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.

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

Background Recent clinical and experimental studies have confirmed the effects of Xinfuli Granule （XG）, a compound Chinesemedicine in the prevention and treatment of heart failure （HF）. This study aimed to investigate the effects and the mechanisms of XG onventricular reconstruction in rats with acute myocardial infarction （AMI）. Methods Sprague-Dawley rats were subjected to left anteriordescending 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 under-went 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 shorteningfraction （LVFS）, left ventricular internal diameter at systole （LVIDS） were measured by ultrasound; HE staining, Masson staining, and Siriusred staining were used to assess the myocardial pathological and physiological changes as well as myocardial fibrosis area and non-infarctzone Ⅰ/Ⅲ collagen ratio. Expression of Smad3 were detected and analyzed by Western blot, immunohistochemistry and immunofluorescenceP-Smad3, Smad2 and Smad7 in the TGF-13/Smads signaling pathway were also analyzed by Western blot. Results The LVIDS （P 〈 0.01）,HW/BW （P 〈 0.05）, type UIII 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 moleculesof the TGF-β/Smads signaling pathway, including Smad3, P-Smad3 and Smad2 protein were decreased, while the expression of Smad7 in-creased in both XG and carvedilol treatment groups as compared to those of the MI group （all P 〈 0.01）. Immunohistochemistry and im-munofluorescence further confirmed the down-regulated Smad3 expression. Conclusion XG can improve ventricular reconstruction andinhibit 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.

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.

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.

Inhibitory effects of oxyresveratrol on ERK and Smad1/2 phosphorylation and HSC activation in preventing carbon tetrachloride-induced rat liver fibrosis

Oxyresveratrol(ORes,trans-2,4,3′,5′-tetrahydroxy stilbene)naturally exists in mulberry,grapes,peanuts and other plants.It belongs to stilbene polyphenolic family and has an extra hydroxyl group at 2-position comparing with resveratrol(Res).Hence,ORes has stronger antioxidant activity than resveratrol.In present study,we employed a rat hepatic fibrosis model induced by carbon tetrachloride(CCl_(4))and administrated ORes via gavage feeding to study the protective effects and potential mechanisms of ORes against hepatic fibrosis.We demonstrated that rat liver oxidative damage induced by CCl_(4)was significantly alleviated after ORes feeding.Furthermore,the mRNA transcription levels ofα-smooth muscle actinn(˛-SMA),desmin,and two MMPs(MMP2 and MMP9)were reduced and the expression levels of transforming growth factorβ1(TGF-β1),p-small mother against decapen-taplegic protein(Smad)1/2 and p-extracellular signal-regulated kinases(ERK)1/2 in the liver tissue down-regulated dramatically.In a parallel study with Res,ORes showed more efficacious protective effect than Res against rat liver fibrosis,which is attributed to extended conjugation system due to the extra hydroxyl group at 2-position on ORes making it more electron-rich and susceptible to oxidation than Res.Therefore,dietary consumption of mulberry and other fruits containing ORes may be beneficial in the prevention of liver fibrosis.

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.

Pilose antler aqueous extract promotes the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells by stimulating the BMP-2/Smad1, 5/Runx2 signaling pathway

Peptides from Pilose antler aqueous extract(PAAE) have been shown to stimulate the proliferation and differentiation of bone marrow mesenchymal stem cells(BMSCs). However, the underlying molecular mechanisms are not well understood. Here, PAAE was isolated and purified to explore the molecular mechanisms underlying PAAE’s effects on BMSCs as well as its osteoprotective effects in ovariectomized rats. Our results showed that PAAE promoted proliferation and differentiation of BMSCs to become osteoblasts by enhancing ALP activity and increasing extracellular matrix mineralization. The trabecular microarchitecture of ovariectomized rats was also found to be protected by PAAE. Quantitative reverse transcription-polymerase chain reaction(Quantitative RT-PCR) results suggest that PAAE also increased the expression of osteogenic markers including, alkaline phosphatase(ALP), runt-related transcription factor 2(Runx2), osteocalcin(OCN), bone morphogenetic protein-2(BMP-2), and collagen I(COL-I). Immunoblotting results indicated that PAAE upregulated the levels of BMP-2 and Runx2 and was associated with Smad1/5 phosphorylation. PAAE A at the concentration of 200μg·mL^-1 showed the strongest effect on proliferation and osteogenic differentiation of BMSCs after 48 h. Using matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF MS), we identified the molecular weight of PAAE A and found that it is less than 3000 Da and showed several significant peaks. In conclusion, PAAE activates the BMP-2/Smad1, 5/Runx2 pathway to induce osteoblastic differentiation and mineralization in BMSCs and can inhibit OVX-induced bone loss. These mechanisms are likely responsible for its therapeutic effect on postmenopausal osteoporosis.

