Effect Xuefu Zhuyu decoction on endothelial-to-mesenchymal transition of pulmonary artery endothelial cells and its mechanism / 中国药理学通报

Aim To investigate the effect of Xuefu Zhuyu decoction on transforming growth factor-β1(TGF-β1 ) -induced endothelial-to-mesenchymal transition (EndMT) of pulmonary microvascular endothelial cells ( PMVEC), and further analyze the mechanism related to the TGF-β1/Smad signaling pathway. Method To construct an EndMT cell model, PMVEC was treated with TGF-β1 (5 μg · L

Intervention effect and mechanism of breviscapine on hepatic fibrosis in rats / 中国药房

OBJECTIVE To investigate the intervention effect and potential mechanism of breviscapine on hepatic fibrosis （HF） in rats based on the transforming growth factor-β（1 TGF-β1）/Smad2/extracellular signal-regulated protein kinase 1（ERK1） and Kelch-like epichlorohydrin-associated protein 1（Keap1）/nuclear factor-erythroid 2-related factor 2（Nrf2）/heme oxygenase-1（HO-1） pathways. METHODS Totally 60 rats were randomly divided into normal control group， model group， breviscapine low-dose， medium-dose and high-dose groups （5.4， 10.8， 21.6 mg/kg）， and colchicine group （positive control， 0.45 mg/kg）， with 10 rats in each group， half male and half female. Except for the normal control group， HF model of the other groups was induced by carbon tetrachloride. Subsequently， each drug group was given corresponding medicine by gavage once a day for 28 days. The liver appearance of rats in each group was observed and their liver coefficients were calculated. The levels of alanineaminotransferase （ALT） and aspartate aminotransferase （AST）in serum， those of ALT， AST， superoxide dismutase （SOD），malondialdehyde （MDA） and glutathione peroxidase （GSH- Px） in liver tissue were detected. The liver tissue inflammatory and fibrotic changes were observed. The protein and mRNA expressions of TGF-β1， Smad2， ERK1， Nrf2， Keap1 and HO-in liver tissue were detected. RESULTS Compared with the normal control group， the model group showed large areas of white nodular lesions in the liver， obvious inflammatory cell infiltration and collagen fiber deposition. The body weight， the levels of SOD and GSH-Px in liver tissue， the protein and mRNA expressions of Nrf2 and HO-1 were significantly lowered in the model group （P＜0.05）； the liver coefficient， the percentage of Masson staining positive area， ALT and AST levels of serum and liver tissue， MDA level of liver tissue， the protein and mRNA expressions of TGF-β1， Smad2， ERK1 and Keap1 were significantly increased （P＜0.05）. Compared with the model group， the liver lesions of rats in each drug group were improved， and the above quantitative indexes were generally reversed （P＜0.05）. CONCLUSIONS Breviscapine has a good intervention effect on HF rats， which may be related to inhibiting TGF-β1/Smad2/ERK1 pathway for anti-fibrosis and regulating Keap1/Nrf2/HO-1 pathway to inhibit oxidative stress.

Cytokine receptor-like factor 1 (CRLF1) promotes cardiac fibrosis via ERK1/2 signaling pathway / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

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.

Effect mechanism of acupuncture for anti-asthmatic airway remodeling based on TGF-β1 / Smad3 signaling pathway / 中国针灸

OBJECTIVE@#To observe the effect of acupuncture at "Feishu" (BL 13) + "Dingchuan" (EX-B 1) and "Kongzui" (LU 6) + "Yuji" (LU 10) for the airway remodeling in asthma rats based on the transforming growth factor-β1 (TGF-β1)/ Smad family member 3 (Smad3) signaling pathway; and explore the efficacy difference between the two acupoint combinations.@*METHODS@#Forty SPF male SD rats, aged 4 weeks, were randomly divided into a blank group (n = 10) and a modeling group (n = 30). The ovalbumin (OVA) sensitization method was used to establish asthma model in the modeling group. After successful model preparation, the rats of the modeling group were randomized into a model group, an acupuncture at "Feishu" (BL 13) + "Dingchuan" (EX-B 1) (AAF) group, and acupuncture at "Kongzui" (LU 6)+"Yuji" (LU 10) (AAK) group, with 10 rats in each one. Starting from day 15 of the experiment, 5 min after motivating, acupuncture was applied to "Feishu" (BL 13) + "Dingchuan" (EX-B 1) and "Kongzui" (LU 6)+"Yuji" (LU 10) in the AAF group and the AAK group respectively. The intervention was delivered for 30 min each time, once daily, lasting 3 weeks consecutively. Using lung function detector, the airway resistance (RL) and dynamic compliance (Cdyn) of the lungs were detected. The histomorphology of lung tissues was detected with HE staining and Masson staining, and the mRNA and protein expression of TGF-β1 and Smad3 in lung tissues was detected with the real-time PCR and Western blot methods.@*RESULTS@#Compared with the blank group, RL was increased and Cdyn was decreased in the rats of the model group (P<0.01); and RL was reduced and Cdyn was increased in the AAF group and the AAK group when compared with those in the model group (P<0.01, P<0.05). The rats of the model group had bronchial lumen stenosis, inflammatory cell infiltration, collagen fibre hyperplasia and thickened smooth muscle in the lung tissues when compared with those in the blank group; and in comparison with the model group, all of the above morphological changes were attenuated in the AAF group and the AAK group. Besides, these morphological changes of the lung tissues were more alleviated in the AAF group when compared with those in the AAK group. In comparison with the blank group, the mRNA and protein expression of TGF-β1 and Smad3 of the lung tissues was increased in the model group (P<0.01), and it was reduced in the AAF group and the AAK group when compared with that in the model group (P<0.05, P<0.01). The mRNA expression of TGF-β1 and Smad3 was lower in the AAF group when compared with that in the AAK group (P<0.05).@*CONCLUSION@#Acupuncture at either "Feishu" (BL 13)+"Dingchuan" (EX-B 1) or "Kongzui" (LU 6)+"Yuji" (LU 10) reduces the airway remodeling in the rats with asthma, which may be related to the down-regulation of mRNA and protein expression of TGF-β1 and Smad3. The better efficacy is obtained with acupuncture at "Feishu" (BL 13)+"Dingchuan" (EX-B 1).

