （P<0.05， P<0.01）. In in vitro experiments， 2.0 g·L^-1 was determined as the optimal concentration of Kangxian Yixin prescription for subsequent intervention. Compared with the normal group， the Ang Ⅱ group showed significantly increased CF viability， significantly upregulated expression of circ0006719， Col1A1 mRNA， α-smooth muscle actin （α-SMA） mRNA， and Col1A1， α-SMA， TGF-β₁， Smad3， and Smad4 proteins in CFs， and significantly downregulated miR-6852-5p expression and Smad7 protein expression （P<0.01）. Compared with the Ang Ⅱ group， the Ang Ⅱ+CAP group and the Ang Ⅱ+Kangxian Yixin prescription group showed markedly decreased CF viability and fibrosis-related mRNA and protein expression， markedly increased miR-6852-5p expression， and markedly attenuated TGF-β₁/Smad pathway activation （P<0.05， P<0.01）. Compared with the Ang Ⅱ+Kangxian Yixin prescription group， the Ang Ⅱ+Kangxian Yixin prescription+miR-6852-5p inhibitor group showed significantly increased CF viability （P<0.01）， markedly increased expression of circ0006719， Col1A1 mRNA， and Col1A1， TGF-β₁， and Smad4 proteins in CFs， and markedly decreased miR-6852-5p expression （P<0.05， P<0.01）. RNA IP and dual-luciferase reporter assays showed a targeting binding relationship between circ0006719 and miR-6852-5p （P<0.01）. ConclusionKangxian Yixin prescription may alleviate myocardial collagen deposition and myocardial fibrosis， improve cardiac function in DCM rats， and inhibit Ang Ⅱ-induced abnormal activation of CFs. Its mechanism may be associated with downregulation of circ0006719， restoration of miR-6852-5p expression， and inhibition of the TGF-β₁/Smad pathway.

ObjectiveTo investigate the mechanism by which Kangxian Yixin prescription regulates the circular RNA 0006719 （circ0006719）/microRNA 6852-5p （miR-6852-5p）/transforming growth factor-β₁ （TGF-β₁）/Smad axis to ameliorate myocardial fibrosis in rats with dilated cardiomyopathy （DCM） and angiotensin Ⅱ （Ang Ⅱ）-induced activation of cardiac fibroblasts （CFs）. MethodsA DCM rat model was established by administration of furazolidone. The rats were divided into normal group， model group， captopril （CAP， 10.1 mg·kg^-1·d^-1） group， and low-， medium-， and high-dose Kangxian Yixin prescription groups （0.9， 1.8， 3.6 g·kg^-1·d^-1）， with 15 rats in each group. After modeling， the treatment groups received corresponding drug solutions by gavage， whereas the normal and model groups received an equal volume of normal saline for 60 consecutive days. Echocardiography was used to measure left ventricular end-systolic diameter （LVESD）， left ventricular end-diastolic diameter （LVEDD）， left ventricular ejection fraction （LVEF）， and left ventricular fractional shortening （LVFS）. Transmission electron microscopy （TEM）， hematoxylin-eosin （HE） staining， and Masson staining were used to observe myocardial ultrastructure， pathological changes， and collagen deposition. Immunohistochemistry （IHC） was used to detect the protein expression of collagen type Ⅰ alpha 1 （Col1A1） and collagen type Ⅲ（Col Ⅲ） in myocardial tissue. Real-time quantitative polymerase chain reaction （Real-time PCR）， Western blot， and fluorescence in situ hybridization （FISH） were used to detect the expression and localization of circ0006719， miR-6852-5p， fibrosis-related molecules， and TGF-β₁/Smad pathway-related molecules in myocardial tissue and CFs. CFs from neonatal rats were isolated and cultured in vitro. Cell Counting Kit-8 （CCK-8） assay was used to determine the optimal concentration of Kangxian Yixin prescription for subsequent intervention. An Ang Ⅱ-induced CF activation model was established， and the cells were divided into normal group， Ang Ⅱ group， Ang Ⅱ+CAP group， Ang Ⅱ+Kangxian Yixin prescription group， and Ang Ⅱ+Kangxian Yixin prescription+miR-6852-5p inhibitor group. CF viability and fibrosis-related molecular expression were detected. RNA immunoprecipitation （RNA IP） and dual-luciferase reporter assays were used to verify the targeting relationship between circ0006719 and miR-6852-5p. ResultsCompared with the normal group， the model group showed significantly increased LVESD and LVEDD， significantly decreased LVEF and LVFS， aggravated myocardial ultrastructural and histopathological injury， and significantly increased collagen volume fraction （CVF） in myocardial tissue （P<0.01）. The expression of circ0006719， Col1A1 mRNA， Col ⅢmRNA， and Col1A1， Col Ⅲ， TGF-β₁， Smad3， and Smad4 proteins in myocardial tissue was significantly upregulated， whereas miR-6852-5p expression and Smad7 protein expression were significantly downregulated （P<0.01）. Compared with the model group， the CAP group and the high- and medium-dose Kangxian Yixin prescription groups showed varying degrees of improvement in cardiac function， myocardial fibrosis， circ0006719/miR-6852-5p expression， and TGF-β₁/Smad pathway-related indicators， with more pronounced improvements in the CAP group and the high-dose Kangxian Yixin prescription group （P<0.05， P<0.01）. In in vitro experiments， 2.0 g·L^-1 was determined as the optimal concentration of Kangxian Yixin prescription for subsequent intervention. Compared with the normal group， the Ang Ⅱ group showed significantly increased CF viability， significantly upregulated expression of circ0006719， Col1A1 mRNA， α-smooth muscle actin （α-SMA） mRNA， and Col1A1， α-SMA， TGF-β₁， Smad3， and Smad4 proteins in CFs， and significantly downregulated miR-6852-5p expression and Smad7 protein expression （P<0.01）. Compared with the Ang Ⅱ group， the Ang Ⅱ+CAP group and the Ang Ⅱ+Kangxian Yixin prescription group showed markedly decreased CF viability and fibrosis-related mRNA and protein expression， markedly increased miR-6852-5p expression， and markedly attenuated TGF-β₁/Smad pathway activation （P<0.05， P<0.01）. Compared with the Ang Ⅱ+Kangxian Yixin prescription group， the Ang Ⅱ+Kangxian Yixin prescription+miR-6852-5p inhibitor group showed significantly increased CF viability （P<0.01）， markedly increased expression of circ0006719， Col1A1 mRNA， and Col1A1， TGF-β₁， and Smad4 proteins in CFs， and markedly decreased miR-6852-5p expression （P<0.05， P<0.01）. RNA IP and dual-luciferase reporter assays showed a targeting binding relationship between circ0006719 and miR-6852-5p （P<0.01）. ConclusionKangxian Yixin prescription may alleviate myocardial collagen deposition and myocardial fibrosis， improve cardiac function in DCM rats， and inhibit Ang Ⅱ-induced abnormal activation of CFs. Its mechanism may be associated with downregulation of circ0006719， restoration of miR-6852-5p expression， and inhibition of the TGF-β₁/Smad pathway.

