Research progress of cryptotanshinone on anti-fibrosis and its mechanism / 药学实践杂志

As a natural compound with high efficiency and low toxicity, cryptotanshinone (CTS) has a good anti-fibrosis effect in various organs and tissues. However, its mechanism of action has not been clearly defined, and there is no systematic literature review to describe its potential anti-fibrosis mechanism. The efficacy and mechanism of cryptotanshinone in the treatment of fibrosis in various organs were summarized and the use prospects were put forward in this paper.

Myosin Heavy Chain 7 Gene-derived miRNA-208b-3p Enhances the Fibrosis-related Gene Expression in Cardiac Fibroblasts / 中山大学学报(医学科学版)

ObjectiveTo investigate the effect of myosin heavy chain 7 gene-derived miRNA-208b-3p on the fibrotic phenotype of cardiac fibroblasts. MethodsmiRNA chip array was performed to detect the dysregulated miRNAs in the myocardium of diabetic db/db mice and db/m control mice. Neonatal mouse ventricular cardiomyocytes （NMVCs） and cardiac fibroblasts （CFs） were isolated from C57BL/6 mice and cultured. Real-time quantitative PCR （RT-qPCR） was conducted to determine the expression of miR-208b-3p in mouse CFs and NMVCs subjected to angiotensinⅡ（AngⅡ） and high glucose plus glucose oxidase （G/Go） treatment， respectively. Cell counting kit 8（CCk8） assay， flow cytometry and determination of fibrosis-related protein， including COL1A1， COL3A1and α-SMA， were performed in mCFs transfected with miR-208b-3p. Dual luciferase reporter assay was performed to confirm the interaction between miR-208b-3p and the 3'-UTR of metal response element binding transcription factor 2 （Mtf2） and progesterone receptor membrane component 1（Pgrmc1）， respectively. The expressions of Mtf2 and Pgrmc1 at the mRNA and protein levels in mCFs after miR-208b-3p mimic transfection were determined using RT-qPCR and Western blot assay， respectively. The small interfering RNA （siRNA） was used to inhibit Mtf2 and Pgrmc1 expression in mCFs， and the effects of Mtf2 siRNA， Pgrmc1 siRNA and miR-208b-3p on fibrosis-related protein expression in mCFs were investigated. ResultsResults of miRNA chip array and RT-qPCR assay showed that miR-208b-3p was up-regulated in the myocardium of the diabetic db/db mice. miR-208b precursor and the host gene of Myh7 were consistently increased in db/db mice. miR-208b-3p and Myh7 mRNA were expressed in mCFs and NMVCs， but the levels of miR-208b-3p and Myh7 mRNA in NMVCs were much higher than those in mCFs. miR-208b-3p was up-regulated in mCFs and NMVCs subjected to Ang Ⅱ and G/Go treatment， respectively. miR-208b-3p could significantly enhance fibrosis-related protein， including COL1A1， COL3A1 and α-SMA， in mCFs， without affecting the proliferation activity and cell cycle distribution of mCFs. Dual luciferase reporter assay revealed the interactions of miR-208b-3p with the 3'-UTR of Mtf2 and Pgrmc1. The results of RT-qPCR and Western blotting confirmed that miR-208b-3p inhibited Mtf2 and Pgrmc1 expression at the post- transcriptional level. Transfection with miR-208b-3p mimic， Mtf2 siRNA and Pgrmc1 siRNA could consistently enhance the fibrosis-related protein expression in the cardiac fibroblasts. ConclusionsmiR-208b-3p enhances fibrosis-related gene expression by targeting Mtf2 and Pgrmc1in mCFs.

