Electroacupuncture improves myocardial fibrosis in heart failure rats by attenuating ECM collagen deposition through modulation of TGF-β1/Smads signaling pathway

Background: To explore the effects of electroacupuncture on cardiac function and myocardial fibrosis in rat models of heart failure, and to elucidate the underlying mechanism of electroacupuncture in heart failure treatment. Methods: Healthy male Sprague-Dawley rats were allocated into three groups: Sham group, Model group, and electroacupuncture (Model + EA) group, with each group comprising 8 rats. The model underwent a procedure involving the ligation of the left anterior descending coronary artery to induce a model of heart failure. The Model + EA group was used for 7 consecutive days for electroacupuncture of bilateral Shenmen (HT7) and Tongli (HT5), once a day for 30 min each time. Left ventricular parameters in rats were assessed using a small-animal ultrasound machine to analyze changes in left ventricular end-diastolic volume, left ventricular end-systolic volume, left ventricular ejection fraction, and left ventricular fractional shortening. Serum interleukin-1β (IL-1β), cardiac troponin (cTn), and N-terminal brain natriuretic peptide precursor levels were measured using ELISA. Histopathological changes in rat myocardium were observed through HE staining, while collagen deposition in rat myocardial tissue was assessed using the Masson staining method. Picro sirius red staining, immunohistochemical staining, and RT-qPCR were utilized to distinguish between the various types of collagen deposition. The expression level of TGF-β1 and SMAD2/3/4/7 mRNA in rat myocardial tissues was determined using RT-qPCR. Additionally, western blot analysis was conducted to assess the protein expression levels of TGF-β1, SMAD3/7, and p-SMAD3 in rat myocardial tissues. Results: Compared with the Sham group, the left ventricular ejection fraction and left ventricular fractional shortening values of the Model group were significantly decreased (P < 0.01);the left ventricular end-diastolic volume and left ventricular end-systolic volume values were remarkably increased (P < 0.01);serum N-terminal brain natriuretic peptide precursor content was increased (P < 0.01);serum IL-1β and cTn levels were increased (P < 0.01);myocardial collagen volume fraction were increased (P < 0.01);and those of the expression of TGF-β1 and SMAD2/3/4 mRNA was increased (P < 0.01);the expression of SMAD7 mRNA was decreased (P < 0.01);the protein expression levels of TGF-β1, SMAD3, and p-Smad3 were increased (P < 0.01);the protein expression level of SMAD7 was decreased (P < 0.01) in the Model group. Compared to the Model group, the expression levels of the proteins TGF-β1, SMAD3, and p-Smad3 in myocardial tissue were found to be decreased (P < 0.01), and the expression level of the protein SMAD7 was found to be increased (P < 0.01) in the Model + EA group;the collagen volume fraction and deposition of type Ⅰ /Ⅲ collagen were decreased (P < 0.01) in the Model + EA group. Conclusion: Electroacupuncture alleviates myocardial fibrosis in rats with heart failure, and this effect is likely due to attributed to the modulation of the TGF-β1/Smads signaling pathway, which helps reduce collagen deposition in the extracellular matrix.

Berberine Attenuates Cigarette Smoke Extract-induced Airway Inflammation in Mice:Involvement of TGF-β1/Smads Signaling Pathway

Although several studies confirmed that berberine may attenuate airway inflammation in mice with chronic obstructive pulmonary disease(COPD),its underlying mechanisms were not clear until now.We aimed to establish an experiment mouse model for COPD and to investigate the effects of berberine on airway inflammation and its possible mechanism in COPD model mice induced by cigarette smoke extract(CSE).Twenty SPF C57BL/6 mice were randomly divided into PBS control group,COPD model group,low-dose berberine group and high-dose berberine group,5 mice in each group.The neutrophils and macrophages were examined by Wright's staining.The levels of inflammatory cytokines TNF-α and IL-6 in bronchoalveolar lavage fluid(BALF)were detennined by enzyme-linked immunosorbent assay.The expression levels of TGF-β1,Smad2 and Smad3 mRNA and proteins in lung tissues were respectively detected by quantitative real-time polymerase chain reaction and Western blotting.It was found that CSE increased the number of inflammation cells in BALF,elevated lung inflammation scores,and enhanced the TGF-β1/Smads signaling activity in mice.High-dose berberine restrained the alterations in the COPD mice induced by CSE.It was concluded that high-dose berberine ameliorated CSE-induced airway inflammation in COPD mice.TGF-β1/Smads signaling pathway might be involved in the mechanism.These findings suggested a therapeutic potential of high-dose berberine on the CSE-induced airway inflammation.

