Mechanism of Youguiwan Regulating BMP-2/Smad Signaling Pathway to Promote Bone Formation in Postmenopausal Rats with Osteoporosis / 中国实验方剂学杂志

ObjectiveTo observe the effects of Youguiwan on bone metabolism and bone morphogenetic protein-2 （BMP-2）/Smad signaling pathway in ovaries-removed rats with osteoporosis and study the mechanism of Youguiwan in the prevention and treatment of osteoporosis. MethodA postmenopausal rat model of osteoporosis was prepared by bilateral ovariectomy. The 40 female SD rats were randomly divided into five groups， including sham operation group， model group， alendronate sodium group （0.1 mg·kg-1）， and high-dose and low-dose （5.36 and 2.68 g·kg-1） groups of Youguiwan. The drug was given seven days after modeling， once a day for 12 weeks. After the treatment， the changes in femur tissue structure were observed by micro-CT， including bone mineral density （BMD）， bone volume/total volume （BV/TV）， trabecular number （Tb.N）， trabecular thickness （Tb.Th）， bone surface/bone volume （BS/BV）， and trabecular separation （Tb.Sp）. Ossification was observed by saffrane-solid green staining， and serum levels of bone metabolism markers， including bone alkaline phosphatase （BALP）， osteocalcin （BGP）， type Ⅰ procollagen amino terminal propeptide （PINP）， and tartrate-resistant acid phosphatase 5b （TRACP-5b）， were determined by enzyme-linked immunosorbent assay （ELISA）. The protein and mRNA expression levels of Runx2， BMP-2， and Smad1 in rat femur were detected by Western blot and Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. ResultCompared with the sham operation group， bone trabecula in the model group was sparse. BMD， BV/TV， Tb.N， and Tb.Th were decreased （P<0.05， P<0.01）. BS/BV （P<0.05） and Tb.Sp were increased. The content of BGP， BALP， PINP， and TRACP-5b in serum was significantly increased （P<0.01）. The mRNA and protein expressions of Runx2， BMP-2， and Smad1 in rat femur were significantly decreased （P<0.05， P<0.01）. Compared with the model group， the number of bone trabeculae in the high-dose and low-dose groups of Youguiwan was increased， and the bone microstructure was improved. BMD， BV/TV， Tb.N， and Tb.Th were increased significantly （P<0.05， P<0.01）， and BS/BV and Tb.Sp were increased. The content of bone metabolic markers decreased （P<0.05， P<0.01）. ConclusionYouguiwan has certain preventive and therapeutic effects on postmenopausal osteoporosis， and its mechanism may be related to promoting bone formation by regulating the BMP-2/Smad signaling pathway.

Regulation mechanism of macrophage transition in renal fibrosis / 器官移植

Renal fibrosis is a common pathological change from development to end-stage renal diseases in all progressive chronic kidney diseases. Renal fibrosis after kidney transplantation will severely affect the renal graft function. Macrophages are characterized with high heterogeneity and plasticity. During the process of kidney injury, macrophages are recruited, activated and polarized by local microenvironment, and participate in the process of renal tissue injury, repair and fibrosis through multiple mechanisms. Recent studies have shown that macrophages may transit into myofibroblasts and directly participate in the formation of renal fibrosis. This process is known as macrophage-myofibroblast transition. Nevertheless, the regulatory mechanism remains elusive. In this article, the role of macrophages in renal fibrosis, the characteristics of macrophage-myofibroblast transition and the possible regulatory mechanism were reviewed, aiming to provide reference for relevant research of renal fibrosis.