虫草素调控eIF2α/TGF-β/Smad信号通路改善肾间质纤维化的机制

目的:观察冬虫夏草主要有效成分——虫草素(cordycepin)对肾间质纤维化及其e IF2α/TGF-β/Smad信号通路的干预作用和机制。方法:将15只C57BL/6小鼠随机分为对照组、模型组和虫草素组,建立小鼠单侧输尿管结扎(unilateral ureteral obstruction,UUO)肾间质纤维化模型,虫草素组小鼠经皮下注射虫草素(5 mg·kg-1·d-1),同时,以生理盐水干预对照组和模型组,共5 d。实验结束后,取小鼠结扎侧的肾脏,观察肾间质纤维化病理特征,并测定肾组织I型胶原(collagen type I,Col I)和α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)核酸表达(Northern blot);另外,在体外培养大鼠肾小管上皮细胞(NRK-52E细胞),在转化生长因子(transforming growth factor,TGF)-β联合或不联合虫草素干预后,观察I型胶原、IV型胶原(collagen type IV,Col IV)核酸表达的变化(Northern blot),并检测e IF2α、磷酸化e IF2α(phosphorylated e IF2α,p-e IF2α)以及Smad2/3、磷酸化Smad2/3(phosphorylated Smad2/3,p-Smad2/3)蛋白表达的变化(Western blot)。结果:虫草素在体内改善模型鼠肾间质纤维化,包括纤维化的病理特征以及I型胶原、α-SMA核酸的表达;虫草素在体外促进NRK-52E细胞p-e IF2α的高表达,抑制TGF-β诱导的p-Smad2/3以及I型、IV型胶原的表达水平。结论:虫草素在体内有改善肾间质纤维化的作用,它可能是通过诱导e IF2α磷酸化,抑制TGF-β/Smad信号通路中的关键信号分子——p-Smad2/3表达,减少肾组织胶原和α-SMA表达等途径而改善肾间质纤维化。

Huoxin Pill(活心丸)Attenuates Cardiac Fibrosis by Suppressing TGF-β1/Smad2/3 Pathway in Isoproterenol-Induced Heart Failure Rats

Objective:To evaluate the effects of Huoxin Pill(活心丸,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-β1(TGF-β1)/Smads pathway was further explored using immunohistochemistry(IHC)and Westernblot 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 collagenⅠand collagenⅢprotein expression in cardiac tissues of ISO-induced HF rats.Furthermore,HXP treatment significantly down-regulated the increase of TGF-β1 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-β1/Smad2/3 pathway.

The Smad pathway in transforming growth factor-β signaling

The Smad pathway is involved in transforming growth factor-b (TGF-b) signal transduction. The Smad complex binds with the promoter of target gene to modulate gene transcription. Various transcriptional coactivators and corepressors associate directly with Smads for appropriate binding of Smads to target promoters and regulation of Smads transcriptional activities. The ultimate degradation of Smads mediated by the ubiquitin-proteasome pathway (UPP) has been established as a mechanism to shut off the Smad pathway. In addition to the Smad pathway, TGF-?can also activate other signaling pathway such as the MAPK pathway. The cross-talk of the Smad pathway with other signaling pathways constitutes an important mechanism for the regulatory network of TGF-b signaling.

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 role of Smad6 in immunity of the pearl oyster Pinctada fucata martensii

Inhibitory Smads(I-Smads),which belong to the Smad family and inhibit bone morphogenic protein 2(BMP2)signaling by a variety of mechanisms,can suppress innate immunity responses in vertebrates.However,there are no reports for the role of Smad6 in immunity in mollusks.In this study,we showed that Smad6 of the pearl oyster Pinctada fucata martensii was located in the Smad6 cluster of the phylogenetic tree;mRNA expression of Smad6 and Smad3 was up-regulated after lipopolysaccharide and polyinosinic:polycytidylic challenge;and transcript levels of Smad6 and Smad3 showed opposite patterns during wound healing.Under salinity stress,water inflow and outflow in the gills appear to be regulated by BMP2-Smads signals,and BMP2-Smads signaling may be closely related to the immune response.Our results indicate that Smad6 is involved in immunity,that it plays a positive role in the response to immune challenge and an inhibitory role during wound healing,and that Smad6 and Smad3 may work against each other.

