Role of TGF-β/Smad signaling pathway in diabetic kidney disease and research progress of traditional Chinese medicine intervention / 中国中药杂志

Diabetic kidney disease is an important microvascular complication of diabetes and the leading cause of end-stage renal disease. Its pathological characteristics mainly include epithelial mesenchymal transition(EMT) in glomerulus, podocyte apoptosis and autophagy, and damage of glomerular filtration barrier. Transforming growth factor-β(TGF-β)/Smad signaling pathway is specifically regulated by a variety of mechanisms, and is a classic pathway involved in physiological activities such as apoptosis, proliferation and differentiation. At present, many studies have found that TGF-β/Smad signaling pathway plays a key role in the pathogenesis of diabetic kidney disease. Traditional Chinese medicine has significant advantages in the treatment of diabetic kidney disease for its multi-component, multi-target and multi-pathway characteristics, and some traditional Chinese medicine extracts, traditional Chinese medicines and traditional Chinese medicine compound prescription improve the renal injury of diabetic kidney disease by regulating TGF-β/Smad signaling pathway. This study clarified the mechanism of TGF-β/Smad signaling pathway in diabetic kidney disease by expounding the relationship between the key targets of the pathway and diabetic kidney disease, and summarized the research progress of traditional Chinese medicine in the treatment of diabetic kidney disease by interfering with TGF-β/Smad signaling pathway in recent years, to provide reference for drug research and clinical treatment of diabetic kidney disease in the future.

Amygdalin Ameliorates Liver Fibrosis through Inhibiting Activation of TGF-β/Smad Signaling / 中国结合医学杂志

OBJECTIVE@#To observe the effect of amygdalin on liver fibrosis in a liver fibrosis mouse model, and the underlying mechanisms were partly dissected in vivo and in vitro.@*METHODS@#Thirty-two male mice were randomly divided into 4 groups, including control, model, low- and high-dose amygdalin-treated groups, 8 mice in each group. Except the control group, mice in the other groups were injected intraperitoneally with 10% carbon tetrachloride (CCl4)-olive oil solution 3 times a week for 6 weeks to induce liver fibrosis. At the first 3 weeks, amygdalin (1.35 and 2.7 mg/kg body weight) were administered by gavage once a day. Mice in the control group received equal quantities of subcutaneous olive oil and intragastric water from the fourth week. At the end of 6 weeks, liver tissue samples were harvested to detect the content of hydroxyproline (Hyp). Hematoxylin and eosin and Sirius red staining were used to observe the inflammation and fibrosis of liver tissue. The expressions of collagen I (Col-I), alpha-smooth muscle actin (α-SMA), CD31 and transforming growth factor β (TGF-β)/Smad signaling pathway were observed by immunohistochemistry, quantitative real-time polymerase chain reaction and Western blot, respectively. The activation models of hepatic stellate cells, JS-1 and LX-2 cells induced by TGF-β1 were used in vitro with or without different concentrations of amygdalin (0.1, 1, 10 µmol/L). LSECs. The effect of different concentrations of amygdalin on the expressions of liver sinusoidal endothelial cells (LSECs) dedifferentiation markers CD31 and CD44 were observed.@*RESULTS@#High-dose of amygdalin significantly reduced the Hyp content and percentage of collagen positive area, and decreased the mRNA and protein expressions of Col-I, α-SMA, CD31 and p-Smad2/3 in liver tissues of mice compared to the model group (P<0.01). Amygdalin down-regulated the expressions of Col-I and α-SMA in JS-1 and LX-2 cells, and TGFβ R1, TGFβ R2 and p-Smad2/3 in LX-2 cells compared to the model group (P<0.05 or P<0.01). Moreover, 1 and 10 µmol/L amygdalin inhibited the mRNA and protein expressions of CD31 in LSECs and increased CD44 expression compared to the model group (P<0.05 or P<0.01).@*CONCLUSIONS@#Amygdalin can dramatically alleviate liver fibrosis induced by CCl4 in mice and inhibit TGF-β/Smad signaling pathway, consequently suppressing HSCs activation and LSECs dedifferentiation to improve angiogenesis.