Mechanism of HMGB1 in scarring after glaucoma drainage valve implantation / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To explore the dynamic expression of high mobility group box 1(HMGB1)in scar tissues after glaucoma drainage valve implantation, and to further reveal the role and possible mechanism of HMGB1 in scarring after glaucoma surgery.METHODS: A total of 60 New Zealand white rabbits were randomly divided into control group(n=20), model group(n=20, silicone implantation under conjunctival sac)and model with drug administration group(n=20, silicone implantation under conjunctival sac combined with 5-fluorouracil injection). The conjunctival tissues were collected at 4 and 8 wk after surgery. HE staining and Masson staining were used to detect the proliferation and distribution of fibroblasts and collagen fibers in conjunctival tissues. Immunohistochemistry was utilized to detect the distribution and changes of HMGB1, transforming growth factor(TGF)-β1, Smad3 and α-smooth muscle actin(SMA)in conjunctival tissues. RT-PCR and Western blot were adopted to detect the mRNA and protein expression of HMGB1, TGF-β1, Smad3 and α-SMA in conjunctival tissues.RESULTS: HE staining and Masson staining showed that the proliferation of inflammatory cells, fibroblasts and collagen fibers in the model group was significantly higher than that in the control group at both 4 and 8 wk. Meanwhile, the proliferation of fibroblasts and collagen fibers in the model with drug administration group was significantly lower than that in the model group. Immunohistochemical staining showed that the expression of HMGB1, TGF-β1, Smad3 and α-SMA protein was observed in the conjunctival tissues of the model group both 4 and 8 wk, with brown and significantly deeper staining of the model group at 8 wk. Meanwhile, the positive staining in the model with drug administration group at both 4 and 8 wk was significantly lower than that in the model group. There was positive correlations between the number of fibroblasts stained with HE and the expression of HMGB1 in the conjunctival tissue of the model group at both 4 and 8 wk(r=0.602, 0.703, all P&#x0026;#x003C;0.05). RT-PCR and Western blot revealed that the mRNA and protein expression levels of HMGB1, TGF-β1, Smad3 and α-SMA in the model group were significantly higher than those in the control group at both 4 and 8 wk(all P&#x0026;#x003C;0.05). Meanwhile, the mRNA and protein expression levels of HMGB1, TGF-β1, Smad3 and α-SMA in the model with drug administration group were significantly lower than those in the model group(all P&#x0026;#x003C;0.05). There was positive correlations between mRNA expressions of HMGB1 and TGF-β1, Smad3 in the model group and the model with drug administration group(all P&#x0026;#x003C;0.05).CONCLUSION: The expression of HMGB1 increased at a time-dependent manner after glaucoma valve implantation. HMGB1 acts an indispensable role in the initiation and progression of scar formation after glaucoma surgery, which may be involved in the regulation of TGF-β/Smad signaling pathway.

Recombinant Schistosoma japonicum egg ribonuclease SjCP1412 inhibits the activation of LX-2 hepatic stellate cells in vitro / 中国血吸虫病防治杂志