Effects of liquorice extract on cardiac fibroblasts fibrosis induced by TGF-β1 / 中国临床药理学与治疗学

AIM: To investigate the effect of liquorice extract on TGF-β1-induced myocardial fibroblast (CFs) fibrosis. METHODS: 10 ng/mL TGF-β1 induced CFs to establish myocardial fibrosis cell model. Fibrotic cells were treated with liquorice extract and the cell activity was detected by MTT assay. CCK-8 was used to detect the effect of liquorice extract on CFs proliferation. The expression of smooth muscle actin (α-SMA) was detected by immunofluorescence. Western blot was used to detect TGF-β1/Smad signaling pathway related proteins and p-Smad2, p-Smad3 expression levels. The mRNA expression levels of Smad2, Smad3 and Smad4 were detected by RT-PCR. RESULTS: Compared with the control group, there were statistically significant differences in cell activity (P<0.05). The cell proliferation rate of glycyrrhiza uralensis extract groups was significantly lower than that of TGF-β1 group (P<0.05). The expression levels of α-SMA and TGF-β1/Smad signaling pathway related proteins in 100 μg/mL liquorice extract were significantly lower than those in TGF-β1 group (P<0.05). CONCLUSION: Glycyrrhiza extract can improve the occurrence and development of TGF-β1-induced myocardial fibrosis, and its mechanism maybe related to the inhibition of TGF-β1/Smad signaling pathway.

The protective effects of naringenin on liver fibrosis by regulating TGF-fil/smad pathway / 中国药理学通报

Aim To study the effects of naringenin on MCD diet-induced liver fibrosis and its related mechanisms.Methods LX2 cells were incubated with TGF-β1 for 24 h to establish the in vitro fibrosis model.LX2 cells were treated with NGN at the same time.Male C57BL/6 mice were fed with MCD diet for six weeks to induce liver fibrosis.100 mg·kg-1·d-1 NGN was administered by gavage simultaneously.The protein expressions of α-SMA, col1, TGF-β1, p-smad2 and p-smad3 were evaluated by Western blot.The mRNA expressions of α-SMA, col1 and col3 were detected by qRT-PCR.The degree of liver fibrosis was evaluated by Sirius red staining.Results Both in in vivo and in vitro experiments, compared with model group, the mRNA levels of α-SMA, col1 and col3 and protein levels of α-SMA, TGF-β1, p-smad2 and p-smad3 significantly decreased in NGN treatment group.The results of HE staining and Sirius red staining also indicated that NGN significantly decreased liver fibrosis induced by MCD diet.Conclusions Naringin can significantly inhibit liver fibrosis induced by MCD diet, which may be related to TGF-β1/Smad pathway.

