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 study the expressions of osteopontin (OPN),caspase-3 and mt-P53 proteins, and their relationship in gliomas. Methods Seventy gliomas specimens of patients (glioma group) were selected, and 10 samples of non-glioma brain tissue were used as control group. The SP method was used to detect the positive rates of protein expressions of OPN, caspase-3 and mt-P53 between two groups. The relationship between protein expressions of OPN, caspase-3 and mt-P53 in gliomas and grade of gliomas were detected by Western blot assay. Spearman rank correlation was compared between the positive expression of OPN, caspase-3 and rate mt-P53. Results The positive expression rates of OPN and mt-P53 were significantly higher in glioma group (64.29%and 60%) than those of control group (no positive expression), but the positive expression rate of caspase-3 was significantly lower than that of control group (47.14%vs. 90%, P<0.05). There were no significant differences in OPN, caspase-3 and mt-P53 expressions between different gender, age, tumor size and tumor position (P>0.05). The higher the WHO classification, the higher the positive expression rates of OPN and mt-P53 (P<0.001), and the lower the positive expression rate of caspase-3 (P<0.001). With the increased level of glioma grade, OPN and mt-P53 protein levels were increased, but caspase-3 protein expression level was decreased. There was a negatively correlation between OPN and the positive expression of caspase-3, but there was a positive correlation between OPN and the expression of mt-P53 (rs=-0.720 and 0.722, P<0.05). There was a negative correlation between caspase-3 and mt-P53 expressions (rs=-0.556, P<0.05). Conclusion The higher the WHO classification, the higher the positive expression rates of OPN and mt-P53, while the lower the positive expression rate of caspase-3. The study reveals that OPN, caspase-3 and mt-P53 expressions are associated with the occurrence and the progress of gliomas. The combined detection of them can contribute to the judgment of biological behavior of gliomas.