Assessment of the effect of cinacalcet on right ventricular function in rats with pulmonary arterial hypertension by echocardiography: an experimental study / 中华超声影像学杂志

Objective:To evaluate the effect of cinacalcet on right ventricular function in rats with MCT-induced arterial pulmonary hypertension by echocardiography.Methods:Thirty male SD rats were randomly divided into control group, PAH group, and cinacalcet group, with 10 rats in each group. Rats in the cinacalcet group were given intraperitoneal injection of cinacalcet hydrochloride with 30 mg/kg, and the control group and the PAH group were given equal-volume of solvent. Echocardiographic parameters: right ventricular wall thickness (RVWT), right ventricular basal dimension (RVD), left ventricular eccentricity index (EI), tricuspid annular plane systolic excursion (TAPSE), right ventricular fractional area change (RVFAC), tricuspid lateral annular systolic velocity (s′), right ventricular global longitudinal strain (RV4CSL), and right ventricular free wall longitudinal strain (RVFWSL), etc. Histopathological parameters: pulmonary arteriole wall thickness (WT), right ventricular cardiomyocyte mean diameter (RV cell-D), collagen volume fraction (CVF) and right ventricular hypertrophy index (RVI). Echocardiographic and pathological parameters were compared among three groups, and the correlation between right ventricular pathological changes and strain parameters was analyzed.Results:①Compared with the control group, WT, RV cell-D, CVF and RVI in PAH group were increased (all P<0.01), the size of right ventricle and thickness of RV wall were increased (all P<0.05), and the right ventricular longitudinal strain was reduced ( P<0.01). ②Compared with the PAH group, rats in the cinacalcet group showed reduced WT, RV cell-D, CVF and RVI (all P<0.01), as well as improved structure and function of the right ventricle (all P<0.05). There was no statistical difference of the above parameters between cinacalcet and control group (all P>0.05). ③Correlation analysis: the right chamber remodeling parameters CVF and RV cell-D were positively correlated with WT ( rs=0.706 3, 0.629 4; both P<0.05); and RVFWSL correlated well with CVF, RV cell-D ( rs=-0.685 3, r=-0.767 2; both P<0.05). Conclusions:The right ventricular inverse remodeling of PAH rats with the intervention of cinacalcet was retained, suggesting that cinacalcet had a protective effect on the structure and function of the right ventricle in rats with PAH.

Cardiac Fibrosis in right Atrial tissue is not different in male Pasifika and Pākehā Cardiac surgery patients in Aotearoa

@#Cardiovascular disease (CVD) is the leading cause of death worldwide and in New Zealand. However, a significant inequality in the burden of CVD amongst different ethnic groups exists with a 2 - 3-times higher CVD mortality rate in Pasifika compared to Pākehā. It is unknown whether a difference in cardiac fibrosis might underly this ethnic inequality in CVD mortality. To address this, we determined cardiac fibrosis, myocardial fat infiltration, and the expression of some key miRNAs (miR-15a, miR-15b, miR- 34a and miR-153) in right atrial appendages of Pacific Islanders and New Zealand European patients (n=21) undergoing cardiopulmonary bypass surgery. Cardiac fibrosis was measured by total collagen deposition identified by Picro Sirius Red staining, whereas fat accumulation was determined via Oil-Red-O staining. No differences in cardiac fibrosis were observed between ethnic groups (Collagen: Pasifika 23.4±12.5% vs. New Zealand European 29.4±13.2%, one-way ANOVA, p=0.17). Similarly, no differences were observed in accumulation of lipid nor the expression of the miRNAs examined (-15a, - 15b, -34a and -153) between different groups. In conclusion, the earlier requirements for surgical intervention for CVD of Pasifika in Aotearoa might not be explained by differences in miRNAs associated with cardiomyocyte loss, fibrosis or myocardial lipid infiltration. New and Noteworthy Despite the established significant inequality in the burden of CVD amongst the Pasifika compared to the Pākehā (New Zealand European) populations in Aotearoa, we found no difference inhistopathological (cardiac fibrosis, lipid infiltration, or associated pro- or anti-f

Discovery of novel 4-phenylquinazoline-based BRD4 inhibitors for cardiac fibrosis

