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.

Cinobufotalin prevents bone loss induced by ovariectomy in mice through the BMPs/SMAD and Wnt/β-catenin signaling pathways

Background:Osteoporosis is a chronic bone disease characterized by bone loss and decreased bone strength.However,current anti-resorptive drugs carry a risk of various complications.The deep learning-based efficacy prediction system(DLEPS)is a forecasting tool that can effectively compete in drug screening and prediction based on gene expression changes.This study aimed to explore the protective effect and potential mechanisms of cinobufotalin(CB),a traditional Chinese medicine(TCM),on bone loss.Methods:DLEPS was employed for screening anti-osteoporotic agents according to gene profile changes in primary osteoporosis.Micro-CT,histological and morphological analysis were applied for the bone protective detection of CB,and the osteogenic differentiation/function in human bone marrow mesenchymal stem cells(hBMMSCs)were also investigated.The underlying mechanism was verified using qRT-PCR,Western blot(WB),immunofluorescence(IF),etc.Results:A safe concentration(0.25mg/kg in vivo,0.05μM in vitro)of CB could effectively preserve bone mass in estrogen deficiency-induced bone loss and promote osteogenic differentiation/function of hBMMSCs.Both BMPs/SMAD and Wnt/β-catenin signaling pathways participated in CB-induced osteogenic differentiation,further regulating the expression of osteogenesis-associated factors,and ultimately promoting osteogenesis.Conclusion:Our study demonstrated that CB could significantly reverse estrogen deficiency-induced bone loss,further promoting osteogenic differentiation/function of hBMMSCs,with BMPs/SMAD and Wnt/β-catenin signaling pathways involved.

Calcitriol attenuates liver fibrosis through hepatitis C virus nonstructural protein 3-transactivated protein 1-mediated TGF β1/Smad3 and NF-κB signaling pathways

BACKGROUND Hepatic fibrosis is a serious condition,and the development of hepatic fibrosis can lead to a series of complications.However,the pathogenesis of hepatic fibrosis remains unclear,and effective therapy options are still lacking.Our group identified hepatitis C virus nonstructural protein 3-transactivated protein 1(NS3TP1) by suppressive subtractive hybridization and bioinformatics analysis,but its role in diseases including hepatic fibrosis remains undefined.Therefore,additional studies on the function of NS3TP1 in hepatic fibrosis are urgently needed to provide new targets for treatment.AIM To elucidate the mechanism of NS3TP1 in hepatic fibrosis and the regulatory effects of calcitriol on NS3TP1.METHODS Twenty-four male C57BL/6 mice were randomized and separated into three groups,comprising the normal,fibrosis,and calcitriol treatment groups,and liver fibrosis was modeled by carbon tetrachloride(CCl4).To evaluate the level of hepatic fibrosis in every group,serological and pathological examinations of the liver were conducted.TGF-β1 was administered to boost the in vitro cultivation of LX-2 cells.NS3TP1,α-smooth muscle actin(α-SMA),collagen I,and collagen Ⅲ in every group were examined using a Western blot and real-time quantitative polymerase chain reaction.The activity of the transforming growth factor beta 1(TGFβ1)/Smad3 and NF-κB signaling pathways in each group of cells transfected with pcDNA-NS3TP1 or siRNA-NS3TP1 was detected.The statistical analysis of the data was performed using the Student’s t test.RESULTS NS3TP1 promoted the activation,proliferation,and differentiation of hepatic stellate cells(HSCs)and enhanced hepatic fibrosis via the TGFβ1/Smad3 and NF-κB signaling pathways,as evidenced by the presence of α-SMA,collagen I,collagen Ⅲ,p-smad3,and p-p65 in LX-2 cells,which were upregulated after NS3TP1 overexpression and downregulated after NS3TP1 interference.The proliferation of HSCs was lowered after NS3TP1 interference and elevated after NS3TP1 overexpression,as shown by the luciferase assay.NS3TP1 inhibited the apoptosis of HSCs.Moreover,both Smad3 and p65 could bind to NS3TP1,and p65 increased the promoter activity of NS3TP1,while NS3TP1 increased the promoter activity of TGFβ1 receptor I,as indicated by coimmunoprecipitation and luciferase assay results.Both in vivo and in vitro,treatment with calcitriol dramatically reduced the expression of NS3TP1.Calcitriol therapy-controlled HSCs activation,proliferation,and differentiation and substantially suppressed CCl4-induced hepatic fibrosis in mice.Furthermore,calcitriol modulated the activities of the above signaling pathways via downregulation of NS3TP1.CONCLUSION Our results suggest that calcitriol may be employed as an adjuvant therapy for hepatic fibrosis and that NS3TP1 is a unique,prospective therapeutic target in hepatic fibrosis.