MiR-146a-5p targeting SMAD4 and TRAF6 inhibits adipogenensis through TGF-β and AKT/mTORC1 signal pathways in porcine intramuscular preadipocytes

Background: Intramuscular fat(IMF) content is a vital parameter for assessing pork quality. Increasing evidence has shown that microRNAs(miRNAs) play an important role in regulating porcine IMF deposition. Here, a novel miRNA implicated in porcine IMF adipogenesis was found, and its effect and regulatory mechanism were further explored with respect to intramuscular preadipocyte proliferation and differentiation.Results: By porcine adipose tissue miRNA sequencing analysis, we found that miR-146a-5p is a potential regulator of porcine IMF adipogenesis. Further studies showed that miR-146a-5p mimics inhibited porcine intramuscular preadipocyte proliferation and differentiation, while the miR-146a-5p inhibitor promoted cell proliferation and adipogenic differentiation. Mechanistically, miR-146a-5p suppressed cell proliferation by directly targeting SMAD family member 4(SMAD4) to attenuate TGF-β signaling. Moreover, miR-146a-5p inhibited the differentiation of intramuscular preadipocytes by targeting TNF receptor-associated factor 6(TRAF6) to weaken the AKT/mTORC1 signaling downstream of the TRAF6 pathway.Conclusions: MiR-146a-5p targets SMAD4 and TRAF6 to inhibit porcine intramuscular adipogenesis by attenuating TGF-β and AKT/mTORC1 signaling, respectively. These findings provide a novel miRNA biomarker for regulating intramuscular adipogenesis to promote pork quality.

Cetirizine regulates scleroderma skin fibrosis in mice via the TGF-β1/Smad3 signaling pathway

Objective:To investigate the effect of cetirizine on the fibrosis of skin tissue in systemic sclerosis(SSc)mice and its mechanism of action.Methods:Thirty-two BALB/C mice were randomly divided into a blank group,a model group,a cetirizine low-dose group,and a cetirizine high-dose group,with eight in each group.The blank group was injected with normal saline on the back,and the other three groups were injected with bleomycin on the back to prepare SSc mouse models.The mice were injected once a day for 28 consecutive days,while the normal group and the model group were given saline.The dose group was administrated intragastrically at 2 mg/kg and 5 mg/kg,respectively,for 28 consecutive days.Detect the thickness of the dermis by taking the skin tissue in the back injection area of each group.Hematoxylin-eosin staining(HE)and Masson staining.Sample hydrolysis method to detect hydroxyproline(HYP)content in skin tissue.Immunohistochemical detection ofα-smooth muscle actin(α-SMA)expression in skin tissues.Enzyme-linked immunosorbent assay(ELISA)to detect serum interleukin(IL-6,IL-10)and transforming growth factor(TGF-αand TGF-β1).Quantitative real-time PCR(qRT-PCR)was used to detect the expression levels of collagen type I(COL1A1),type III collagen(COL3A1),Smad homolog 3(Smad3),and TGF-β1 mRNA.Western blot was used to detect the expression levels of COL1A1,COL3A1 and p-Smad3.Results:Compared with the blank group,the dermis thickness and HYP content of the model group increased,the skin tissue lesions and fibrosis were more severe,theα-SMA positive expression intensity in the skin tissue was higher,and the serum IL-6,IL-10,TGF-α,TGF-β1 content increased,COL1A1,COL3A1,Smad3,TGF-β1 mRNA expression levels increased in skin tissues,COL1A1,COL3A1,p-Smad3 protein expression increased,the differences were statistically significant(P<0.05).Compared with the model group,the dermal thickness and HYP content of the low and high dose cetirizine groups were reduced,the degree of skin tissue lesions and fibrosis was improved,the expression ofα-SMA in skin tissues was weakened,the levels of IL-6,IL-10,TGF-α,TGF-β1 in serum were reduced,the expression levels of COL1A1,COL3A1,Smad3 and TGF-β1 in skin tissues were reduced,and the expression levels of COL1A1,COL3A1,and p-Smad3 proteins were reduced,the decrease in the high-dose group was more significant,and the differences were statistically significant(P<0.05).Conclusion:Cetirizine can improve the degree of fibrosis of skin tissue in SSc mice and reduce the immune inflammation response.The mechanism of action is related to the TGF-β1/Smad3 signaling pathway.