Fuganlin oral liquid ameliorates airway remodeling through TGF-β1/Smad3 signaling pathway in asthmatic mice / 中国药理学通报

Aim To explore the effect of Fuganlin on airway remodeling in obese asthmatic mice and its mechanism.Methods A model of chronic airway inflammation in C57 BL/6 mice with obese asthma induced by OVA and high-fat diet was established,and treated with Fuganlin 5.86,11.72 and 23.44 g·kg-1 by gavage.After the last challenge,the respiratory system resistance(Rrs),respiratory system elasticity(Ers),and respiratory system compliance(Crs)were measured with a lung function oscillator; the total number of white blood cells in whole blood was measured; tissue HE and MASSON staining were employed to observe the pathological changes.ELISA was used to detect the levels of IgE in serum and the levels of TGF-β1,Smad3 and SP in lung tissues; IHC was used to detect the expression levels of Smad3,SARA and protein in lung tissues.Results Fuganlin reduced the increase in the number of white blood cells in blood and inhibited the content of IgE in serum.Fuganlin could reduce the Rrs and Ers,enhance the Crs and regulate the respiratory function.Histopathological results showed that Fuganlin could reduce inflammatory cell infiltration and collagen deposition in the chronic airway inflammation model of obese mice,and inhibit bronchial mucosal proliferation; ELISA results showed that Fuganlin inhibited the expression of TGF-β1,Smad3,and SP; IHC results showed that Smad3 and SARA protein expression decreased.Conclusions The anti-obesity asthma effect of Fuganlin may help to improve respiratory function,control airway inflammation,and antagonize airway remodeling.

Effects of novel Fufang Biejia Ruangan Tablets with sheep placenta as substitute for Hominis Placenta on CCl / 中草药·英文版

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

Inhibition of ginsenoside Rb 1 on the epithelial-mesenchymal transition of renal tubular epithelial cells by regulating TGF-β1/Smad3 signaling pathway / 中国药房

OBJECTIVE To study the effects of ginsenoside Rb 1（G-Rb1）on epithelial-mesenchymal transition （EMT）of renal tubular epithelial cells and its potential mechanism. METHODS The growth factor β1（TGF-β1）10 ng/mL was used to induce EMT of human renal tubular epithelial cells HK- 2. The morphological changes of HK- 2 cells were observed after treated with 10， 20，30 μmol/L G-Rb1 for 48 h. The transcriptional activities of biovector SBE in human embryonic kidney cell HEK 293 were determined after 24 h treatment with 1.0，2.5，5.0，10，20，30 μmol/L G-Rb1. Effects of above concentration of G-Rb 1 on the viability of HK- 2 cells were determined after 24 h of treatment. mRNA expressions of α-smooth muscle actin （α-SMA），collagen Ⅰ （COL-Ⅰ）and fibronectin （FN）in HK- 2 cells were detected after treated with 10，20，30 μmol/L G-Rb1 for 24 h. The expressions of α-SMA，Smad3，p-Smad3，COL-Ⅰ，FN and E-cadherin were detected after treated with 10，20，30 μmol/L G-Rb1 for 24 h. RESULTS G-Rb1 of 10-30 μmol/L significantly inhibited TGF-β1-induced EMT in HK- 2 cells and the increase of transcriptional activities of biovector SBE induced by TGF-β1（P＜0.05），but had no effects on relative activities of HK- 2 cells（P＞0.05）. The protein and mRNA expressions of α-SMA，COL-Ⅰ and FN ， the protein expressions of Smad 3 and p-Smad 3 were significantly up-regulated induced by TGF-β1（P＜0.05），while the protein expression of E-cadherin was significantly down- regulated（P＜0.05）；G-Rb1 could effectively reverse aboveprotein or mRNA expressions. CONCLUSIONS G-Rb1 can protect renal tubular epithelial cells from EMT induced byxiezhishen TGF-β1 to a certain extent ，which may be related to inhibiting the activation of TGF-β1/Smad3 signaling pathway.

Tangshen formula promotes cellular cholesterol efflux in HG-induced mTECs by suppression of TGF-β1/Smad3 pathway / 中国临床药理学与治疗学

AIM: To observe the effect of Tangshen formula (TSF) treatment on TGF-β1/Smad3 pathway and cellular cholesterol efflux and explore its potential mechanism in HG-induced mouse tubular epithelial cells (mTECs). METHODS: After 25 mmol/L high glucose induced mTECs, TSF and Smad3 inhibitor SIS3 were given to intervene respectively. The lipid content in the cells was detected by ELISA kit; TGF-β1/Smad3 pathway and PGC-1α, LXR, ABCA1, ABCG1 were detected by Western blot and real-time PCR. RESULTS: TSF diminished the levels of TC, TG, LDL-C and increased the levels of HDL-C in HG-induced mTECs. Western blot and real-time PCR analysis showed that expression levels of TGF-β1, Smad3, Collagen and Fibronectin were significantly downregulated in the HG-induced mTECs with TSF and SIS3 treatment. And PGC-1α, LXR, ABCA1, ABCG1 expression levels were significantly upregulated in the HG-induced mTECs with TSF and SIS3 treatment. CONCLUSION: TSF can promote the cellular cholesterol efflux in HG-induced mTECs vis suppression of TGF-β1/Smad3 pathway.