Sanwubai San Inhibits TGF-β1 Induced Epithelial Mesenchymal Transition of Human Gastric Cancer SGC-7901 Cells Through TGF-β/ Smad Pathway / 中国实验方剂学杂志

ObjectiveTo study the effect of serum containing Sanwubai San on TGF-β1 induced epithelial mesenchymal transition （EMT） of human gastric cancer SGC-7901 cells and its mechanism in vitro based on transforming growth factor-β/Smad（TGF-β/Smad）signaling pathway. MethodTwenty-eight male SD rats （SPF grade， three months） were randomly divided into blank group and Sanwubai low （0.031 25 g·kg-1·d-1， ig）， medium （0.062 5 g·kg-1·d-1， ig） and high （0.125 g·kg-1·d-1， ig） dose groups， seven in each group. The blank group was given the same volume of ultrapure water （ig）. The gavage was performed once a day for seven consecutive days. The serum containing the drug was taken from the abdominal aorta 45 min after the last administration. Cell counting kit-8 （CCK-8） method was used to detect the effect of serum in Sanwubai San high dose group on the activity of SGC-7901 cells. Changes of cell morphology after treatment with TGF-β1 and serum containing Sanwubai San were observed by microscopy， and the migration rate and invasion rate of the SGC-7901 cells were detected by cell scratch assay and transwell assay， respectively. Western blot was used to detect the expression of E-cadherin， snail， TGF-β1， Smad3， p-Smad3 and Smad7 proteins. ResultCompared with the blank group， 10%， 15% and 20% high-dose Sanwubai San inhibited the activity of SGC-7901 cells in a concentration and time dependent manner. Compared with the conditions in the blank group， the cells in the model group lost spindle shape， and most cells became round and long. Compared with the model group， the Sanwubai San groups had decreased pseudopodia and small cells with the morphology returning to normal. Compared with the conditions in the blank group， enhanced ability of cell migration and invasion （P<0.01）， lowered expression of E-cadherin and Smad7 （P<0.01）， and increased expression of Snail， p-Smad3 and TGF-β1 （P<0.01） were found in the model group， with the total protein level of Smad3 remaining unchanged. Compared with the conditions in the model group， the cell migration ability was inhibited in the Sanwubai San high and medium dose groups （P<0.01） after 24 h， and the ability was inhibited in all three Sanwubai San groups after 48 h （P<0.01）， while the invasion ability was enhanced. In addition， the Sanwubai San high and medium dose groups had elevated expression of E-cadherin （P<0.01） and Smad7 （P<0.01）， and decreased expression of Snail （P<0.01）， and the expression of TGF-β1 and p-Smad3 was down-regulated in the three Sanwubai San groups （P<0.01）. ConclusionSanwubai San could inhibit TGF-β1 induced EMT in SGC-7901 cells， and its mechanism might be related to the regulation of TGF-β/Smad signaling pathway.

Functions of key genes involved in TGF-β/Smad signaling pathway in progression of pulmonary fibrosis / 环境与职业医学

Background Although transforming growth factor-β (TGF-β)/Smad signaling pathway is important in regulating the occurrence and development of pulmonary fibrosis, the pathogenesis of pulmonary fibrosis remains elusive. Objective To explore the functions of genes associated with TGF-β/Smad signaling pathway in the progression of pulmonary fibrosis. Methods A NIH-3T3 fibroblast model induced by TGF-β1 was established. The experiment samples were divided into a control group and a TGF-β1 treatment group. The control group was exposed to normal saline, while the TGF-β1 treatment group was exposed to 10 ng·mL−1 TGF-β1 for 12 h. The RNAs of the two groups were extracted, sequenced, and analyzed by bioinformatics methods to identify seven key genes in TGF-β pathway, including Dcn, Smad3, Smad7, Fbn1, Thbs1, TGF-β1, and TGF-β3. The gene expression levels of five markers [Collagen1α1, Collagen1α2, α-smooth muscle actin (α-SMA), TGF-β1, and TGF-β3] and the seven key genes were detected by quantitative real-time PCR (qRT-PCR). The proteins of the two groups were extracted. The important marker protein expression levels of Smad3, the phosphorylation of Smad3 (P-Smad3), and α-SMA were detected by Western blotting. At the same time, 30 healthy SPF-grade C57BL/6 mice were randomly divided into three groups, with 10 mice in each group: a control group, a SiO2 inhalation exposure group for 28 d (10 mice), and a SiO2 inhalation exposure group for 56 d (10 mice). The mice in the two treatment groups were exposed to a natural SiO2 environment for 4 h per day with a 10-min pause for breathing fresh air at 2 h intervals. The lung tissues of the mice were taken after execution. The changes of pulmonary fibrosis were detected by Masson staining, and mRNAs and proteins were extracted to detect the expression of the above key genes and proteins. Results The expression levels of the five marker genes Collagen1α1, Collagen1α2, α-SMA, TGF-β1, and TGF-β3 were significantly increased in the TGF-β1-induced NIH-3T3 fibroblasts than those in the control group (P < 0.01); the expression levels of P-Smad3 and α-SMA proteins increased significantly (P < 0.01); the expression results of the seven key genes screened in the TGF pathway were that Dcn and Smad3 were obviously down-regulated (P < 0.01), and Smad7, Fbn1, Thbs1, TGF-β1, and TGF-β3 were obviously up-regulated (P < 0.01). The changes in gene expression levels of the transcriptome sequencing showed the same trend. The results of Masson staining showed that the content of collagen fibers in the lung tissues also increased in the SiO2 inhalation exposure groups over time. In the mouse experiment, five marker genes were obviously up-regulated compared with the control group (P < 0.01); no obvious change was found in the expression of Smad3 protein, and the expression levels of P-Smad3 and α-SMA were obviously higher in the SiO2 exposure groups than those in the control group (P < 0.01); the expression levels of Dcn and Smad3 showed a down-regulated trend, while the expression levels of Smad7, Fbn1, Thbs1, TGF-β1, and TGF-β3 showed an up-regulated trend with the increase of SiO2 inhalation exposure days (P < 0.01). The expression levels of the above five marker genes, three important marker proteins, and seven key genes were consistent with the expression trends of TGF-β1-induced NIH-3T3 fibroblasts. Conclusion The expression levels of pulmonary fibrosis-related marker genes and proteins change significantly in TGF-β1-induced fibroblast cells, and the lung tissues of mice under natural SiO2 inhalation exposure has obvious fibrosis characteristics. Seven genes (Dcn, Smad3, Smad7, Fbn1, Thbs1, TGF-β1, and TGF-β3) may be involved in the regulation of pulmonary fibrosis by the TGF-β/Smad signaling pathway.