Total flavonoids of Scutellaria barbata improving podocyte injury induced by high glucose through Smad4/PKM2/HIF‑1α pathway

Objective: To observe the effect of total flavonoids of Scutellaria barbata (TF‑SB) on the injury of high glucose induced podocytes (MPC‑5) and the influence of Smad4/PKM2/HIF‑1α pathway. Methods: Firstly, CCK8 was used to analyze the safety and efficacy concentration of TF‑SB on MPC‑5 cells. Then, MPC‑5 was then divided into the control group, model group and TF‑SB group. In addition to the control group, model group and TF‑SB group were induced by high glucose to establish MPC‑5 cell injury model. The effects of TF‑SB on ATP, apoptosis and ROS levels of MPC‑5 cells were detected respectively. The contents of IL‑1β, TNF‑α, and MCP‑1 were determined by ELISA, the expression abundance of glycolytic genes (GLU1, PFK1 and HK1) were detected by RT‑PCR. Western blot method was used to detect the expression level of related proteins in Smad4/PKM2/HIF‑1α pathway. Results: Compared with the blank group, ATP content, GLU1, PKF1 and HK1 expression abundance of MPC‑5 cells in the model group decreased significantly, apoptosis, ROS level and IL‑1 β、 TNF‑ α And MCP‑1 significantly increased (P<0.01);Compared with model group, ATP content, GLU1, PKF1 and HK1 expression abundance, apoptosis, ROS level and IL‑1β in TF‑SB group were significantly increased , TNF‑ α The contents of MCP‑1 and MCP‑1 decreased significantly (P<0.01). In addition, compared with the blank group, the model group Smad4 and HIF‑1 α The protein expression and PKM2 expression in nucleus were significantly increased, while PKM2 expression in cytoplasm was significantly decreased (P<0.01);Compared with model group, TF‑SB group Smad4, HIF‑1 α The expression of PKM2 in the nucleus and expression of PKM2 were significantly decreased, while the expression of PKM2 in the cytoplasm was significantly increased (P<0.01). Conclusion: TF‑SB promotes the mitochondrial activity of MPC‑5 cells to induce glycolysis, and then inhibits the secretion of inflammation, which may play a role in treating diabetes nephropathy by inhibiting Smad4/PKM2/HIF‑1α signaling pathway.

Role of transforming growth factor-beta1-smad signal transduction pathway in patients with hepatocellular carcinoma