Mechanism of Fuzheng Qufeng Prescription in Suppressing Epithelial-mesenchymal Transition of Podocytes in Membranous Nephropathy Rats via TGF-β1/Smad Pathway / 中国实验方剂学杂志

Objective:To study the effect of Fuzheng Qufeng prescription （FZQP） on transforming growth factor-<italic>β</italic>₁ （TGF-<italic>β</italic>₁）/Smad signaling pathway and epithelial-mesenchymal transition of podocyte in membranous nephropathy （MN） rats and to explore its molecular mechanism for podocyte protection. Method:The rats were randomly divided into normal control group （NC） and modeling group. Rats in modeling group induced by bovine serum albumin （C-BSA） were randomly divided into model group （MN）， losartan potassium group （LP， 0.05g·kg^-1）， and FZQP high dose （FZQPH， 41 g·kg^-1）， medium dose （FZQPM， 20.5 g·kg^-1）， and low dose （FZQPL， 10.25 g·kg^-1） groups. The administration lasted for 4 weeks. In week 0， 2， and 4 of administration， the levels of 24 hours urine protein （24 h-Upro） were tested. At the end of 4th week， the levels of blood urea nitrogen （BUN） and serum creatinine （SCr） were detected， and the rats in each group were sacrificed and the renal pathological morphology changes were observed by light microscope with hematoxylin-eosin （HE）， Masson and periodic acid-silver metheramine （PASM） staining. The deposition of immune complex， the thickening of glomerular basement membrane （GBM） and podocyte foot process were observed by transmission electron microscope （TEM）. The distribution and expression intensity of Desmin in renal tissues were detected by immunohistochemistry （IHC）. The mRNA and protein expression levels of TGF-<italic>β</italic>₁， Smad2/3， phospho（p）-Smad2/3， Smad7 and Desmin in renal tissues were respectively detected by Western blot （WB） and Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. Result:Compared with NC group， the levels of 24 h-Upro， BUN and SCr significantly increased in model group （<italic>P</italic><0.01）， with increased deposition of immune complex， significantly thickened GBM and fusion of foot processes， significantly increased Desmin mRNA and protein expression （<italic>P</italic><0.01） and increased TGF-<italic>β</italic>₁， Smad2， and Smad3 mRNA and protein expression （<italic>P</italic><0.05）， and decreased Smad7 mRNA and protein expression （<italic>P</italic><0.05，<italic>P</italic><0.01）. Compared with model group， 24 h-Upro and BUN decreased in FZQP groups and LP group （<italic>P</italic><0.05）， levels of serum SCr in FZQPM group decreased （<italic>P</italic><0.05）， deposition of immune complex， thickening of GBM and fusion of foot process were all alleviated in FZQP groups and LP group. Distribution of Desmin along GBM decreased in FZQPH group， FZQPM group and LP group （<italic>P</italic><0.05）. Both mRNA and protein expression levels of TGF-<italic>β</italic>₁ and p-Smad2/Smad2 in FZQPM group decreased， while mRNA and protein expression levels of Smad7 increased （<italic>P</italic><0.05）. Both mRNA and protein expression levels of p-Smad3/Smad3 in FZQPH group decreased （<italic>P</italic><0.05）. Both mRNA and protein expression levels of Desmin in podocyte in FZQPH group， FZQPM group and LP group decreased （<italic>P</italic><0.05）. Conclusion:FZQP might realize podocyte protection effect in MN via suppressing EMT mediated by overactivated TGF-<italic>β</italic>₁/Smad signaling pathway.

Effect of Bushen Shugan Prescription on Oxidative Stress and TGF-β1/Smad Signaling Pathway in Hippocampus of Senile Depressed Mice / 中国实验方剂学杂志