Objective To investigate the effect of recombinant Schistosoma japonicum egg ribonuclease SjCP1412 (rSjCP1412) on proliferation, cell cycle, apoptosis and activation of human hepatic stellate cells LX-2 in vitro, and explore the underlying mechanisms. Methods The rSjCP1412 protein was expressed in Escherichia coli BL21 by prokaryotic expression, and the highly purified soluble rSjCP1412 protein was prepared by Ni NTA affinity chromatography and urea gradient refolding dialysis. Yeast RNA was digested using 12.5, 25.0, 50.0 µg rSjCP1412 proteins at 37 °C for 2, 3, 4 h, and the enzymatic products were electrophoresed on 1.5% agarose gel to observe the RNAase activity of rSjCP1412 protein. The proliferation of LX-2 cells stimulated by different doses of rSjCP1412 protein for 48 hours was measured using CCK-8 assay, and the apoptosis of LX-2 cells stimulated by different doses of rSjCP1412 protein for 48 hours was detected using the Annexin V-FITC/PI double staining, while the percentage of LX-2 cells at G0/G1, S and G2/M phases of cell cycle following stimulation with different doses of rSjCP1412 protein for 48 h was detected by DAPI staining. The type I collagen, type III collagen and α-smooth muscle actin (α-SMA) mRNA expression was quantified using quantitative florescent real-time PCR (qPCR) assay and Western blotting at transcriptional and translational levels in LX-2 cells following stimulation with different doses of rSjCP1412 protein for 48 h, while soluble egg antigen (SEA) served a positive control and PBS without rSjCP1412 protein as a normal control in the above experiments. The expression of collagen I, α-SMA and Smad4 protein was determined using Western blotting in LX-2 cells following stimulation with rSjCP1412 protein, transforming growth factor-β1 (TGF-β1) alone or in combination, to examine the signaling for the effect of rSjCP1412 protein on LX-2 cells. Results The rSjCP1412 protein was successfully expressed and the highly purified soluble rSjCP1412 protein was prepared, which had a RNase activity. Compared with the normal group, the survival rates of LX-2 cells significantly decreased post-treatment with 12.5, 25.0, 50.0 µg/mL rSjCP1412 protein and SEA for 48 h (F = 22.417 and 20.448, both P values < 0.05). The apoptotic rates of LX-2 cells significantly increased post-treatment with 12.5, 25.0, 50.0 µg/mL rSjCP1412 protein for 48 h (F = 11.350, P < 0.05), and treatment with 12.5, 25.0, 50.0 µg/mL rSjCP1412 protein for 48 h resulted in arrest of LX-2 cells in G0/G1 phase (F = 20.710, P < 0.05). Treatment with 12.5, 25.0, 50.0 µg/mL rSjCP1412 protein for 48 h caused a significant reduction in relative expression levels of collagen I (F = 11.340, P < 0.05), collagen III (F = 456.600, P < 0.05) and α-SMA mRNA (F = 23.100, P < 0.05) in LX-2 cells, and both rSjCP1412 protein and SEA treatment caused a significant reduction in collagen I (F = 1 302.000, P < 0.05), α-SMA (F = 49.750, P < 0.05) and Smad4 protein expression (F = 52.420, P < 0.05) in LX-2 cells. In addition, rSjCP1412 protein treatment inhibited collagen I (F = 66.290, P < 0.05), α-SMA (F = 31.300, P < 0.05) and Smad4 protein expression (F = 27.010, P < 0.05) in LX-2 cells activated by TGF-β1. Conclusion rSjCP1412 protein may induce apoptosis of LX-2 cells and inhibit proliferation, cell cycle and activation of LX-2 cells through down-regulating Smad4 signaling molecules.

The relationship between angiopoietin 1 and Smadhomolog 9 gene expression in cancer tissues and tumor metastasis, invasion behavior and prognosis in patients with lung adenocarcinoma / 中国医师进修杂志

Objective:To explore the relationship between the expression of angiopoietin 1 (ANGPT1) and Smadhomolog 9 (Smad9) genes in cancer tissues and tumor metastasis, invasion behavior and prognosis in patients with lung adenocarcinoma.Methods:Sixty patients with lung adenocarcinoma in Chengwu Hospital Affiliated to Shandong First Medical University from October 2018 to December 2019 were selected as the research objects. The expressions of ANGPT1 and Smad9 mRNA in cancer tissues and adjacent tissues were compared, as well as the expressions of ANGPT1 and Smad9 mRNA in cancer tissues of patients with different tumor metastasis and invasion behaviors. The relationship between ANGPT1 and Smad9 mRNA expression and tumor metastasis and invasion behavior of lung adenocarcinoma were analyzed, and the 1-year survival rate of patients with lung adenocarcinoma was calculated. The 1-year survival rate of patients with different ANGPT1 and SMAD9 mRNA expression levels were compared.Results:The relative expression of ANGPT1 mRNA and Smad9 mRNA in cancer tissues were lower than those in adjacent tissues: 2.45 ± 0.26 vs. 11.18 ± 0.93, 4.23 ± 0.31 vs. 7.58 ± 0.65, the differences were statistically significant ( P<0.05). There were significant differences in the relative gene expression of ANGPT1 and Smad9 mRNA in different clinical stages, tumor diameter, degree of differentiation, lymph node metastasis and pleural invasion ( P<0.05). Spearman correlation analysis showed that the expression of ANGPT1 and Smad9 mRNA were negatively correlated with clinical stage, tumor diameter, lymph node metastasis and pleural invasion ( r = - 0.517, - 0.539, - 0.606, - 0.679, P<0.05), and positively correlated with the degree of differentiation ( r = 0.628, P<0.05). The 1-year survival rate of 58 patients was 72.41%. Kaplan-Meier curve analysis showed that the 1-year survival rate of patients with low expression of ANGPT1 and Smad9 mRNA in cancer tissues were were lower than those in patients with high expression ( P<0.05). Conclusions:Down-regulation of ANGPT1 and Smad9 genes in cancer tissues will accelerate the metastasis and invasion behavior of lung adenocarcinoma. Up-regulating the expression of both genes can be a potential way to improve survival.