Bromodomain containing protein 4 (BRD4), as an epigenetic reader, can specifically bind to the acetyl lysine residues of histones and has emerged as an attractive therapeutic target for various diseases, including cancer, cardiac remodeling and heart failure. Herein, we described the discovery of hit 5 bearing 4-phenylquinazoline skeleton through a high-throughput virtual screen using 2,003,400 compound library (enamine). Then, structure-activity relationship (SAR) study was performed and 47 new 4-phenylquinazoline derivatives toward BRD4 were further designed, synthesized and evaluated, using HTRF assay set up in our lab. Eventually, we identified compound C-34, which possessed better pharmacokinetic and physicochemical properties as well as lower cytotoxicity against NRCF and NRCM cells, compared to the positive control JQ1. Using computer-based molecular docking and cellular thermal shift assay, we further verified that C-34 could target BRD4 at molecular and cellular levels. Furthermore, treatment with C-34 effectively alleviated fibroblast activation in vitro and cardiac fibrosis in vivo, which was correlated with the decreased expression of BRD4 downstream target c-MYC as well as the depressed TGF-β1/Smad2/3 signaling pathway. Taken together, our findings indicate that novel BRD4 inhibitor C-34 tethering a 4-phenylquinazoline scaffold can serve as a lead compound for further development to treat fibrotic cardiovascular disease.

The potential role of long non-coding RNA Dnm3os in the activation of cardiac fibroblasts / 生物医学工程学杂志

Long non-coding RNA (lncRNA) Dnm3os plays a critical role in peritendinous fibrosis and pulmonary fibrosis, but its role in the process of cardiac fibrosis is still unclear. Therefore, we carried out study by using the myocardial fibrotic tissues obtained by thoracic aortic constriction (TAC) in an early study of our group, and the

Low-intensity pulsed ultrasound ameliorates angiotensin II-induced cardiac fibrosis by alleviating inflammation via a caveolin-1-dependent pathway / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

OBJECTIVES@#Cardiac hypertrophy and fibrosis are major pathological manifestations observed in left ventricular remodeling induced by angiotensin II (AngII). Low-intensity pulsed ultrasound (LIPUS) has been reported to ameliorate cardiac dysfunction and myocardial fibrosis in myocardial infarction (MI) through mechano-transduction and its downstream pathways. In this study, we aimed to investigate whether LIPUS could exert a protective effect by ameliorating AngII-induced cardiac hypertrophy and fibrosis and if so, to further elucidate the underlying molecular mechanisms.@*METHODS@#We used AngII to mimic animal and cell culture models of cardiac hypertrophy and fibrosis. LIPUS irradiation was applied in vivo for 20 min every 2 d from one week before mini-pump implantation to four weeks after mini-pump implantation, and in vitro for 20 min on each of two occasions 6 h apart. Cardiac hypertrophy and fibrosis levels were then evaluated by echocardiographic, histopathological, and molecular biological methods.@*RESULTS@#Our results showed that LIPUS could ameliorate left ventricular remodeling in vivo and cardiac fibrosis in vitro by reducing AngII-induced release of inflammatory cytokines, but the protective effects on cardiac hypertrophy were limited in vitro. Given that LIPUS increased the expression of caveolin-1 in response to mechanical stimulation, we inhibited caveolin-1 activity with pyrazolopyrimidine 2 (pp2) in vivo and in vitro. LIPUS-induced downregulation of inflammation was reversed and the anti-fibrotic effects of LIPUS were absent.@*CONCLUSIONS@#These results indicated that LIPUS could ameliorate AngII-induced cardiac fibrosis by alleviating inflammation via a caveolin-1-dependent pathway, providing new insights for the development of novel therapeutic apparatus in clinical practice.

Huoxin Pill () Attenuates Cardiac Fibrosis by Suppressing TGF-β1/Smad2/3 Pathway in Isoproterenol-Induced Heart Failure Rats / 中国结合医学杂志

