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.

AIM: To develop LC-MS /MS method for simultaneous determination of simvastatin and simvastatin acid concentrations in rat plasma, and investigate the pharmacokinetic effects of Wuzhi capsule (WZC) on simvastatin and simvastatin acid concentrations in rats. METHODS: The method was based on simple liquid liquid extraction (LLE) with lovastatin as internal standard. Agilent Eclipse-C

[ ABSTRACT] AIM: To investigate the regulatory mechanism of histone acetylation on cardiac hypertrophy in -duced by phenylephrine , and to provide a new idea for preventing and curing hypertrophic cardiomyopathy .METHODS:Phenylephrine was given by continuous subcutaneous injection for modeling cardiac hypertrophy in C 57BL/6 mice.The le-vel of histone H3 lysine 27 acetylation (H3K27ac) in the promoter of cardiac nuclear transcription factor GATA4 and the mRNA expression of GATA4 were identified by chromatin immunoprecipitation (ChIP) and real-time PCR, respectively. Meanwhile, the protein expression of histone H3K27ac, atrial natriuretic peptide (ANP) and α-myosin heavy chain (α-MHC) was determined by Western blot .Cardiac hypertrophy in the mice was observed by HE staining and echocardio-graphy.RESULTS:The results of Western blot showed that the level of histone H 3K27ac in phenylephrine group was sig-nificantly increased compared with normal saline group (P<0.05), and ChIP-qPCR data showed that the level of histone H3K27ac in the promoter of GATA4 was increased significantly in the same samples (P<0.05).The expression levels of GATA4 mRNA and ANP protein in phenylephrine group were apparently increased compared with normal saline group ( P<0.05).However, histone acetylase inhibitor anacardic acid attenuated histone H 3K27 hyperacetylation induced by pheny-lephrine, and downregulated the over-expression of GATA4 and ANP in the heart of the mice (P<0.05).HE staining and echocardiography data showed that phenylephrine apparently increased left ventricular posterior wall thickness and de -creased left ventricular end-systolic diameter and left ventricular end-diastolic diameter , while anacardic acid also reversed these indexes that mentioned above and attenuated cardiac hypertrophy induced by phenylephrine in the mice .CONCLU-SION:Histone acetylation modification imbalance is involved in cardiac hypertrophy induced by phenylephrine , and the histone acetylase inhibitor anacardic acid decreases histone hyperacetylation induced by phenylephrine and attenuates car -diac hypertrophy in the mice .

Werner syndrome (WS) is a premature aging disorder that mainly affects tissues derived from mesoderm. We have recently developed a novel human WS model using WRN-deficient human mesenchymal stem cells (MSCs). This model recapitulates many phenotypic features of WS. Based on a screen of a number of chemicals, here we found that Vitamin C exerts most efficient rescue for many features in premature aging as shown in WRN-deficient MSCs, including cell growth arrest, increased reactive oxygen species levels, telomere attrition, excessive secretion of inflammatory factors, as well as disorganization of nuclear lamina and heterochromatin. Moreover, Vitamin C restores in vivo viability of MSCs in a mouse model. RNA sequencing analysis indicates that Vitamin C alters the expression of a series of genes involved in chromatin condensation, cell cycle regulation, DNA replication, and DNA damage repair pathways in WRN-deficient MSCs. Our results identify Vitamin C as a rejuvenating factor for WS MSCs, which holds the potential of being applied as a novel type of treatment of WS.
Animals ; Ascorbic Acid ; pharmacology ; Cell Cycle Checkpoints ; drug effects ; Cell Line ; Cellular Senescence ; drug effects ; DNA Damage ; DNA Repair ; drug effects ; DNA Replication ; drug effects ; Disease Models, Animal ; Heterochromatin ; metabolism ; pathology ; Humans ; Mesenchymal Stem Cells ; metabolism ; pathology ; Mice ; Nuclear Lamina ; metabolism ; pathology ; Reactive Oxygen Species ; metabolism ; Telomere Homeostasis ; drug effects ; Werner Syndrome ; drug therapy ; genetics ; metabolism

A LC-MS method was established for the determination of the protein binding rates of oleanolic acid in human plasma and serum albumin. The equilibrium dialysis combined with LC-MS to determine the total concentration in plasma and free drug concentration of oleanolic acid was carried out. The human plasma protein binding rates of oleanolic acid at three concentrations were 79.6%, 81.9% and 63.3%, respectively. The human serum albumin protein binding rates of oleanolic acid at three concentrations were 53.5%, 56.6% and 47.7%, respectively. The method is shown to be simple, accurate, sensitive and specific for the determination of biological samples. The protein binding rates in human plasma and serum albumin were of high strength.