Protective mechanism of Tangshenbao on kidney of diabetic nephropathy rats / 国际中医中药杂志

Objective:To investigate the protective effect and possible mechanism of Tangshenbao on renal damage in diabetic nephropathy (DN) rats.Methods:Totally 36 SPF male SD rats were randomly divided into normal group ( n=6) and model group ( n=30). The DN rat model was prepared by single high-dose intraperitoneal injection of STZ. According to the random number table method, the rats were divided into model group, irbesartan group and Tangshenbao low-, medium- and high-dosage groups, with 6 rats in each group. Drug intervention lasted for 8 weeks. The general condition and body weight of rats in each group were recorded. The blood glucose, kidney index, 24 h urine protein (24 h UTP), SCr and BUN levels were detected. The pathological morphology of renal tissue was observed by PAS staining and transmission electron microscopy. The mRNA and protein expressions of Ets-1, TGF-β1, Smad2 and Smad3 in renal tissue were detected by real-time fluorescence quantitative PCR and Western blot. Results:Compared with model group, the body weight of Tangshenbao low, medium and high dose groups and irbesartan group significantly increased ( P<0.01). The kidney index decreased ( P<0.05 or P<0.01). The contents of 24 hUTP, BUN and SCr significantly decreased ( P<0.05 or P<0.01). Glomerular volume was significantly reduced ( P<0.05 or P<0.01), the mRNA expressions of Ets-1 (1.59 ± 0.06, 1.47 ± 0.04, 1.31 ± 0.03, 1.39 ± 0.03 vs. 1.64 ± 0.04), TGF-β1 (1.65 ± 0.05, 1.59 ± 0.03, 1.38 ± 0.05, 1.49 ± 0.04 vs. 1.77 ± 0.08), Smad2 (1.48 ± 0.05,1.39 ± 0.05, 1.22 ± 0.03, 1.31 ± 0.04 vs. 1.54 ± 0.05), Smad3 (1.57 ± 0.04, 1.48 ± 0.03, 1.28 ± 0.03, 1.39 ± 0.02 vs. 1.64 ± 0.05) in renal tissue of rats significantly decreased ( P<0.05 or P<0.01), the protein expressions of Ets-1 (1.33 ± 0.32, 1.16 ± 0.38, 0.77 ± 0.06, 0.84 ± 0.06 vs. 1.97 ± 0.43), TGF-β1 ( 1.35 ± 0.14, 1.24 ± 0.22, 0.94 ± 0.13, 1.07 ± 0.06 vs. 1.63 ± 0.20), Smad2 (1.24 ± 0.26, 1.14 ± 0.31, 0.77 ± 0.28, 0.85 ± 0.19 vs. 1.72 ± 0.34) and Smad3 (1.29 ± 0.14, 1.19 ± 0.21, 0.85 ± 0.39, 0.90 ± 0.37 vs. 1.76 ± 0.21) decreased ( P<0.05 or P<0.01). Conclusion:Tangshenbao can improve renal damage in DN rats, and its mechanism may be related to the inhibition of Ets-1 expression and TGF-β1/Smads signaling pathway.

miR-9500 Regulates Migration and Invasion of Lung Adenocarcinoma Cells by Targeting SMAD2 / 肿瘤防治研究