Objective To investigate the effect of Astragalus polysaccharides(APS)on ventricular remodeling in mice with dilated cardiomyopathy(DCM)based on the cyclic guanosine monophosphate-adenosine monophosphate synthase(cGAS)/stimu-lator of interferon gene(STING)pathway.Methods The cTnTR141w transgenic DCM mice were randomly assigned into DCM group,APS low-dose group,APS high-dose group,Captopril group,and APS high-dose+cG AS STING pathway activator(Roc A)group,each with 12 mice.A total of 12 C57BL/6J mice were designated as control group.Each group was administered the corresponding drugs,once a day for 8 weeks.The left ventricular end diastolic diameter(LVEDD),left ventricular ejection frac-tion(LVEF),and left ventricular end systolic diameter(LVESD)of mice were measured.ELISA was used to detect serum crea-tine kinase(CK)and troponin T(cTn-T).The mouse heart mass index and left ventricular mass index were measured.Masson staining was used to detect myocardial tissue fibrosis.ELISA was used to test interleukin(IL)-10,tumor necrosis factor-a(TNF-α),and IL-6 in myocardial tissue.Western blot was used to test cGAS and STING proteins in myocardial tissue.Results The a-mount of blue stained myocardial fibers generated in the myocardium was increased,the LVEDD,LVESD,serum CK and cTn-T levels,cardiac mass index,left ventricular mass index,myocardial tissue TNF-α and IL-6 levels,and cGAS and STING proteins were elevated,while the LVEF and myocardial tissue IL-10 were decreased in the DCM group as compared with those in the control group(all P＜0.05).Compared with the DCM group,the generation of blue stained myocardial fibers in the myocardium of mice decreased,the LVEDD,LVESD,serum CK and cTn-T levels,cardiac mass index,left ventricular mass index,myocardial tissue TNF-α and IL-6 levels,and cGAS and STING proteins were reduced,while the LVEF and myocardial tissue IL-10 were increased in the APS low-and high-dose groups,and Captopril group(all P＜0.05).Compared with the high-dose APS group,the generation of blue stained myocardial fibers in the myocardium of mice increased,the LVEDD,LVESD,serum CK and cTn-T levels,cardiac mass index,left ventricular mass index,myocardial tissue TNF-α and IL-6 levels,and cGAS and STING pro-teins were elevated,while the LVEF and myocardial tissue IL-10 were reduced in the high-dose APS+Roc A group(all P＜0.05).Conclusion The mechanism by which APS inhibits inflammation and improves ventricular remodeling in DCM mice may be related to the inhibition of cGAS/STING pathway.