Objective:To observe the changes in oxidative stress and transforming growth factor-<italic>β</italic>₁ （TGF-<italic>β</italic>₁）/Smad signaling pathway in hippocampal tissue of senile depressed mice after chronic unpredictable mild stress and to explore the possible anti-depression mechanism of Bushen Shugan prescription. Method:Ninety five-month-old mice were randomly divided into six groups， namely the normal group， senile depression model group， high-， medium-， and low-dose Bushen Shugan prescription groups， and fluoxetine group， with 15 in each group. Mice in all groups， except for the normal group， were exposed to chronic unpredictable mild stress （CUMS） for inducing the senile depression. Since the first day of modeling， the mice in the high-， medium- and low-dose Bushen Shugan prescription groups were gavaged with 19.5， 9.75， 4.87 g·kg^-1 Bushen Shugan prescription， the ones in the fluoxetine group with 0.033 g·kg^-1fluoxetine， and those in the normal and senile depression model groups with an equal volume of normal saline for 21 successive days. The behavioral responses of mice in each group were evaluated in the open field test （OFT）， and the hippocampal tissues of mice were collected for testing the relevant indexes. The superoxide dismutase （SOD） content was determined by WST-1 method， malondialdehyde （MDA） content by TBA method， glutathione （GSH） content by micro enzyme-linked immunosorbent assay （ELISA）， and mRNA expression of TGF-<italic>β</italic>₁， Smad2， Smad3， and Smad7 by Real-time polymerase chain reaction （Real-time PCR）. Result:Compared with the normal group， the senile depression model group exhibited significantly lowered horizontal and vertical scores in OFT， decreased SOD and GSH contents in hippocampal tissues， elevated MDA （<italic>P</italic><0.05）， up-regulated TGF-<italic>β</italic>₁， Smad2， and Smad3 mRNA expression， and down-regulated Smad7 （<italic>P</italic><0.05）. Compared with the senile depression model group， Bushen Shugan prescription at the high， medium， and low doses and fluoxetine all increased SOD and GSH contents in mouse hippocampal tissues， decreased the MDA content （<italic>P</italic><0.05）， down-regulated the mRNA expression of TGF-<italic>β</italic>₁， Smad2， and Smad3 in hippocampal tissues， and up-regulated the Smad7 mRNA expression （<italic>P</italic><0.05）. The comparison with the high-dose Bushen Shugan prescription group showed that the SOD and GSH contents in hippocampal tissues of mice in the medium- and low-dose Bushen Shugan prescription groups declined significantly， while the MDA contents rose significantly （<italic>P</italic><0.05）. Besides， the mRNA expression levels of TGF-<italic>β</italic>₁， Smad2 and Smad3 in the hippocampal tissues of mice in the medium- and low-dose Bushen Shugan prescription groups were significantly up-regulated， and those of Smad7 were significantly down-regulated （<italic>P</italic><0.05）. Conclusion:Bushen Shugan prescription alleviates the depression symptoms in aged SAPM8 mice possibly by regulating the hippocampal oxidative stress and TGF-<italic>β</italic>₁/Smad signaling pathway.

Effect of pirfenidone on the proliferation of human glomerular mesangial cells induced by serum IgA1 of IgA nephropathy patients / 中华肾脏病杂志

Objective:To investigate the effect of pirfenidone (PFD) on the proliferation of human glomerular mesangial cells (HMC) stimulated by serum IgA1 in patients with IgA nephropathy (IgAN) and its possible mechanism.Methods:Serum IgA1 of IgAN patients was purified by Jacalin affinity chromatography combined with Sephacryl S-200 gel filtration, and then heated to aggregated form (aIgA1). CCK8 method was used to confirm the concentration and time of PFD. The cells were divided into blank control group, IgA1 (0.5 mg/ml) group and IgA1 (0.5 mg/ml)+PFD (2 mmol/L) group. The CCK8 method was used to detect proliferation of mesangial cells. The cell cycle was detected by flow cytometry, and the proliferation index of mesangial cells was calculated. The expression levels of transforming growth factor β1 (TGF-β1), Smad4, Smad7, fibronectin (FN) and collagen Ⅳ protein and mRNA were detected through Western blotting and real-time PCR.Results:Compared with blank control group, the proliferation of HMC was promoted significantly by aIgA1 ( P<0.05). After PFD treatment, the proliferation of HMC was significantly inhibited ( P<0.01). Compared with the blank control group, the number of G1 phase cells decreased, the number of S phase cells and cell proliferation index increased in IgA1 group (all P<0.05). Compared with IgA1 group, the number of cells in G1 phase increased significantly, the number of cells in S phase and G2/M phase decreased significantly, and the cell proliferation index decreased in IgA1+PFD group (all P<0.05). Western blotting and real-time PCR results showed that compared with the blank control group, the protein and mRNA expressions of collagen Ⅳ, FN and Smad4 in HMC stimulated by aIgA1 were significantly increased, while TGF-β1 protein expression was increased and Smad7 protein expression was decreased (all P<0.05). After PFD treatment, the protein and mRNA expression of collagen Ⅳ, FN and Smad4 in HMC was significantly decreased, while TGF-β1 protein expression was obviously decreased, and Smad7 protein was up-regulated (all P<0.05). There was no significant difference in the mRNA expression of TGF-β1 and Smad7 in each group before and after PFD treatment (all P>0.05). Conclusions:PFD can increase the arrest of HMC in G1 phase, inhibit the proliferation of HMC induced by aIgA1 of IgAN patients, and reduce the production of extracellular matrix. The mechanism may be related to up-regulation of Smad7 expression and down-regulation of TGF-β1/Smad4 pathway.