OBJECTIVE@#To evaluate the effects of Huoxin Pill (, HXP) on cardiac fibrosis and heart failure (HF) in isoproterenol (ISO)-induced HF rats.@*METHODS@#Thirty Wistar rats were randomly divided into 5 groups including control, HF, isosorbide mononitrate (ISMN), HXP low (HXP-L), and HXP high (HXP-H) groups (n=6 for each group) according to the complete randomization method. Rats were pretreated with ISMN (5 mg/kg daily), low concentration of HXP (10 mg/kg daily) or high concentration of HXP (30 mg/kg daily) or equal volume of saline by intragastric administration for 1 week, followed by intraperitoneal injection of ISO (10 mg/kg, 14 days), and continually intragastric administrated with above medicines or saline for additional 6 weeks. The effects of HXP treatment on the cardiac function, heart weight index (HWI), pathological changes, and collagen content were further assessed. Moreover, the role of HXP on activation of transforming growth factor- β 1 (TGF-β 1)/Smads pathway was further explored using immunohistochemistry (IHC) and Western-blot assay.@*RESULTS@#HXP treatment significantly alleviated the decrease of ejection fraction (EF) and fractional shortening (FS), while decreased the elevation of left ventricular end-systolic volume (LVESV) in ISO-induced HF rats (P<0.05). Moreover, HXP treatment obviously attenuated the increase of HWI and serum level of creatine kinase MB (CK-MB, P<0.05), as well as pathological changes in ISO-induced HF rats. Further determination indicated that HXP treatment alleviated the elevation of collagen I and collagen III protein expression in cardiac tissues of ISO-induced HF rats. Furthermore, HXP treatment significantly down-regulated the increase of TGF-β 1 and p-Smad2/3 protein expression in cardiac tissues of HF rats (P<0.05), while did not affect the expression of total Smad2/3.@*CONCLUSIONS@#HXP attenuated heart failure and cardiac fibrosis in ISO-induced HF rats by suppression of TGF-β 1/Smad2/3 pathway.

Therapeutic silencing miR-146b-5p improves cardiac remodeling in a porcine model of myocardial infarction by modulating the wound reparative phenotype

Fibrotic remodeling is an adverse consequence of immune response-driven phenotypic modulation of cardiac cells following myocardial infarction (MI). MicroRNA-146b (miR-146b) is an active regulator of immunomodulation, but its function in the cardiac inflammatory cascade and its clinical implication in fibrotic remodeling following MI remain largely unknown. Herein, miR-146b-5p was found to be upregulated in the infarcted myocardium of mice and the serum of myocardial ischemia patients. Gain- and loss-of-function experiments demonstrated that miR-146b-5p was a hypoxia-induced regulator that governed the pro-fibrotic phenotype transition of cardiac cells. Overexpression of miR-146b-5p activated fibroblast proliferation, migration, and fibroblast-to-myofibroblast transition, impaired endothelial cell function and stress survival, and disturbed macrophage paracrine signaling. Interestingly, the opposite effects were observed when miR-146b-5p expression was inhibited. Luciferase assays and rescue studies demonstrated that the miR-146b-5p target genes mediating the above phenotypic modulations included interleukin 1 receptor associated kinase 1 (IRAK1) and carcinoembryonic antigen related cell adhesion molecule 1 (CEACAM1). Local delivery of a miR-146b-5p antagomir significantly reduced fibrosis and cell death, and upregulated capillary and reparative macrophages in the infarcted myocardium to restore cardiac remodeling and function in both mouse and porcine MI models. Local inhibition of miR-146b-5p may represent a novel therapeutic approach to treat cardiac fibrotic remodeling and dysfunction following MI.

The potential role of calnexin in the activation of cardiac fibroblasts / 生物医学工程学杂志

Calnexin is a lectin-like molecular chaperone protein on the endoplasmic reticulum, mediating unfolded protein responses, the endoplasmic reticulum Ca homeostasis, and Ca signals conduction. In recent years, studies have found that calnexin plays a key role in the heart diseases. This study aims to explore the role of calnexin in the activation of cardiac fibroblasts. A transverse aortic constriction (TAC) mouse model was established to observe the activation of cardiac fibroblasts , and the cardiac fibroblasts activation model was established by transforming growth factor β1 (TGFβ1) stimulation. The adenovirus was respectively used to gene overexpression and silencing calnexin in cardiac fibroblasts to elucidate the relationship between calnexin and cardiac fibroblasts activation, as well as the possible underlying mechanism. We confirmed the establishment of TAC model by echocardiography, hematoxylin-eosin, Masson, and Sirius red staining, and detecting the expression of cardiac fibrosis markers in cardiac tissues. After TGFβ1 stimulation, markers of the activation of cardiac fibroblast, and proliferation and migration of cardiac fibroblast were detected by quantitative PCR, Western blot, EdU assay, and wound healing assay respectively. The results showed that the calnexin expression was reduced in both the TAC mice model and the activated cardiac fibroblasts. The overexpression of calnexin relieved cardiac fibroblasts activation, in contrast, the silencing of calnexin promoted cardiac fibroblasts activation. Furthermore, we found that the endoplasmic reticulum stress was activated during cardiac fibroblasts activation, and endoplasmic reticulum stress was relieved after overexpression of calnexin. Conversely, after the silencing of calnexin, endoplasmic reticulum stress was further aggravated, accompanying with the activation of cardiac fibroblasts. Our data suggest that the overexpression of calnexin may prevent cardiac fibroblasts against activation by alleviating endoplasmic reticulum stress.