Objective To investigate the effect of Astragalus polysaccharides(APS)on ventricular remodeling in mice with dilated cardiomyopathy(DCM)based on the cyclic guanosine monophosphate-adenosine monophosphate synthase(cGAS)/stimu-lator of interferon gene(STING)pathway.Methods The cTnTR141w transgenic DCM mice were randomly assigned into DCM group,APS low-dose group,APS high-dose group,Captopril group,and APS high-dose+cG AS STING pathway activator(Roc A)group,each with 12 mice.A total of 12 C57BL/6J mice were designated as control group.Each group was administered the corresponding drugs,once a day for 8 weeks.The left ventricular end diastolic diameter(LVEDD),left ventricular ejection frac-tion(LVEF),and left ventricular end systolic diameter(LVESD)of mice were measured.ELISA was used to detect serum crea-tine kinase(CK)and troponin T(cTn-T).The mouse heart mass index and left ventricular mass index were measured.Masson staining was used to detect myocardial tissue fibrosis.ELISA was used to test interleukin(IL)-10,tumor necrosis factor-a(TNF-α),and IL-6 in myocardial tissue.Western blot was used to test cGAS and STING proteins in myocardial tissue.Results The a-mount of blue stained myocardial fibers generated in the myocardium was increased,the LVEDD,LVESD,serum CK and cTn-T levels,cardiac mass index,left ventricular mass index,myocardial tissue TNF-α and IL-6 levels,and cGAS and STING proteins were elevated,while the LVEF and myocardial tissue IL-10 were decreased in the DCM group as compared with those in the control group(all P＜0.05).Compared with the DCM group,the generation of blue stained myocardial fibers in the myocardium of mice decreased,the LVEDD,LVESD,serum CK and cTn-T levels,cardiac mass index,left ventricular mass index,myocardial tissue TNF-α and IL-6 levels,and cGAS and STING proteins were reduced,while the LVEF and myocardial tissue IL-10 were increased in the APS low-and high-dose groups,and Captopril group(all P＜0.05).Compared with the high-dose APS group,the generation of blue stained myocardial fibers in the myocardium of mice increased,the LVEDD,LVESD,serum CK and cTn-T levels,cardiac mass index,left ventricular mass index,myocardial tissue TNF-α and IL-6 levels,and cGAS and STING pro-teins were elevated,while the LVEF and myocardial tissue IL-10 were reduced in the high-dose APS+Roc A group(all P＜0.05).Conclusion The mechanism by which APS inhibits inflammation and improves ventricular remodeling in DCM mice may be related to the inhibition of cGAS/STING pathway.

Objective To explore the effect of Kangxian Yixin decoction on PKA/CaMKⅡ Signal Pathway and Mitochondrial membrane potential of cardiomyocyte hypertrophy in dilated cardiomyopathy rats.Methods Drinking furazolidone to duplicate the DCM rats model for 10 weeks,then the rats were checked by ultrasound,the successfully established model rats were randomly divided into model group,low dose Kangxian Yixin decoction group,middle dose Kangxian Yixin decoction group,high dose Kangxian Yixin decoction group and captopril group.Normal group was separated.After 4 weeks of drug intervention,This experiment was over,ATP,Ca2+-Mg2+-ATP enzymatic activity,CaMKⅡ,PKA and UCP2 mRNA were tested.H9c2 cardiomyocyte hypertrophy model was set up by norepinephrine,after 24 h of drug intervention,mitochondrial membrane potential was tested.Results Compared with the normal group,cardiomyocyte mitochondria were damaged in each model group,ATP,Ca2+-Mg2+-ATP enzymatic activity and PKA mRNA were lower,UCP2 mRNA and CaMKⅡ mRNA were higer,mitochondrial membrane potential obviously decreased(P<0.05 or P<0.01).The mitochondria were protected in every drug group,the tested indexes get well in different degree,especially in hige dose Kangxian Yixin decoction group(P<0.05 or P<0.01).Conclusion Kangxian Yixin decoction can reduce myocardial cell mitochondrial damage in model rats with DCM,improve myocardial cell energy metabolism and cardiac systolic and diastolic functions,one of the mechanisms may be optimizing PKA/CaMKⅡ signal pathway and improving mitochondrial membrane potential.

This study was aimed to observe the influence of Kang-Xian Y i-Xin (KXYX) formula on the heart size and cardiac function of patients with dilated cardiomyopathy (DCM). A total of 85 cases were randomly divided into the treatment group (43 cases) and the control group (42 cases, with 1 death case). Both groups were treated with routine western medicine. And the KXYX formula was combined in the treatment group once a day. Six months later, changes of the left ventricular end diastolic diameter (LVEDD), left atrial diameter (LAd), ejection fraction (EF) and fractional shortening (FS) were observed by ultrasonography. The results showed that both groups can reduce the LVEDD and Lad. And the effect was obvious in the treatment group (P< 0.05). There was significant difference in the LVEDD of both groups after treatment (P< 0.05). The EF and FS were raised obviously in both groups. There was significant difference before and after treatment in the treatment group (P < 0.01). There was statistical differ-ence between groups after treatment (P< 0.05). It was concluded that the KXYX formula can decrease the LVEDD, Lad, enhance EF and FS, in order to promote the cardiac function of patients with DCM.