Study on the Effect and Mechanism of Total Flavonoids from Marchantia convoluta on the Anti-hepatic Fibrosis in Mice / 中国药房

OBJECTIVE：To study the effect and potential mechanism of the total flavonoids from Marchantia convoluta on anti-hepatic fibrosis in the mice. METHODS ：Seventy-two mice were randomly divided into blank group ，model group ，positive control group （colchicine 0.2 mg/kg）and M. convoluta total flavonoids high-dose ，medium-dose and low-dose groups （300，150， 75 mg/kg），with 12 mice in eac group. Except for blank group ，other groups were subcutaneously given 25％ CCl4-peanut oil solution on the back to induce liver fibrosis model. At the same time ，blank group and model group were given water intragastrically，while other groups were given relevant medicine intragastrically 20 mL/kg，once a day ，for consecutive 10 weeks. After last administration ，the serum levels of ALT and AST were detected . Histopathological changes of liver tissue in mice was observed. The levels of COL- Ⅰ，COL-Ⅲ and TGF-β1 in liver tissue were detected . The protein expression levels of α-SMA and TGF-β1，Smad2，Smad4 and Smad 7 in liver tissue were detected . The expression levels of TGF-β1，Smad2，Smad4 and Smad 7 mRNA in liver tissue were detected . RESULTS ：Compared with blank group ，the serum levels of ALT and AST in model group，the levels of COL- Ⅰ，COL-Ⅲ and TGF-β1 in liver tissue，protein expression levels of α-SMA，TGF-β1，Smad2 and Smad 4，mRNA expression levels of TGF-β1，Smad2 and Smad4 were increased significantly （P＜0.05 or P＜0.01）.The mRNA and protein expression levels of Smad 7 in liver tis sue were decreased significantly （P＜0.05）. The degree of liver tissue injury and collagen fiber hyperplasia were serious. Compared with model group ，above indexes of mice were reversed significantly in positive control group and M. convoluta total flavonoids high-dose group （P＜0.05 or P＜0.01）. Serum level of ALT ，the levels of COL- Ⅰ，mRNA and protein expression of TGF-β1，Smad2 and Smad 4 in liver tissue were decreased significantly in M. convoluta total flavonoids medium-dose group （P＜0.05 or P＜0.01）. Protein expression of Smad 2 and Smad 4 in liver tissue were decreased significantly in M. convoluta total flavonoids low-dose group （P＜0.05）. The liver injury and fibrosis of mice were relieved in administration groups. CONCLUSIONS ：M. convoluta total flavonoids possess the effect of anti-hepatic fibrosis ，the mechanism of which is related to the regulation of mRNA and protein expression of TGF-β1，Smad2，Smad4 and Smad 7 in the signaling pathway of TGF-β/Smad.