Effects of Cigu Xiaozhi Formula on miR-378a-3p Expression and Hh Signaling Pathway in TGF-β1 Induced LX2 Cells

[Objectives]To observe the effects of Cigu Xiaozhi Formula on miR-378a-3p expression and Hh signaling pathway in TGF-β1 induced and activated LX2 cells.[Methods]Cells were divided into control group,induction group,drug-containing serum group,miR-378a-3p inhibitor group,and miR inhibitor NC group.CCK-8 method was used to detect the cell viability of each group,and flow cytometry was used to detect the apoptosis rate of each group.RT-qPCR was used to detect the expression of miR-378a-3p in each group s cells,and RT-qPCR and Western blot were used to detect mRNA and protein expression of Shh,Gli1,Gli2,Col-I,andα-SMA in each group s cells.[Results]Compared with the control group,the cell viability and expression of Shh,Gli1,Gli2,Col-I,andα-SMA mRNA and protein in induction group increased(P<0.01),while the expression of miR-378a-3p decreased(P<0.01).Compared with the induction group,the cell viability and expression of Shh,Gli1,Gli2,Col-I,α-SMA mRNA andα-SMA and Gli2 protein decreased in drug-containing serum group(P<0.05),while cell apoptosis rate and miR-378a-3p expression increased(P<0.01).In miR-378a-3p inhibitor group,cell viability and the expression of Shh,Gli1,Gli2,Col-I,α-SMA mRNA and Gli1,Gli2,α-SMA protein increased(P<0.05,P<0.01),while the apoptosis rate and miR-378a-3p expression decreased(P<0.05,P<0.01).[Conclusions]Cigu Xiaozhi Formula containing serum can upregulate miR-378a-3p expression and downregulate the expression of Gli2 andα-SMA in TGF-β1 induced LX2 cells,thereby inhibiting the activation of LX2 cells and exerting the effects of anti liver fibrosis.

Xinfuli Granule improves post-myocardial infarction ventricular remodelingand myocardial fibrosis in rats by regulating TGF-β/Smads signaling pathway

Recent clinical and experimental studies have confirmed the effects of Xinfuli Granule (XG), a compound Chinese medicine in the prevention and treatment of heart failure (HF). This study aimed to investigate the effects and the mechanisms of XG on ventricular reconstruction in rats with acute myocardial infarction (AMI).MethodsSprague-Dawley rats were subjected to left anterior descending branch ligation. The rats that survived 24 h were randomly assigned to five groups: medium-dose of XG group (MI+XGM), high-dose of XG group (MI+XGH), carvedilol group (MI+C), medium-dose of XG + carvedilol group (MI+C+XGM). Fourteen rats underwent identical surgical procedures without artery ligation, serving as sham controls. At 28 days, left ventricular weight to body weight (LVW/BW) and heart weight to body weight (HW/BW) were calculated; left ventricular ejection fraction (LVEF), left ventricular shortening fraction (LVFS), left ventricular internal diameter at systole (LVIDS) were measured by ultrasound; HE staining, Masson staining, and Sirius red staining were used to assess the myocardial pathological and physiological changes as well as myocardial fibrosis area and non-infarct zone I/III collagen ratio. Expression of Smad3 were detected and analyzed by Western blot, immunohistochemistry and immunofluorescence. P-Smad3, Smad2 and Smad7 in the TGF-β/Smads signaling pathway were also analyzed by Western blot.ResultsThe LVIDS (P < 0.01), HW/BW (P < 0.05), type I/III collagen ratio (P < 0.01) and myocardial collagen (P < 0.01) decreased significantly while the LVW/BW, LVFS (P < 0.05) increased significantly in MI+XGM group as compared with those in other groups. The expression of key signal molecules of the TGF-β/Smads signaling pathway, including Smad3, P-Smad3 and Smad2 protein were decreased, while the expression of Smad7 increased in both XG and carvedilol treatment groups as compared to those of the MI group (all P < 0.01). Immunohistochemistry and immunofluorescence further confirmed the down-regulated Smad3 expression.ConclusionXG can improve ventricular reconstruction and inhibit myocardial fibrosis in rats with AMI by regulating TGF-β/Smads signaling pathway.