Piperine inhibits the transformation of endothelial cells into fibroblasts / 中华心血管病杂志

Objective@#To investigate the role of piperine on the transformation of endothelial cells into fibroblasts.@*Methods@#Cultured human umbilical vein endothelial cells (HUVECs, 4-6 passage) were used for the main experiments. The transformation models of endothelial cells into fibroblasts were induced by transforming growth factor β (TGF-β) stimulation. HUVECs were divided into 6 groups: control group, TGF-β group and 4 groups treated with various concentrations of piperine (1, 5, 10, 20 μmol/L). CKK-8 was used to detect cell proliferation. The CD31/α-smooth muscle actin (α-SMA) expression level was detected by fluorescent staining. The vascular endothelial cadherin (VE-cadherin)/vimentin expression was detected by immunofluorescence staining. RT-PCR was used detect the mRNA expressions of transformation markers. Western blot was used to detect the protein expression of snail and twist.@*Results@#TGF-β increased HUVECs proliferation (P<0.05), which could be significantly inhibited by 10 and 20 μmol/L of piperine, but not by 1 and 5 μmol/L of piperine. Immunofluorescence results demonstrated that TGF-β increased HUVECs transformation to fibroblasts as shown by downregulated expression of endothelial markers CD31, VE-cadherin, and upregulated expression of α-SMA and vimentin, again, these effects could be attenuated by 10 and 20 μmol/L piperine. The expression levels of collagen type Ⅰ and type Ⅲ were significantly higher in TGF-β group than in control group (P<0.05), significantly lower in TGF-β+10 μmol/L piperine group and TGF-β+20 μmol/L piperine group than in TGF-β group (P<0.05).In addition, RT-PCR results showed that TGF-β increased mRNA expression of transformation markers (snail1, snail2, twist1, twist2), while 10 and 20 μmol/L of piperine could significantly downregulated the mRNA expressions of these markers. The protein expression levels of snail and twist were significantly higher in TGF-β group than in control group (both P<0.05), which was significantly lower in TGF-β+20 μmol/L piperine group than in TGF-β group (both P<0.05).@*Conclusions@#Piperine can inhibit the transformation of endothelial cells into fibroblasts. This effect might be viewed as one of the potential mechanisms of reduced myocardial fibrosis post piperine treatment.

Emodin alleviates cardiac fibrosis by suppressing activation of cardiac fibroblasts upregulating metastasis associated protein 3

Excess activation of cardiac fibroblasts inevitably induces cardiac fibrosis. Emodin has been used as a natural medicine against several chronic diseases. The objective of this study is to determine the effects of emodin on cardiac fibrosis and the underlying molecular mechanisms. Intragastric administration of emodin markedly decreased left ventricular wall thickness in a mouse model of pathological cardiac hypertrophy with excess fibrosis induced by transaortic constriction (TAC) and suppressed activation of cardiac fibroblasts induced by angiotensin II (AngII). Emodin upregulated expression of metastasis associated protein 3 (MTA3) and restored the MTA3 expression in the setting of cardiac fibrosis. Moreover, overexpression of MTA3 promoted cardiac fibrosis; in contrast, silence of MTA3 abrogated the inhibitory effect of emodin on fibroblast activation. Our findings unraveled the potential of emodin to alleviate cardiac fibrosis upregulating MTA3 and highlight the regulatory role of MTA3 in the development of cardiac fibrosis.