Study on the Protective Effects of Schisandrin A on Hepatic Fibrosis Induced by Carbon Tetrachloride in Mice and Its Mechanism / 中国药房

OBJECTIVE：To study the pr otective effect of schisandrin A （SA）on CCl 4-induced liver fibrosis model mice and its mechanism. METHODS ：Mice were randomly divided into blank control group ，model group ，silymarin group （positive control，100 mg/kg），SA low-dose and high-dose groups （20，40 mg/kg），with 10 mice in each group. Except for blank control group，other groups were given CCl 4 subcutaneously to induce liver fibrosis model. After successful modeling ，administration groups were given relevant medicine intragastrically ，once a day ，for consecutive 6 weeks；blank control group and model group were given constant volume of 0.5％sodium carboxymethyl cellulose solution intragastrically by the same way. HE staining was used to observe the pathological changes of liver tissue in mice. UV spectrophotometry and ELISA assay were adopted to detect the serum levels of liver injury indexes （ALT and AST ）and the contents of inflammatory factors （TNF-α，IL-1β，IL-6）. Western blotting assay was used to detect the expression of NOD like receptor protein 3（NLRP3）/NF-κB and TGF-β/Smad signaling pathway protein. RESULTS ：Compared with blank control group ，obvious pathological changes of liver fibrosis were observed in model group. The serum levels of liver injury indexes and contents of inflammatory factors were significantly increased （P＜0.01）. The expression of NLRP 3，apoptosis associated spot-like protein ，Caspase-1 and IL- 1β，TGF-β1 and ratios ofp-NF-κB p65/NF-κB p65，p-IκBα/IκBα，p-Samd3/Smad3 were increased significantly （P＜0.01）. Compared with model group ，SA could significantly relieve hepatic fibrosis in mice ，reduce serum levels of liver injury indexes and contents of inflammatory factors ，as well as the expression of NLRP 3/NF-κB and TGF-β/Smad signaling pathway protein and phosphorylation level（P＜0.01）. CONCLUSIONS ： SA can effectively relieve liver injury and inflammation of CCl 4-induced hepatic fibrosis model mice ，which may be through the regulation of NLRP 3/NF-κB and TGF-β/Smad3 signaling pathways ，thus inhibiting the process of liver fibrosis.

Recent advances in signaling pathways related to choroidal neovascularization / 国际眼科杂志(Guoji Yanke Zazhi)

@#Choroid neovascularization is the characteristic pathological change of many fundus diseases and is the most common cause for severe vision loss and metamorphopsia. Among the pathogenic factors, VEGF is considered to be the most important and treatment targeting VEGF showed promising results. However, anti-VEGF agents need to be administrated frequently and they are usually expensive. Also, some patients got no response to this treatment. These facts force us to find other pathway that involves in the formation of CNV. This article reviews the latest research on CNV-related signaling pathways so as to provide a deeper look into CNV and hopefully point out new directions for treating diseases that share similar pathogenesis.

Pirfenidone inhibits TGF-β/Smad signaling pathway to alleviate hepatic cirrhosis induced by carbon tetrachloride / 中国免疫学杂志

Objective: This research aimed to explore the therapeutic effect and its mechanism of pirfenidone in liver cirrhosis induced by carbon tetrachloride in mice. Methods: Sixty male C57 BL/6 mice were randomly divided into the control group, model group and different doses of pirfenidone group, twelve rats in each group. Mice were intraperitoneally injected with 20％ CCl4 soybean oil solution ( 5 ml/kg), twice a week for 7 weeks. And these mice were free to drink 20％ ethanol solution in the third week after building the model. The low, medium and high dose groups were respectively given 50, 100 and 200 mg/kg of pirfenidone solution according to the body weights, while the model group and control group were given equal volume of blank solvent after building the model, once a day for 2 weeks. The serum level of ALT and AST, liver index, spleen index, the gene or protein expression level of TGF-β1 and Smad3 were analyzed before and after the treatment of pirfenidone. Results: The serum level of ALT, AST increased significantly in the model group ( P＜0. 05), while decreased significantly in different doses of pirfenidone group ( P＜0. 05). The liver and spleen index in the model group was significantly higher than that in the control group ( P＜0. 05). However, after treating with pirfenidone, the liver and spleen index were significantly lower than that in the model group ( P＜0. 05). The number of TGF-β1 positive cells in the model group was significantly more than that in the control group, but it was significantly decreased in the pirfenidone group. The gene expression level of Smad3 in the model group was significantly higher than that in the control group ( P＜0. 05). The gene expression level of TGF-β1 and Smad3 in different doses of pirfenidone group were significantly lower than that in the model group ( P＜ 0. 05). Meanwhile, the protein level of TGF-β1 and Smad3 were significantly increased in the model group, while decreased in the pirfenidone group. Conclusion: Pirfenidone relieves liver cirrhosis caused by carbon tetrachloride in mice by inhibiting the TGF-β1/Smad3 signaling pathway.