Objective To explore the molecular mechanism underlying miR-9500 regulating the migration and invasion of lung adenocarcinoma cells by targeting SMAD2. Methods The core target genes of miR-9500 were screened out by bioinformatics analysis, and their GO function analysis, KEGG signaling pathway enrichment, and survival analysis were performed. The targeted binding sites between miR-9500 and SMAD2 were predicted, and the direct targeting relationship between miR-9500 and SMAD2 was verified by dual-luciferase reporter assay. qRT-PCR and Western blot were used to assess the effect of miR-9500 on the mRNA and protein expression levels of SMAD2. Wound healing assay, Transwell assay, and Matrigel invasion assay were used to determine the effect of miR-9500 on the migration and invasion of lung adenocarcinoma cells. Results The core target genes of miR-9500 were mainly enriched in the cancer pathway, TGF-β signaling pathway, and focal adhesion. However, only the expression levels of VAMP2, SMAD2, and RXRA among the top 10 core target genes were significantly correlated with the overall survival of patients with lung adenocarcinoma. miR-9500 targeted SMAD2 and down-regulated the expression levels of SMAD2, and overexpression of miR-9500 significantly inhibited the migration and invasion of lung adenocarcinoma cells and markedly decreased the expression levels of MMP2 and MMP9. Conclusion miR-9500 can inhibit the migration and invasion of lung adenocarcinoma cells by targeting SMAD2, which may play an important role in the tumorigenesis and development of lung adenocarcinoma as a tumor suppressor.

Exploration of Mechanism of Xiaonang Tiaojing Decoction in Treating PCOS-IR Model Rats Based on AMH/AMHR ⅡSignaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of Xiaonang Tiaojing decoction（XNTJD）in improving polycystic ovary syndrome with insulin resistance（PCOS-IR）model rats based on anti-Müllerian hormone（AMH）/AMH type Ⅱ receptor（AMHRⅡ）signaling pathway. MethodForty-eight adult female SD rats were randomly divided into the blank group， model group， XNTJD group（11.4 g·kg-1·d-1） and Diane-35 group（0.21 g·kg-1·d-1）， PCOS-IR model was established by high-fat and high-sugar diet combined with letrozole in rats of all groups except the blank group， rats in the administration groups were given the corresponding dose of drugs by gavage for 15 days with an interval of 1 d every 4 d， normal saline of the same volume was given to the blank group and the model group. Estrous cycle was recorded daily during treatment. At the end of treatment， body weight and Lee's index were recorded， AMH， luteinizing hormone（LH）， LH/follicle stimulating hormone（FSH）， testosterone（T）and estradiol（E2）levels were measured by enzyme-linked immunosorbent assay（ELISA）， fasting plasma glucose（FPG）was measured by glucometer， fasting insulin（FINS） level was measured by radioimmunoassay（RIA）， and the insulin resistance index（HOMA-IR） and insulin sensitivity index（QUICKI）were calculated， triglyceride（TG）and total cholesterol（TC）levels were measured by automatic biochemical analyzer， hematoxylin-eosin（HE）staining was used to observe the morphological changes of the ovary， the levels of AMHRⅡ， bone morphogenetic protein-15（BMP-15）and Smad5 in ovarian tissues were detected by immunohistochemistry（IHC），Western blot was used to analyze the protein expression levels of AMHRⅡ， BMP-15 and Smad5. ResultCompared with the blank group， a large number of leukocytes were observed in the vaginal exfoliated cells of rats in the model group， mainly in diestrus， the levels of body weight， Lee's index， glucose-lipid metabolism indexes（FPG， FINS， HOMA-IR， TG， TC）， AMH and sex hormones（LH， LH/FSH， T）were significantly increased（P<0.01）， and QUICKI and E2 levels were significantly decreased（P<0.01）， there were more cystic bulges on the ovarian surface， more wet weight， more atretic and cystic dilated follicles in the ovarian tissues， and the thickness of granulosa cell layer was reduced without oocytes， the expression level of AMHRⅡ protein in ovarian tissues was significantly increased（P<0.01）， and the expression levels of BMP-15 and Smad5 proteins were significantly decreased（P<0.01）. Compared with the model group， the exfoliated cells in the vagina of rats treated with XNTJD group showed keratinocytes from the 5th to 6th day of treatment， and a stable estrous cycle gradually appeared， body weight， Lee's index， glucose-lipid metabolism indexes（FPG， FINS， HOMA-IR， TG， TC）， AMH and sex hormones（LH， LH/FSH， T）levels were significantly decreased（P<0.05， P<0.01）， QUICKI and E2 levels were significantly increased（P<0.01）， ovarian surface was smoother and lighter in wet weight， oocytes and mature follicles were observed in ovarian tissues， the thickness of granulosa cell layer increased and the morphology was intact， the expression levels of BMP-15 and Smad5 proteins were significantly increased（P<0.01）and the expression level of AMHRⅡ protein was significantly decreased（P<0.01）in ovarian tissues. ConclusionXNTJD may mediate the up-regulation of BMP-15 and Smad5 in ovarian tissues of PCOS-IR rats by down-regulating AMH/AMHRⅡ， thereby improving ovarian function， sex hormones and glucose-lipid metabolism levels in PCOS-IR rats.