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.

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.

Signaling cross-talk between TGF-β/BMP and other pathways

Transforming growth factor-beta （TGF-β）/bone morphogenic protein （BMP） signaling is involved in the vast majority of cellular processes and is fundamentally important during the entire life of all metazoans. Deregulation of TGF-β/ BMP activity almost invariably leads to developmental defects and/or diseases, including cancer. The proper functioning of the TGF-β/BMP pathway depends on its constitutive and extensive communication with other signaling pathways, leading to synergistic or antagonistic effects and eventually desirable biological outcomes. The nature of such signaling cross-talk is overwhelmingly complex and highly context-dependent. Here we review the different modes of cross-talk between TGF-β/BMP and the signaling pathways of Mitogen-activated protein kinase, phosphatidylinositol-3 kinase/ Akt, Wnt, Hedgehog, Notch, and the interleukin/interferon-gamma/tumor necrosis factor-alpha cytokines, with an emphasis on the underlying molecular mechanisms.

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.

Phospho-control of TGF-β superfamily signaling

Members of the transforming growth factor-β （TGF-β） family control a broad range of cellular responses in metazoan organisms via autocrine, paracrine, and endocrine modes. Thus, aberrant TGF-β signaling can play a key role in the pathogenesis of several diseases, including cancer. TGF-β signaling pathways are activated by a short phospho-cascade, from receptor phosphorylation to the subsequent phosphorylation and activation of downstream signal transducers called R-Smads. R-Smad phosphorylation state determines Smad complex assembly/disassembly, nuclear import/export, transcriptional activity and stability, and is thus the most critical event in TGF-β signaling. Dephosphorylation of R-Smads by specific phosphatases prevents or terminates TGF-β signaling, highlighting the need to consider Smad （de）phosphorylation as a tightly controlled and dynamic event. This article illustrates the es- sential roles of reversible phosphorylation in controlling the strength and duration of TGF-β signaling and the ensu- ing physiological responses.

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.

Role of TGF-βl Signaling in Heart Valve Calcification Induced by Abnormal Mechanical Stimulation in a Tissue Engineering Model