AIM： To explore the role of transforming growth factorbeta1 （TGF-β1）-smad signal transduction pathway in patients with hepatocellular carcinoma. METHODS： Thirty-six hepatocellular carcinoma specimens were obtained from Qidong Liver Cancer Institute and Department of Pathology of the Second Affiliated Hospital of Nanjing Medical University. All primary antibodies （polyclonal antibodies） to TGF-β1, type H Transforming growth factor-beta receptor （TβR-Ⅱ）, nuclear factor-kappaB （NF-KB）, CD34, smad4 and smad7, secondary antibodies and immunohistochemical kit were purchased from Zhongshan Biotechnology Limited Company （Beijing, China）. The expressions of TGF-β1, TβR-Ⅱ, NF- KB, smad4 and smad7 proteins in 36 specimens of hepatocellular carcinoma （HCC） and its adjacent tissue were separately detected by immunohistochemistry to observe the relationship between TGF-β1 and TβR-Ⅱ, between NF-KB and TGF-β1, between smad4 and smad7 and between TGF-β1 or TβR-Ⅱ and microvessel density （MVD）. MVD was determined by labelling the vessel endothelial cells with CD34. RESULTS： The expression of TGF-β1, smad7 and MVD was higher in HCC tissue than in adjacent HCC tissue （P〈0.01, P〈0.05,P〈0.01 respectively）. The expression of TβR-Ⅱ and smad4 was lower in HCC tissue than in its adjacent tissue （P〈0.01, P〈0.05 respectively）. The expression of TGF-β1 protein and NF-KB protein was consistent in HCC tissue. The expression of TGF-β1 and MVD was also consistent in HCC tissue. The expression of TIER- Ⅱ was negatively correlated with that of MVD in HCC tissue. CONCLUSION： The expressions of TGF-IB1, TβR- Ⅱ, NF-KB, smad4 and smad7 in HCC tissue, which are major up and down stream factors of TGF-β1-smad signal transduction pathway, are abnormal. These factors are closely related with NVD and may play an important role in HCC angiogenesis. The inhibitory action of TGF-β1 is weakened in hepatic carcinoma cells because of abnormality of TGF-β1 receptors （such as TIBR- Ⅱ） and postreceptors （such as smad4 and smad7）. NF-KB may cause activation and production of TGF-β1.

Celastrol Protects TGF-β1-induced Endothelial-mesenchymal Transition

The endothelial-to-mesenchymal transition（End MT） 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 End MT 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 EndM T as evidenced by the inhibition of the mesenchymal markers,including collagen Ⅰ,Ⅲ,α-SMA,fibronectin m RNA expression,and the increase in the m RNA 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 End MT 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,twist1,twist2,ZEB1 and ZEB2.These results indicate that CEL blocks TGF-β1-induced End MT through TGF-β1/Smads signalling pathway and suggest that it may be a feasible therapy for cardiac fibrosis diseases.

The Effects of Singal Protein SMADs on Rat Cardiocyte Hypertrophy

Objectives To investigate the role of signal protein SMADs in rat cardiac hypertrophy. Methods The rat models of cardiac hypertrophy were produced by constriction of the abdominal aorta. The left vertricular mass index （LVMI） was investigated. The expression of transforming growth factor-β1 mRNA （TGF-β1） and Smad 2,3,7 mRNA were assessed by RT-PCR. Reslutes The LVMI and the expression of TGF-β1 and Smad 2,3,7mRNA in hypertrophic left ventricule were increased on day 3 after the operation and continued to 4th weeks. The peak expression of TGF-β1 and Smad 2,3 mRNA were in 2 weeks after operation. The expression of Smad 7 was increased in 3 day after operation, but the peak was in 1 week after operation, then decreased. Conclusions The TGF-β1 and signal protein Smad 2,3,7 were included in the progress of rat cardiac hypertrophy produced by constriction of abdominal aorta.

Cytokine receptor-like factor 1(CRLF1)promotes cardiac fibrosis via ERK1/2 signaling pathway

Cardiac fibrosis is a cause of morbidity and mortality in people with heart disease.Anti-fibrosis treatment is a significant therapy for heart disease,but there is still no thorough understanding of fibrotic mechanisms.This study was carried out to ascertain the functions of cytokine receptor-like factor 1(CRLF1)in cardiac fibrosis and clarify its regulatory mechanisms.We found that CRLF1 was expressed predominantly in cardiac fibroblasts.Its expression was up-regulated not only in a mouse heart fibrotic model induced by myocardial infarction,but also in mouse and human cardiac fibroblasts provoked by transforming growth factor-β1(TGF-β1).Gain-and loss-of-function experiments of CRLF1 were carried out in neonatal mice cardiac fibroblasts(NMCFs)with or without TGF-β1 stimulation.CRLF1 overexpression increased cell viability,collagen production,cell proliferation capacity,and myofibroblast transformation of NMCFs with or without TGF-β1 stimulation,while silencing of CRLF1 had the opposite effects.An inhibitor of the extracellular signal-regulated kinase 1/2(ERK1/2)signaling pathway and different inhibitors of TGF-β1 signaling cascades,comprising mothers against decapentaplegic homolog(SMAD)-dependent and SMAD-independent pathways,were applied to investigate the mechanisms involved.CRLF1 exerted its functions by activating the ERK1/2 signaling pathway.Furthermore,the SMAD-dependent pathway,not the SMAD-independent pathway,was responsible for CRLF1 up-regulation in NMCFs treated with TGF-β1.In summary,activation of the TGF-β1/SMAD signaling pathway in cardiac fibrosis increased CRLF1 expression.CRLF1 then aggravated cardiac fibrosis by activating the ERK1/2 signaling pathway.CRLF1 could become a novel potential target for intervention and remedy of cardiac fibrosis.