Effect of microRNA-133b on Myocardial Fibrosis / 中国医学科学院学报

Objective To investigate the effect of microRNA-133b(miR-133b)on cardiac fibrosis and its mechanism.Methods Human cardiac fibroblasts(CFs)were harvested.The proliferation of CFs was detected by CCK8 during the overexpression and knock-down of miR-133b.The expressions of connective tissue growth factor(CTGF),α-smooth muscle actin(α-SMA),collagen Ⅰ,and collagen Ⅲ were detected with qRT-PCR and Western blot analysis after miR-133b overexpression or downexpression.Target genes of miR-133b were predicted by bioinformatics software.Dual-luciferase activity assay were used to verify a target gene of miR-133b.Results qRT-PCR showed that the expression level of miR-133b in the miR-133b mimic group was significantly higher than that in the negative control group(=26.219,=0.000).The expression level of miR-133b in the miR-133b inhibitor group was significantly lower than that in the negative control group(=6.738,=0.003).After 21,45,69,93,and 117 hours of transfection,the proliferation ability of CFs significantly decreased in the miR-133b mimic group but significantly increased in the miR-133b group(all <0.05,compared with the negative control group).After overexpression of miR-133b,the mRNA and protein levels of CTGF(=9.213,=0.001;=8.195,=0.001),α-SMA(=6.511, =0.003;=4.434,=0.011),collagenⅠ(=3.172,=0.034;=4.053,=0.015)and collagen Ⅲ(=6.404,=0.003;=5.319,=0.006)were significantly down-regulated.After the expression of miR-133b was knocked down,the mRNA and protein levels of CTGF(=9.439,=0.001;=14.100,=0.000),α-SMA(=4.519,=0.011;=4.377,=0.012),collagen Ⅰ(=5.966,=0.004;=5.514,=0.005)and collagen Ⅲ(=4.622,=0.010;=4.996,=0.008)were significantly increased.The relative luciferase activity of the cells co-transfected with miR-133b mimic and WT 3'UTR expression vector was significantly lower than that of the cells co-transfected with mimic control and WT 3'UTR expression vectors(=5.654,=0.005);however,there was no significant difference in relative luciferase activity between cells co-transfected with miR-133b mimic and MUT 3'UTR expression vectors and cells co-transfected with mimic control and MUT 3'UTR expression vectors(=0.380,=0.724).Conclusion miR-133b may affect the activation and proliferation of CFs by targeting CTGF and thus improve cardiac fibrosis.

Myocardial amyloid deposition in patients aged over 85 years with heart failure and preserved ejection fraction / 中华心血管病杂志

Objective@#To determine the frequency and extent of left ventricular amyloid deposition in patients aged over 85 years with heart failure and preserved ejection fraction (HFpEF).@*Methods@#A total of 43 patients aged 85 to 100 years old were enrolled in this study based on the autopsy database of Beijing Hospital from February 1, 2003 to October 31, 2016. The frequency and extent of left ventricular amyloid deposition and myocardial fibrosis were determined in left ventricular specimens from patients with antemortem diagnosis of HFpEF without clinically apparent amyloid (n=28) and from control subjects (n=15) post Congo red staining and Masson's trichrome staining. Kappa test was used to evaluate the consistency of the myocardial amyloidosis and fibrosis.@*Results@#The heart weight of the patients in HFpEF group and in control group were similar((452.7±107.7)g vs. (415.0±70.8)g, t=-1.218, P=0.23)). Positive Congo-red staining was found in 24 examples (24/28) in HFpEF group and 5 examples (5/15) in the control group; severe amyloid deposition was found in 7 examples (7/28) in HFpEF group, but not in the control group. Amyloid deposition was more severe in HFpEF group than in control group (χ2=12.205, P<0.01). Masson's trichrome staining evidenced moderate to severe fibrosis in 19 cases (19/28) in HFpEF group and 8 cases (8/15) in control group (χ2=1.019, P=0.35). A consistent evaluation of the degree of myocardial fibrosis and the degree of myocardial amyloid deposition in all selected participants was performed and results showed that these two parameters were not consistent (Kappa value=0.2, P=0.820).@*Conclusion@#Amyloid deposition is common in the elderly patients with heart failure and preserved ejection fraction, suggesting that myocardial amyloidosis may be related to the development of HFpEF. There is no significant correlation between myocardial amyloidosis and myocardial fibrosis in this cohort.