A tissue engineering model of heart valve calcification induced in a bio-reactor was established to evaluate the calcification induced by abnormal mechanical stimulation and explore the underlying molecular mechanisms.Polyethylene glycol (PEG)-modified decellularized porcine aortic leaflets seeded with human valve interstitial cells (huVICs)were mounted on a Ti-Ni alloy frame to fabricate two-leaflet and three-leaflet tissue engineered valves.The two-leaflet model valves were exposed to abnormal pulsatile flow stimulation with null (group A),low (1000mL/min,group B),medium (2000mL/min,group C),and high velocity (3000mL/min,group D)for 14 days. Morphology and calcification were assessed by yon Kossa staining,alkaline phosphatase (ALP)content,and Runx2 immunostaining.Leaflet calcification and mRNA and protein expression of transforming growth factor (TGF)-β1,bone morphogenetic protein 2 (BMP2),Smadl,and MSX2 were measured at different time points.ALP content was examined in two-leaflet valves seeded with BMP2 shRNA plasmid-infected huVICs and exposed to the same stimulation conditions.The results showed that during 14 days of flow stimulation,huVICs on the leaflet surface proliferated to generate normal monolayer coverage in groups A,B,and C.Under mechanical stimulation,huVICs showed a parallel growth pattern in the direction of the fluid flow,but huVICs exhibited disordered growth in the high-velocity flow environment,yon Kossa staining,ALP measurement,and immunohistochemical staining for Runx2 confirmed the lack of obvious calcification in group A and significant calcification in group D.Expression levels of TGF-β1,BMP2, and MSX2 mRNA and protein were increased under fluid stimulation.ALP production by BMP2 shRNA plasmid-infected huVICs on model leaflets was significantly reduced.In conclusion,abnormal mechanical stimulation in a bioreactor induced calcification in the tissue engineering valve model.The extent of calcification correlated positively with the flow velocity,as did the mRNA and protein levels of TGF-β1,BMP2,and MSX2.These findings indicate that TGF-β1/BMP2 signaling is involved in valve calcification induced bv abnormal mechanical stimulation.

Lignans from Seeds of Herpetospermum caudigerum Wall.Protect Against Alcoholic Liver Injury via TGF-β/Smads Pathway in Rats

[Objectives]This study aimed to investigate the effects of lignans from seeds of Herpetospermum caudigerum Wall.(SHC)on expression of TGF-β/Smads in liver tissue of hepatic alcohol-injuried rats,and to explore its protective mechanism.[Methods]A total of 60 SD rats were randomly divided into six groups.The rats in all the groups except those in the normal group were given with white spirit by gavage for 8 weeks to establish alcoholic liver injury models.After rat models were established successfully,they were administered intragastrically with 400,200 and 100 mg/kg of lignans,respectively.The rats in the normal group were administered intragastrically with 50 mg/kg of silymarin.The administration lasted for 8 weeks,once a day.The changes in the general state,liver tissue pathology and collagen deposition of the rats were observed.The expression of TGF-β,TGF-β1 receptor 1(TβR-I),TGF-β1 receptor 2(TβR-II),Smad2/p-Smad2 and Smad3/p-Smad3 in the hepatic tissue was detected.[Results]The expression levels of TGF-β1,Smad2,p-Smad2,and p-Smad3 significantly declined,and the expression levels of TβR-I,TβR-II and Smad3 did not change significantly in the liver tissue of rats in the lignans groups and the expression levels of TGF-β,Smad2,and p-Smad3 significantly declined.Meanwhile,the expression levels of TβR-I,TβR-II,p-Smad2 and Smad3 did not change significantly in the silymarin group.[Conclusions]The lignans from SHC have significant intervention effects on alcoholic livery injury.The mechanism may be related to the inhibition of hepatic stellate cell(HSC)activation,TGF-βsecretion and p-Smad2,p-Smad3 expression in the signaling pathway.

Deamidation enables pathogenic SMAD6 variants to activate the BMP signaling pathway

The BMP signaling pathway plays a crucial role in regulating early embryonic development and tissue homeostasis.SMAD6 encodes a negative regulator of BMP,and rare variants of SMAD6 are recurrently found in individuals with birth defects.However,we observed that a subset of rare pathogenic variants of SMAD6 consistently exhibited positive regulatory effects instead of the initial negative effects on the BMP signaling pathway.We sought to determine whether these SMAD6 variants have common pathogenic mechanisms.Here,we showed that pathogenic SMAD6 variants accompanying this functional reversal exhibit similar increases in deamidation.Mechanistically,increased deamidation of SMAD6 variants promotes the accumulation of the BMP receptor BMPR1A and the formation of new complexes,both of which lead to BMP signaling pathway activation.Specifically,two residues,N262 and N404,in SMAD6 were identified as the crucial sites of deamidation,which was catalyzed primarily by glutamine-fructose-6-phosphate transaminase 2(GFPT2).Additionally,treatment of cells harboring SMAD6 variants with a deamidase inhibitor restored the inhibitory effect of SMAD6 on the BMP signaling pathway.Conversely,when wild-type SMAD6 was manually simulated to mimic the deamidated state,the reversed function of activating BMP signaling was reproduced.Taken together,these findings show that deamidation of SMAD6 plays a crucial role in the functional reversal of BMP signaling activity,which can be induced by a subset of various SMAD6 variants.Our study reveals a common pathogenic mechanism shared by these variants and provides a potential strategy for preventing birth defects through deamidation regulation,which might prevent the off-target effects of gene editing.