Yin Yang 1(YY1) synergizes with Smad7 to inhibit TGF-β signaling in the nucleus

As a prototype of the TGF-β superfamily cytokines, TGF-β is well known for its diverse roles in embryogenesis and adult tis- sue homeostasis. TGF-β evokes cellular responses by signaling mainly through cell membrane receptors and transcription fac- tor R-Smads and Co-Smad （Smad4）, while an inhibitory Smad, Smad7, acts as a critical negative regulator of TGF-β signaling. Smad7 antagonizes TGF-β signaling by regulating the stability or activity of the receptors or blocking the DNA binding of the functional R-Smad-Smad4 complex in the nucleus. However, the function of Smad7 in the nucleus is not fully understood. Yin Yang 1 （YY1） is a ubiquitously expressed transcription factor with multiple functions. It has been reported that YY1 can inhib- it Smad-dependent transcriptional responses and TGF-β/BMP-induced cell differentiation independently of its DNA binding ability. In this study, we found that Smad7 interacts with YY1 and the interaction is attenuated by TGF-β signaling. Reporter assays and target gene expression analyses revealed that Smad7 and YY1 act in concert to inhibit TGF-β-induced transcription in the nucleus. Furthermore, Smad7 could enhance the interaction of YY1 with the histone deacetylase HDAC1. Consistently, YY 1 and HDAC 1 augmented the transcription repression activity of Smad7 in Gal4-1uciferase reporter analysis. Therefore, our findings define a novel mechanism of Smad7 and YY1 to antagonize TGF-β signaling.

Effects of transforming growth factor-β_1 and signal protein Smad3 on rat cardiomyocyte hypertrophy

Background SMAD proteins have recently been identified as the first family of putative transforming growth factor-β_1(TGF-β_1) signal transducers. This study was to investigate the effects of TGF-β_1 and signal protein Smad3 on rat cardiac hypertrophy.Methods The incorporation of [3H]-leucine was measured to determine the hypertrophy of cardiomyocyte incubated with different doses of TGF-β_1 in cultured neonatal cardiomyocytes. The model of rat cardiac hypertrophy was produced with constriction of the abdominal aorta. At different times after the operation, rats were killed, and their left ventricular mass index (LVMI) determined. The mRNA expression of TGF-β_1 and Smad3 of cultured cells and hypertrophic left ventricles were assessed by RT-PCR. The protein expression of Smad3 was assessed by Western blot.Results In cultured neonatal cardiomyocytes, TGF-β_1 significantly promoted incorporation of [3H]-leucine. With the concentration of 3 pg/L, it increased the expression of Smad3 in mRNA and protein levels after 15 minutes, and continued for up to 8 hours of cultured cardiomyocytes. The LVMI and the expression of TGF-β_1 (mRNA) and Smad3 (mRNA and protein) of hypertrophic left ventricle were increased by day 3 after the operation and continued to the 4th week. The peak expression of these was in the second week after operation.Conclusion TGF-β_1 has positive effects on rat cardiomyocyte hypertrophy. Signal protein Smad3 could be related to the pathologic progression of rat cardiac hypertrophy.