Human umbilical mesenchymal stem cell-derived exosome attenuates car-diac fibrosis in diabetic mice / 中国病理生理杂志

AIM:To explore the protective effects of exosome secreted by human umbilical mesenchymal stem cells on cardiac fibrosis in diabetic mouse model.METHODS:Male C57BL/6 mice at 6～8 weeks of age were divided in-to 3 groups randomly:control group ,diabetes mellitus(DM)group and DM+exosome group.To develop mouse DM mo-del,the mice were fed with high-fat diet for 5 weeks,followed by intraperitoneal injection of 45 mg/kg streptozocin once a week for 5 weeks.It was considered as a successful DM model that the blood glucose of the mice was ≥16.7 mmol/L.The mice in DM+exosome group were injected with exosome via tail vein.The mice in other 2 groups were injected with saline at the same volume.The heart function was evaluated by color Doppler echocardiography for small animals.The blood sam-ples were collected from abdominal aortas.The blood glucose and non-esterified fatty acids were measured by biochemical colorimetric assay.HE staining was performed to observe the structural changes of myocardial fibers ,and Masson staining was used to observe the cardiac fibrosis.RESULTS:The results of echocardiography showed that left ventricular end-dias-tolic dimension(LVIDd)and left ventricular end-systolic dimension(LVIDs)of diabetic mice were larger than those incontrol mice(P<0.05 and P<0.01,respectively).The ejection fraction(EF)and fractional shortening(FS)decreased in the diabetic mice(P<0.01).Exosome treatment significant decreased the LVIDs(P<0.01),but increased the EF and FS(P<0.01).The blood glucose and non-esterified fatty acids were significantly increased in the diabetic mice.The injection of the stem cell exosome significantly decreased the blood glucose and non -esterified fatty acids(P<0.01).HE staining observation showed that cardiomyocyte hypertrophy and fragmentation of cardiomyocyte in DM group were more se -rious than those in control group.Masson staining showed that the area of fibrosis in DM group was larger than that in con-trol group(P<0.01),but that in DM+exosome group was reduced(P<0.01).CONCLUSION:Exosome secreted by human umbilical mesenchymal stem cells protects the DM model mice from cardiac fibrosis.

MicroRNA-199a-3p enhances expressions of fibrosis-associated genes through targeting Smad1 in mouse cardiac fibroblasts / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the role of miR-199a-3p in cardiac fibrosis and the potential target of miR-199a-3p.</p><p><b>METHODS</b>Cardiac fibroblasts were isolated from C57BL/6 mice and cultured. The miR-199a-3p mimic and Smad1 siRNA were transiently transfected into the cardiac fibroblasts via liposome. Dual luciferase reporter assay was performed to confirm the interaction between miR-199a-3p and the 3'-UTR of Smad1. The expressions of Smad1 and fibrosis-related genes at the mRNA and protein levels in the cells after miR-199a-3p mimic transfection were determined using RT-qPCR and Western blotting, respectively. The expressions of Smad1, Smad3 and fibrosis-related genes at the protein level in cells transfected with miR-199a-3p mimic and Smad1 siRNA were detected using Western blotting.</p><p><b>RESULTS</b>Over-expression of miR-199a-3p significantly increased the expression of cardiac fibrosis-related genes in cultured mouse cardiac fibroblasts. Dual luciferase reporter assay revealed the interaction of miR-199a-3p with the 3'-UTR of Smad1. The results of RT-qPCR and Western blotting confirmed that miR-199a-3p inhibited Smad1 expression at the post- transcriptional level. Transfection with miR-199a-3p mimic and siRNA-mediated Smad1 silencing consistently activated the Smad3 signaling pathway and enhanced the expressions of cardiac fibrosis-related genes in the cardiac fibroblasts.</p><p><b>CONCLUSIONS</b>As the target gene of miR-199a-3p, Smad1 mediates the pro-fibrotic effect of miR-199a-3p by activating the Smad3 signaling in cultured mouse cardiac fibroblasts.</p>

Comprehensive Assessment of Authenticating Method of Pathological Staining in Cardiac Fibrosis / 医学研究杂志