Research status of E3 ubiquitin ligase Smurf2 and related signaling pathways in glioma

Glioma is the tumor with the highest incidence in the brain,and it is eager to seek new and efiective treatment.The interaction of ubiquitination and deubiquitination regulates many cell activities in organisms,and participates in tumor occurrence,development,migration,invasion and other processes.This article summarized the progress of E3 ubiquitination ligase smad ubiquitination regulatory factor 2(Smurf2)and glioma-related signaling pathways to assist clinical diagnosis and treatment of glioma.

Smad3 mediates immediate early induction of Idl by TGF-β

Idl is a member of the inhibitor of differentiation （Id） protein family that regulates a wide range of cell functions. Previous studies have shown that expression of the Idl gene is down-regulated by TGF-β in epithelial cells, whereas it is up-regulated by BMP in a variety of cell types. During our study of the biological function of TGF-β1, we found that Idl can be strongly up-regulated by TGF-β1 in the human mammary gland epithelial cell line MCF10A. Quantitative real-time RT-PCR has revealed as high as 7.5-fold induction ofldl mRNA by TGF-β1 in MCF10A cells after 1 h of TGF-β1 stimulation, and this induction does not require de novo protein synthesis. Using Smad knockdown and knockout approaches, we have identified Smad3 as the responsible R-Smad for mediating transcriptional activation of the Idl gene. Chromatin immunoprecipitation assay confirms that Smad3 and Smad4 bind to the upstream region of the Idl gene. Our results demonstrate that Smad3, but not Smad2, mediates TGF-β1-dependent early transcriptional induction of Idl.

Linggui Zhugan Decoction(苓桂术甘汤)Inhibits Ventricular Remodeling after Acute Myocardial Infarction in Mice by Suppressing TGF-β1 Smad Signaling Pathway

Objective:To investigate the inhibitory effect of Linggui Zhugan Decoction(LZD,苓桂术甘汤)on the ventricular remodeling(VR)after acute myocardial infarction(AMI)and related mRNA and proteins expression in transforming growth factor-beta 1(TGF-β1)/Smad signaling pathway,and explain its putative mechanism.Methods:A VR model was generated by ligation of coronary artery in mice.Two weeks after surgery,60 mice were randomly divided into the model group,the sham-operation group(distilled water),the positive control group(2.4 mg/kg simvastatin),and the low-,medium-and high-dose LZD groups(2.1,4.2,8.4 g crude drug/kg,respectively)by a random number table,10 mice in each group.Mice in each group was treated for 4 weeks.Changes of hemodynamics indices and cardiac weight index were detected by the PowerLab data acquisition and analysis recording instrument.Morphology changes of myocardial tissue were observed by hematoxylin-eosin and Masson staining.The expressions of TGF-β1,Smad2,Smad3,p-Smad2 and p-Smad3 in myocardial tissue were detected by Western blotting.The mRNA expressions of TGF-β1,Smad2 and Smad3 were detected by reverse transcription-quantitative polymerase chain reaction(RT-qPCR).The expressions of matrix metalloprotein 2(MMP2),MMP9,collagenⅠand collagen Ⅲ were observed by immunohistochemical methods.Results:VR mice showed significant dysfunction in hemodynamic indices and cardiac structure and function.Compared with the shamoperation group,myocardial tissue damage,interstitial fibrosis occurred in the model mice,left ventricular systolic pressure(LVSP),left ventricular pressure maximum contraction rate(+dp/dtmax)and left ventricular pressure maximum relaxation rate(-dp/dtmax)decreased significantly(all P<0.01),while left ventricular end-diastolic pressure(LVEDP),cardiac weight index and left ventricular weight index elevated significantly,meanwhile TGF-β1,p-Smad2,p-Smad3,Smad2,Smad3,MMP2,MMP9,collagen Ⅰ,collagen Ⅲ protein expressions in myocardial tissue and TGF-β1 Smad2 and Smad3 mRNA expressions increased significantly(all P<0.01).Compared with the model group,LZD could significantly improve the pathological changes of myocardial tissue,increase LVSP,+dp/dtmax and-dp/dtmaxf lower LVEDP,reduce the whole heart weight index and left ventricular weight index and inhibit the over-expressions of TGF-β1f p-Smad2,p-Smad3,Smad2,Smad3,MMP2,MMP9,collagenⅠand collagenⅢproteins in myocardial tissue and mRNA expressions of TGF-β1 Smad2 and Smad3(P<0.05 or P<0.01).Conclusion:LZD can significantly suppress VR induced by AMI,and its underlying mechanism may be associated with its inhibitory effect on the TGF-β1/Smad signaling pathway.