Effects of piceatannol on rat kidney with diabetic nephropathy in early stage / 中国病理生理杂志

AIM: To observe the effect of piceatannol on the kidney of diabetic nephropathy rats in early stage, and to explore the possible mechanisms.METHODS: The rats were randomly divided into 5 groups: control group, model group, low dose of piceatannol treatment group, medium dose of piceatannol treatment group and high dose of piceatannol treatment group.The rat model of diabetic nephropathy was induced accordingly, and the rats received 20 mg/kg, 40 mg/kg or 60 mg/kg of piceatannol by gavage once a day for 4 weeks.Blood glucose was detected by glucometer.The urea nitrogen and creatinine levels in the serum were measured by urease-glutamate dehydrogenase enzymatic and inosine acid oxidase methods, respectively, and 24 h urinary microalbumin was analyzed by immune transmission turbidimetry test.Moreover, the pathological changes of the kidney tissues were observed under microscope with HE staining.The protein expression of TGF-β1 and Smad 7 and the phosphorylation levels of Smad2 and Smad3 were determined by Western blot.RESULTS: Compared with model group, piceatannol treatment significantly decreased the levels of blood glucose, blood urea nitrogen and urinary microalbumin, but had no effects on serum creatinine.Furthermore, HE staining showed that the increased mesangial cells, matrix hyperplasia and degenerated epithelial cells in model group were markedly inhibited after piceatannol treatment.Additionally, piceatannol treatment also reduced the protein expression of TGF-β1 and Smad 7, and the phosphorylation levels of Smad2 and Smad3.CONCLUSION: Piceatannol attenuates pathological progression in the kidney of diabetic nephropathy rats in early stage, which may be through inhibiting TGF-β/Smad signaling pathway.

IL-27 Alleviates the Bleomycin-induced Pulmonary Fibrosis through Regulating TGF-β/Smad Signaling Pathway / 昆明医科大学学报

Objective To explore whether IL-27 inhibited the pulmonary fibrosis through regulating the expression of TGF-β/Smad signaling pathway in the bleomycin-induced pulmonary fibrosis model. Methods Forty male C57/BL6 mice were randomly divided into normal control group(group A),bleomycin-induced pulmonary fibrosis group(group B),bleomycin+IL-27 group(group C)and bleomycin+IL-27 antibody group(group D) with 10 in each. Five mice in each group were sacrificed on days 7 and 28 after with intratracheal bleomycin. TGF-βR1,Smad1 and Smad3 in right lung tissue were measured by Western Blot. Results 1. In the bleomycin-induced pulmonary fibrosis model,the expression of TGF-βR1 was higher on days 7 and 28,which was inhibited by IL-27. 2. The expressions of p-Smad1 and p-Smad3 were highest in group D on days 7 and 28, but were lower in group C on day 7 than those in group B. Conclusion Exogenous IL-27 might alleviate pulmonary fibrosis through inhibiting the related protein phosphorylation in TGF-β/Smad signaling pathway.

Validation of the Effect of Modified Maxingshigan Decoction on the TGF-β/Smad Signaling Pathway in Radioactive Lung Injury / 浙江中医药大学学报

Objective] To prove the modified Maxingshigan Decoction(modified MXSGD) intervening radiation induced lung injury(RILI) through the TGF-β/Smad signaling pathway by selectively silencing the TGF-β1 gene. [Methods] (1)Total of 18 clean SD rats were randomly divided into two groups:Chinese medicine group treated with modified MXSGD and normal control group treated with saline. The serum was made after 3 days of taking drugs to prepare medicated serum. (2) The alveolar typeⅡcells were cultured. The total dose of radiation to cell was 8Gy, frequency 3.64Gy/min. The medicated serum was given to each group respectively. (3) After silencing TGF-β1 gene with RNA interference technology, the level of TGF-β1, PAI-1, CTGFmRNA was analyzed by PCR and the protein expression of Smads was measured by Western-blot. [Results] (1) Compared with rat serum group, the expressions of TGF-β1 mRNA, P-Smad2 in 10%medicated serum group were down-regulation or deceased(P0.05). (4) Compared with rat serum+siRNA, the expression of Smad6 and Smad7 was increased in 10%medicated serum+siRNA group. The differences between the two groups were significant(P<0.05, P<0.01). [Conclusion] Modified MXSGD may intervene the RILI by regulating TGF-β/Smad signaling pathway.