Objective To compare hematoxylin and eosin(HE) staining,Masson's staining and picric sirius red(PSR) staining in evaluation of cardiac fibrosis.Methods Twenty adult sprague dawley (SD) rats were given subcutaneous injection of isoprenaline for 5mg/(kg · d) for 3 weeks,which induced cardiac fibrosis model successfully.In order to observing the fibrotic areas and collagen deposition,we prepared paraffin sections by using the heart specimen of rat,and carried out HE staining,Masson staining and PSR staining respectivelly for the special staining.Results All staining methods could clearly show the collagen fibers and fibrosis stage.We could clearly distinguish the type Ⅰ and type Ⅲ collagen fibers through PSR staining with immunoflurescent techniques,but not Masson and HE staining.Conclusion The results demonstrated that PSR staining is better than HE and Masson staining to be used for the evaluation of degree and type of proliferation of collagen fibers.It has important signification for treatment and prevention of cardiac fibrosis.

Regulation of phenylephrine-induced cardiac fibrosis by ANGPTL4 / 实用医学杂志

Objective To study how ANGPTL4 modulates phenylephrine(PE)-induced cardiac fibrosis. Methods PE was applied to induce cardiac fibrosis. The knockdown effect of ANGPTL4 under PE stimuli was confirmed by Western blot;the proliferation of cardiac fibroblast was detected by CCK-8;mRNA levels of Collagen 1(Col1)and connective tissue growth factor(CTGF)were detected by QPCR to investigate the regula-tion of cardiac fibrosis by ANGPTL4. Results PE induced the proliferation of cardiac fibroblast and the up-regula-tion of mRNA levels of Col1 and CTGF;ANGPTL4 knockdown deteriorated cardiac fibrosis,manifested by the up-regulation of Col1 and CTGF. Conclusion ANGPTL4 inhibits PE-induced cardiac fibrosis.

Celastrol Protects TGF-β1-induced Endothelial-mesenchymal Transition / 华中科技大学学报（医学）（英德文版）

The endothelial-to-mesenchymal transition (EndMT) in endothelial cells contributes to the development of cardiac fibrosis,ultimately leading to cardiac remodeling.In this study,the effects and molecular mechanisms of celastrol (CEL) on transforming growth factor-β1 (TGF-β1)-induced EndMT in human umbilical vein endothelial (HUVEC-12) cells were investigated.The presented data demonstrated.that CEL significantly blocked the morphology change of HUVEC-12 cells induced by TGF-β1 without cell cytotoxicity.In accordance with these findings,CEL blocked TGF-β1-induced EndMT as evidenced by the inhibition of the mesenchymal markers,including collagen Ⅰ,Ⅲ,α-SMA,fibronectin mRNA expression,and the increase in the mRNA expression of endothelial cell marker CD31.These changes were also confirmed by double immunofluorescence staining of CD31 and vimentin.The in vitro scratch assay showed that CEL inhibited the migration capacity of the transitioned endothelial cells induced by TGF-β1.Further experiments showed that the beneficial effect of CEL on blocking the EndMT in HUVEC-12 cells was associated with the suppression of the TGF-β1/Smads signalling pathway,which was also confirmed by the inhibition of its downstream transcription factor snail1,twistl,twist2,ZEB1 and ZEB2.These results indicate that CEL blocks TGF-β1-induced EndMT through TGF-β1/Smads signalling pathway and suggest that it may be a feasible therapy for cardiac fibrosis diseases.

Comprehensive Assessment of Authenticating Method of Pathological Staining in Cardiac Fibrosis / 医学研究杂志

Objective To compare hematoxylin and eosin(HE) staining,Masson's staining and picric sirius red(PSR) staining in evaluation of cardiac fibrosis.Methods Twenty adult sprague dawley (SD) rats were given subcutaneous injection of isoprenaline for 5mg/(kg · d) for 3 weeks,which induced cardiac fibrosis model successfully.In order to observing the fibrotic areas and collagen deposition,we prepared paraffin sections by using the heart specimen of rat,and carried out HE staining,Masson staining and PSR staining respectivelly for the special staining.Results All staining methods could clearly show the collagen fibers and fibrosis stage.We could clearly distinguish the type Ⅰ and type Ⅲ collagen fibers through PSR staining with immunoflurescent techniques,but not Masson and HE staining.Conclusion The results demonstrated that PSR staining is better than HE and Masson staining to be used for the evaluation of degree and type of proliferation of collagen fibers.It has important signification for treatment and prevention of cardiac fibrosis.