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.

Melatonin influences the biological characteristics of keloid fibroblasts through the Erk and Smad signalling pathways

Background:Keloids are abnormal fibrous hyperplasias that are difficult to treat.Melatonin can be used to inhibit the development of certain fibrotic diseases but has never been used to treat keloids.We aimed to discover the effects and mechanisms of melatonin in keloid fibroblasts(KFs).Methods:Flow cytometry,CCK-8 assays,western blotting,wound-healing assays,transwell assays,collagen gel contraction assays and immunofluorescence assays were applied to demonstrate the effects and mechanisms of melatonin in fibroblasts derived from normal skin,hypertrophic scars and keloids.The therapeutic potential of the combination of melatonin and 5-fluorouracil(5-FU)was investigated in KFs.Results:Melatonin significantly promoted cell apoptosis and inhibited cell proliferation,migration and invasion,contractile capability and collagen production in KFs.Further mechanistic studies demonstrated that melatonin could inhibit the cAMP/PKA/Erk and Smad pathways through the membrane receptor MT2 to alter the biological characteristics of KFs.Moreover,the combination of melatonin and 5-FU remarkably promoted cell apoptosis and inhibited cell migration and invasion,contractile capability and collagen production in KFs.Furthermore,5-FU suppressed the phosphorylation of Akt,mTOR,Smad3 and Erk,and melatonin in combination with 5-FU markedly suppressed the activation of the Akt,Erk and Smad pathways.Conclusions:Collectively,melatonin may inhibit the Erk and Smad pathways through the mem-brane receptor MT2 to alter the cell functions of KFs,while combination with 5-FU could exert even more inhibitory effects in KFs through simultaneous suppression of multiple signalling pathways.

TGF-β-induced epithelial to mesenchymal transition

During development and in the context of different morphogenetic events, epithelial cells undergo a process called epithelial to mesenchymal transition or transdifferentiation （EMT）. In this process, the cells lose their epithelial characteristics, including their polarity and specialized cell-cell contacts, and acquire a migratory behavior, allowing them to move away from their epithelial cell community and to integrate into surrounding tissue, even at remote locations. EMT illustrates the differentiation plasticity during development and is complemented by another process, called mesenchymal to epithelial transition （MET）. While being an integral process during development, EMT is also recapitulated under pathological conditions, prominently in fibrosis and in invasion and metastasis of carcinomas. Accordingly, EMT is considered as an important step in tumor progression. TGF-β signaling has been shown to play an important role in EMT. In fact, adding TGF-β to epithelial cells in culture is a convenient way to induce EMT in various epithelial cells. Although much less characterized, epithelial plasticity can also be regulated by TGF-β-related bone morphogenetic proteins （BMPs）, and BMPs have been shown to induce EMT or MET depending on the developmental context. In this review, we will discuss the induction of EMT in response to TGF-β, and focus on the under- lying signaling and transcription mechanisms.