Preliminary Study on Improvement Effect of Tiarella polyphylla Ethanol Extract on CCl 4-induced Hepatic Fibrosis in Mice and Its Mechanism / 中国药房

OBJECTIVE：To investigate the improvement effect of Tiarella polyphylla ethanol extract （TPME）on CCl 4-induced hepatic fibrosis in mice ，and to explore its possible mechanism preliminarily. METHODS ：Totally 60 male Kunming mice were randomly divided into normal group ，model group ，positive control group （colchicine 0.1 mg/kg），TPME low-dose ，medium-dose and high-dose groups （250，500，1 000 mg/kg）according to body weight ，with 10 mice in each group. Except for normal group ， other groups were given 20％ CCl4 olive oil solution intraperitoneally to induce hepatic fibrosis ，twice a week ，for consecutive 8 weeks. From the fifth week after modeling ，administration groups were given relevant medicine intragastrically ，normal group and model group were given constant volume of normal saline intragastrically ，once a day ，for consecutive 4 weeks. Twelve hours after last administration ，the liver weight of mice in each group was measured and the liver index was calculated. The serum contents of ALT，AST，SOD，MDA，PC-Ⅲ，C-Ⅳ，LN，TNF-α and IL- 6 were determined. Western blot assay was used to detect the protein expression of α-SMA，TGF-β1 and Smad 3 in liver tissue. HE and Masson staining were used to observe the pathological changes of hepatic tissue. RESULTS ：Compared with normal group ，the liver index ，the activities of ALT and AST and the contents of MDA ， LN，PC- Ⅲ ，C- Ⅳ ，LN，TNF-α and IL- 6 in serum were increased significantly ， while the activity of SOD was 6011） decreased significantly in model group （P＜0.01）；the protein expression of α-SMA，TGF-β1 and Smad 3 in liver tissues were hfjsznd8@126.com increased significantly （P＜0.01）. Obvious fibrosis lesions was observed in liver tissue. Compared with model group ，the live indexes ，the activities of ALT and AST ，the contents of MDA，PC-Ⅲ，C-Ⅳ，LN，TNF-α and IL-6 in serum were decreased significantly in positive control group and TPME groups ， while the activities of SOD were increased significantly （P＜0.05 or P＜0.01）. The protein expression of α-SMA，TGF-β1 and Smad3 in liver tissue were decreased significantly （P＜0.05 or P＜0.01），and liver fibrosis was improved to different extent. Compared with TPME low-dose group ，the contents of PC- Ⅲ，LN and IL- 6 in serum ，protein expression of TGF-β1 and Smad 3 in liver tissue were decreased significantly in TPME high-dose group （P＜0.05）. CONCLUSIONS ：TPME can improve hepatic fibrosis induced by CCl 4 in mice ，the mechanism of which may be associated with the inhibition of collagen synthesis and oxidative stress，the reduction of inflammatory factors ，and the down-regulation of the expression α-SMA and relative proteins of TGF-β1/ Smad signal pathway.

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

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

Human umbilical cord mesenchymal stem cell-derived exosomes alleviate pulmonary fibrosis in mice by inhibiting epithelial-mesenchymal transition / 南方医科大学学报

OBJECTIVE@#To study the anti- fibrotic effect of human umbilical cord mesenchymal stem cell-derived exosomes (hUCMSC-EXOs) and explore the mechanism.@*METHODS@#Twenty-four C57 BL/6 mice were divided into 4 groups (=6), including the control group treated with intratracheal injection of saline (3 mg/kg); lung fibrosis model group with intratracheal injection of 1.5 mg/mL bleomycin solution (prepared with saline, 3 mg/kg); EXOs1 group with intratracheal injection of 1.5 mg/mL bleomycin solution (3 mg/kg) and hUCMSC-EXOs (100 μg/250 μL, given by tail vein injection on the next day after modeling); and EXOs2 group with intratracheal injection of 1.5 mg/mL bleomycin solution (3 mg/kg) and hUCMSC-EXOs (100 μg/250 μL, given by tail vein injection on the 10th day after modeling). At 21 days after modeling, pulmonary index, lung tissue pathology and collagen deposition in the mice were assessed using HE staining and Masson staining. The expression level of TGF-β1 was detected using ELISA, and vimentin, E-cadherin and phosphorylated Smad2/3 (p-Smad2/3) were detected using immunohistochemical staining. CCK8 assay was used to evaluate the effect of hUCMSCEXOs on the viability of A549 cells, and Western blotting was used to detect the expression levels of p-Smad2/3, vimentin, and E-cadherin in the cells.@*RESULTS@#Compared with those in the model group, the mice treated with hUCMSC-EXOs showed significantly reduced the pulmonary index ( < 0.05), collagen deposition, lung tissue pathologies, lowered expressions of TGF-β1 ( < 0.05), vimentin, and p-Smad2/3 and increased expression of E-cadherin. hUCMSC-EXOs given on the second day produced more pronounced effect than that given on the 11th day ( < 0.05). CCK8 assay results showed that hUCMSC-EXOs had no toxic effects on A549 cells ( > 0.05). Western blotting results showed that hUCMSC-EXOs treatment significantly increased the expression of E-cadherin and decreased the expressions of p-Smad2/3 and vimentin in the cells.@*CONCLUSIONS@#hUCMSC-EXOs can alleviate pulmonary fibrosis in mice by inhibiting epithelialmesenchymal transition activated by the TGF-β1/Smad2/3 signaling pathway, and the inhibitory effect is more obvious when it is administered on the second day after modeling.