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.

Crystal Structure of the MH2 domain of Drosophila Mad

The decapentaplegic(Dpp),a member of the TGF-β superfamily,plays a pivotal role in the control of proliferation,global patterning and induction of specific cell fates during Drosophila development.Mother against Dpp(Mad) is the founding member of the conserved Smad protein family which spe-cifically transduces the intracellular TGF-β signaling cascade.Here we report the 2.80 structure of the MH2 domain of Mad(Mad-MH2) that was readily superposed to the mammal Smad-MH2 structures.This unphosphorylated Mad-MH2 forms a symmetric homotrimer in crystals,consistent with the result of the size-exclusion chromatography that Mad-MH2 exhibited a propensity for concentration-dependent oligomerization prior to phosphorylation.Structural analysis revealed that the formation of homotrimeric Mad-MH2 is mainly mediated by contacts involving the extreme C-terminal SSVS motif,and is strengthened by phosphorylation of the last two Ser residues which was confirmed by the gel filtration analysis of the pseudophosphorylated Mad-MH2(DVD).Intriguingly,the homotrimer within an asymmetric unit only possesses two ordered C-terminal tails,reminiscent of the arrangement of the R-Smad/Smad4 complexes,indicating that the subunit with a flexible SSXS motif would be readily replaced by Co-Smad to form a functional heterotrimer.

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.

Xueshuantong for Injection(Lyophilized,注射用血栓通)Alleviates Streptozotocin-Induced Diabetic Retinopathy in Rats

Objective To investigate the ameliorate effect and underlying mechanism of Xueshuantong for Injection(Lyophilized,注射用血栓通,XST)in streptozocin(STZ)-induced diabetic retinopathy(DR)rats.Methods Diabetes mellitus(DM)model was induced by intraperitoneal(i.p.)injection of STZ(60 mg/kg)in Sprague-Dawley rats.Diabetic rats were randomized into 3 groups(n=10)according to a random number table,including DM,XST50 and XST100 groups.XST treatment groups were daily i.p.injected with 50 or 100 mg/kg XST for 60 days,respectively.The control and DM groups were given i.p.injection with saline.Blood glucose level and body weight were recorded every week.Histological changes in the retina tissues were observed with hematoxylin-eosin staining.Apoptosis and inflammation related factors,including cleaved caspase-3,glial fifibrillary acidic protein(GFAP),tumor necrosis factor-α(TNF-α)and intercellular cell adhesion molecule-1(ICAM-1)were detected by Western blot or real-time polymerase chain reaction.Then,the levels of advanced glycation end product(AGE)and its receptor(RAGE)were investigated.Tight junctions proteins(Zonula occludens-1(ZO-1),Occludin and Claudin-5)of blood-retinal barrier were detected by Western blot.The levels of retinal fifibrosis,transforming growth factor-β1(TGF-β1)-Smad2/3 signaling pathway were evaluated at last.Results There was no signifificant difference in the body weight and blood glucose level between XST and DM groups(P>0.05).Compared with the DM group,XST treatment signifificantly increased the retinal thickness of rats(P<0.05 or P<0.01),and suppressed cleaved caspase-3 expression(P<0.01).XST increased the protein expressions of ZO-1,Occludin and Claudin-5 and decreased the mRNA expressions of matrix metalloproteinase 2(MMP-2)and MMP-9(P<0.05 or P<0.01).Moreover,XST signifificantly reduced the productions of AGE and RAGE proteins in the retina of rats(P<0.05 or P<0.01),suppressed the over-expression of TNF-α,and decreased the elevated level of ICAM-1 in retina of rats(P<0.05 or P<0.01).XST signifificantly reduced the levels ofα-smooth muscle actin(α-SMA),connective tissue growth factor(CTGF),TGF-β1 and phosphorylation of Smad2/3 protein in rats(P<0.05 or P<0.01).Conclusions XST had protective effects on DR with possible mechanisms of inhibiting the inflammation and apoptosis,up-regulating the expression of tight junction proteins,suppressing the productions of AGE and RAGE proteins,and blocking the TGF-β/Smad2/3 signaling pathway.XST treatment might play a role for the future therapeutic strategy against DR.