Anti-ageing effects of a new Dimethylaminoethanol-based formulation on DGalactose induced skin ageing model of rat

Background Dimethylaminoethanol has been widely used to fight against wrinkles, in the field of aesthetic medicine there is an increasing demand for safe and effective Dimethylaminoethanol-based products to counteract the ageing process. Objective To evaluate the anti- ageing effects of a new DMAE- based formulation. Methods 30 male rats were randomly allocated into treatment,D-gal ageing modeland control groups, each of which contained ten rats.Treatment group and D- gal ageing model group were subcutaneously injected with D- galactose prepared in normal saline 125mg·kg-1·d-1for 42 d. Control groups were injected with normal saline for42 d with same method and dose. From the 18 th day,after shaving their hair,the treatment grouprats were injected thisnew DMAE-based formulation at a dose of 1ml per week for 4 weeks in the Dermis of two sides hip skin mark zone.Meanwhile,D-gal ageing model group rats were administrated the same volume of normal saline with same method. Skin specimens were obtained 3days after the last treatment. Dermal collagen density and dermal thickness were evaluated by H&E and Massontrichrome staining. And m RNA expressions of TGFβ1, Smad3, Type I,Type III Pro-collagen,TIMP-1,MMP- 1,were assessed by Real- time quantitative polymerase chain reaction. Results Dermal thickness, dermal collagen density and hydroxyproline content in treatment group increased significantly comparing with D- gal ageing model group. No differences were found in m RNA expression of MMP- 1 and Type III Pro- collagen between the treatment group and D- gal ageing model group. In addition, m RNA expression of TGFβ1, Type I Pre-collagen, TIMP1 and smad3 in treatment group were significantly up- regulated in contrast with D- gal ageing model and control group. Conclusion This new DMAE- based formulationcould generate anti- ageing effects by activating collagen synthesisthrough TGF-β1/Smads signaling pathway.

Effects of novel Fufang Biejia Ruangan Tablets with sheep placenta as substitute for Hominis Placenta on CCl4-induced liver fibrosis

Objective:Fufang Biejia Ruangan Tablet(FBRT) is widely used for the treatment of liver fibrosis.However,Hominis Placenta(HP),as an important adjuvant of FBRT,has been restricted for medicinal using due to the limited availability,ethical controversy and safety issues.The present study aimed to investigate the therapeutic effects of novel FBRT(N-FBRT) with sheep placenta(SP) as substitute for HP on liver fibrosis and explore its possible mechanisms.Different dosages of SP in N-FBRT were also evaluated.Methods:Rats were subcutaneously injected with CCl_(4)to induce liver fibrosis and then treated with NFBRT and FBRT.The anti-hepatic fibrosis effect was determined based on biomarkers analysis of liver function and hepatic fibrosis,and the liver pathology was visualized by H&E staining and Masson staining.The oxidative stress and inflammatory cytokines were also detected.Immunohistochemical staining of a-SMA,real time PCR and Western blotting were performed to evaluate hepatic stellate cells(HSCs)activation and TGF-β1/Smad signaling pathway.Results:N-FBRT and FBRT could ameliorate CCl_(4)-induced liver fibrosis and improve liver function,as evidenced by lowering serum biomarkers levels of liver function and hepatic fibrosis,and decreasing hepatic Hyp content and collagen deposition,and improving the hepatic morphology and architecture changes.Moreover,the anti-liver fibrosis effect was better when the dosage of SP used in N-FBRT was 1/2 of HP in FBRT.Administration of N-FBRT markedly alleviated oxidative stress and inflammatory cytokines,and inhibited a-SMA expression.Furthermore,the mRNA expression of Col Ⅰ,Col Ⅲ,a-SMA and TGF-β1,and proteins expression of a-SMA,TGF-β1,Smad2/3 and p-Smad2/3 were significantly down-regulated by N-FBRT treatment.Conclusion:SP can be used as substitute for HP to prepare N-FBRT for the treatment of liver fibrosis and the anti-liver fibrosis effect of N-FBRT is achieved by eliminating oxidative stress and inflammation,and inhibiting HSCs activation and ECM production by blocking TGF-β1/Smad signaling pathway.