Study on the Effects and Mechanism of Matrine on Proliferation and Collagen Synthesis of Hepatic Stellate Cells CFSC-8B Activated by Acetaldehyde / 中国药房

OBJECTIVE：To study the effects of matrine on proliferation and collagen synthesis of rat hepatic stellate cells CFSC-8B activated by acetaldehyde ，and to investigate its possible mechanism. METHODS ：CFSC-8B cells cultured in vitro were divided into blank control group ，model group ，positive control group （2.5 μmol/L colchicine）and matrine low ，medium and high concentration groups （30，60，120 μmol/L）. Except for blank control group ，other groups were activated with 200 μmol/L acetaldehyde for 24 h；medicine groups were intervened with relevant medicine for 24 h（blank control group and model group were intervened with equal volume blank medium ）. Survival rate of cell was detected by CCK- 8 assay. C ells were divided into blank control group ，model group ，positive control group （2.5 μmol/L colchicine），matrine medium and high concentration groups （60，120 μmol/L），then activated and treated with same method. Hydroxyprolin （Hyp）content in cell culture solution was tested by enzyme digestion. The contents of Col- Ⅰ and Col- Ⅲ in cell culture solution were determined by ELISA. mRNA expressionss of α-SMA，TGF-β1，TβR-І，TβR-Ⅱ，Smad3，Smad4 and Smad 7 in cells were detected by RT-PCR. The protein expressions of α-SMA，TGF-β1，TβR-Ⅰ，TβR- Ⅱ，Smad3，Smad4 and Samd 7 in cells were detected by Western blotting. RESULTS ：Compared with blank control group ，survival rate of cells in model group was increased significantly （P＜0.05）；the contents of Hyp ，Col-Ⅰ and Col- Ⅲ in cell culture solution ，mRNA and its protein expressions of α-SMA，TGF-β1，TβR-Ⅰ，TβR-Ⅱ，Smad3，Smad4 in cells were increased significantly in model group （P＜0.05），while the mRNA and protein expression of Smad 7 was decreased significantly（P＜0.05）. Compared with model group ，survival rate of cells ，the contents of Hyp ，Col-Ⅰ and Col- Ⅲ in cell culture solution，the mRNA and protein expressions of α-SMA and Smad 4 were decreased significantly in positive control group and matrine medium and high concentration groups （P＜0.05）， while the mRNA and protein expression of Smad 7 was WF-0099） increased significantly （P＜0.05）；the mRNA and proteinexpressions of TGF-β1，TβR-Ⅰ，TβR-Ⅱ and Smad 3 were decreased significantly in positive control group and matrine high concentration group （P＜0.05）. Compared with matrine medium concentration group ，all above indexes were improved significantly in matrine high concentration group （P＜0.05）. CONCLUSIONS ：Matrine can suppress the proliferation and collagen synthesis of CFSC- 8B cells activated by acetaldehyde ，with a centain concentrlation dependence ，the mechanism of which may be associated with regulating the conduction of TGFβ/Smad signal pathway.

Mechanism of Ethanol Extracts from Sophorae Tonkinensis Radix et Rhizoma on Hypertrophic Scars of Rabbit Ears Through TGF-β1/Smad Pathway / 中国实验方剂学杂志

Objective:To investigate that the effect of ethanol extracts from Sophorae Tonkinensis Radix et Rhizoma on the expression of transforming growth factor-β1 (TGF-β1)and Smad3 in the hypertrophic scars of rabbit ears and elucidate its mechanism to improve hypertrophic scars. Method:The model of hypertrophic ear scar model was established by damaging the inner skin of ears in New Zealand white rabbits.The 49 rabbits were randomly divided into control group, model group, low, medium and high-dose ethanol extracts groups from Sophorae Tonkinensis Radix et Rhizoma (0.4，1.0，2.0 g·kg-1), asiaticoside ointment group(5 mg·kg-1) and compound heparin sodium allantoin gel group(20 mg·kg-1), 7 rabbits per group. Except control group, the different drug about 0.5 mL had been applied the hypertrophic scar of rabbit ears once a day. After 42 days, the tissues of hypertrophic scar were obtained. Hematoxylin-eosin（HE）staining was used to observe the pathological changes of rabbit ear scar tissue and determine the scar hyperplasia index. The expression of TGF-β1 and Smad3 in scar tissue of rabbit ears were detected by immunohistochemistry, Western blot and reverse transcription PCR（RT-PCR）. Result:Compared with control group, the pathological changes of the ear scars in the model group showed obvious hyperplasia and higher hyperplasia index (P<0.01). Meanwhile, the expressions of TGF-β1 and Smad3 in scar tissue of rabbit ears were significantly increased (P<0.01). Compared with model group, the pathological structures of the ear scar tissue were significantly improved and the hyperplasia index of ear scar tissue was clearly reduced in medium and high-dose groups of ethanol extracts from Sophorae Tonkinensis Radix et Rhizoma(P<0.05，P<0.01). The protein and mRNA expression of TGF-β1 and Smad3 in scar tissue were also decreased in different group of ethanol extracts from Sophorae Tonkinensis Radix et Rhizoma compared with the model group (P<0.05，P<0.01). Conclusions:Ethanol extracts from Sophorae Tonkinensis Radix et Rhizoma may play a curative role in inhibiting hypertrophic scars by reducing the expression of TGF-β1 and Smad3 in scar tissue and inhibiting the TGF-β1/Smads signal transduction pathway. These provides the experimental basis for the clinical application of Sophorae Tonkinensis Radix et Rhizoma in the treatment of hypertrophic scars.

Study on neuroprotective mechanism of salidroside after ischemic stroke based on TGF-β1/Smad3 signaling pathway / 中草药

Objective: To investigate the neuroprotective mechanism of salidroside after ischemic stroke and its regulation mechanism in TGF-β1/Smad3 signaling pathway. Methods: A total of 48 SPF SD male rats aged 12-15 weeks were randomly divided into four groups (n = 12): Sham-operated group (sham group), model group, salidroside group (treatment group), and signaling pathway-enhanced intervention group (TGF-β1 group). In the model group, treatment group, and TGF-β1 group, a permanent focal cerebral ischemia rat model was established by suture method, and the sham group was not inserted with nylon thread. 48 h before the modeling operation, the treatment group and the TGF-β1 group were given drug intervention at a fixed time every morning: the treatment group was administered with 10 mg/kg salidroside ventricle, and the TGF-β1 group was treated with 20 mg/kg TGF-β1. The intraventricular injection was administered, and the sham group and the model group were given an equal volume of physiological saline. After 14 d of continuous administration, each group of rats was sacrificed by decapitation. TTC staining, Nissl staining, TUNEL staining, immunofluorescence, and Western blot were used to determine the infarct volume, the number of intact neurons, the cell apoptosis, the expression of Bax and Bcl-2 expression, the expression levels of Bax, Bcl-2, TGF-β1, and p-Smad3. The ultrastructural changes of brain tissue were observed by electron microscopy. Results: Compared with the sham group, the cerebral infarction volume of the model group was significantly increased, the number of intact neurons in the brain tissue was significantly reduced, the apoptosis rate of nerve cells was significantly increased, and the expression of Bax was significantly increased, the expression of Bax was significantly decreased. The expression of TGF-β1 and p-Smad3 was significantly increased, and the difference was statistically significant (P 0.05). Conclusion: Salidroside can activate the TGF-β1/Smad3 signaling pathway after ischemic stroke, thereby alleviating neurological damage and exerting protective effects on nerve cells.

Metformin inhibits collagen production in rat biliary fibroblasts: the molecular signaling mechanism / 南方医科大学学报

OBJECTIVE@#To clarify the molecular signaling mechanism underlying the inhibitory effect of metformin on transforming growth factor-β1 (TGF-β1)-stimulated collagen I production in rat biliary fibroblasts.@*METHODS@#Primary biliary fibroblasts were isolated under aseptic condition from 50 Sprague-Dawley rats (half male and half female), and microscopic observation identified no obvious difference in the morphology or viability of the cells from rats with different sexes or body weight. The cells were treated with TGF-β1 (10 ng/mL), Smad3 siRNA+TGF-β1, CTGF siRNA+TGF-β1, metformin (10 mmol/L)+ TGF-β1, or Compound C (10 μmol/L)+metformin+TGF-β1. The expressions of CTGF and collagen I in the treated cells were determined using ELISA kit or Western blotting; the phorsphorylated and total Smad3 and AMPK expressions were detected using immunoblotting.@*RESULTS@#TGF-β1 time- and dose-dependently induced collagen I production in rat biliary fibroblasts. The activated AMPK by metformin dose-dependently inhibited TGF-β1-induced collagen I production. Pre-incubation of cells with the AMPK inhibitor Compound C restored the inhibitory effect of AMPK on TGF-β1-induced collagen I secretion ( < 0.01). Activation of AMPK by metformin significantly reduced TGF-β1-induced collagen I production by suppressing Smad3-driven CTGF expression ( < 0.01), and the application of Compound C reversed such changes in the fibroblasts ( < 0.01).@*CONCLUSIONS@#Metformin inhibits TGF-β1-stimulated collagen I production by activating AMPK and inhibiting Smad3- driven CTGF expression in rat biliary fibroblasts.