Mechanism of Pachymic Acid in Inhibiting Invasion and Metastasis of Renal Carcinoma Cells via Regulating MMP/TIMP Balance by Smads / 中国实验方剂学杂志

ObjectiveTo investigate the effect and mechanism of pachymic acid （PA） in Poria on the invasion and metastasis of renal carcinoma cells. MethodThe effect of PA （0， 20， 40， 80， 160 μmol·L-1） on cell viability was detected by cell counting kit-8（CCK-8）， and the dose of PA was selected for subsequent experiments. The effect of PA （0， 20， 40， 80 μmol·L-1） on cell proliferation was evaluated by colony formation assay. The effect of PA （0， 20， 40， 80 μmol·L-1） on cell adhesion ability was observed by cell adhesion assay. The effect of PA （0， 20， 40， and 80 μmol·L-1） on cell invasion and metastasis was investigated by Wound healing assay and Transwell invasion assay. The inhibitory effect of PA （0， 20， 40， 80 μmol·L-1） on cell motility was further observed and verified by high-content imaging technology. The effects of PA （0， 20， 40， 80 μmol·L-1） on the expression of matrix metalloproteinase （MMP）/tissue inhibitor of metalloproteinasas （TIMP） related to invasion and metastasis and Smads were detected by Western blot. ResultCCK-8 results showed that compared with the blank group， the PA groups showed decreased cell viability（P<0.01）， with the half-maximal inhibitory concentration （IC50） of ACHN cells of 70.42 μmol·L-1 at 24 h. Colony formation assay showed that the number of cell clonal groups in the PA groups was reduced compared with that in the blank group（P<0.01）. Cell adhesion assay showed that compared with the blank group， the PA groups displayed reduced cell adhesion（P<0.01）. Wound healing assay showed that the wound healing rate of cells in the PA groups was lower than that in the blank group （P<0.05，P<0.01）. Transwell invasion assay showed that compared with the blank group， the number of transmembrane cells in PA groups was reduced（P<0.01）. High-content imaging showed that the cumulative migration distance of cells in the PA groups was shorter than that in the blank group（P<0.01）. The results of Western blot showed that the protein expression of MMP-2 and MMP-9 in the PA groups decreased （P<0.01）， and TIMP-1 protein expression increased （P<0.01） compared with those in the blank group. In addition， compared with the blank group， the PA groups showed decreased protein expression of Smad2 and Smad3 （P<0.01）. ConclusionPA can inhibit the invasion and metastasis of renal carcinoma cells presumably through regulating the homeostasis of MMP/TIMP by Smad2/3.

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.

Effect of Bushen Chushi() decoction combined with platelet-rich plasma on regulating TGF-β1 and Smad-1 expression in serum of knee osteoarthritis patient / 中国骨伤

OBJECTIVE@#To investigate the effect of Bushen Chushi decoction combined with platelet-rich plasma(PRP) to treat knee osteoarthritis(KOA) in early and middle stage and its regulation on TGF-β1 and Smad-1 expression in serum.@*METHODS@#Total of 45 patients with KOA in early and middle stage from May 2020 to April 2022 were treated and divided into control group and observation group. In control group, there were 30 patients including 12 males and 18 females, aged from 43 to 69 years old with an average of(57.3±6.5) years old and disease duration ranged from 1.5 to 5.0 years with an average of(3.8±1.7) years, and there were 8 cases in gradeⅠ, 13 cases in gradeⅡ, and 9 cases in grade Ⅲ according to Kellgren-Lawrence Grade, PRP 5 ml was injected into knee joint on the first day of No1, 3 week together for 2 times. In the observation group, there were 15 cases including 7 males and 8 females, aged from 45 to 70 years old with an average of (56.7±6.2) years old and disease duration ranged from 1.8 to 5.7 years with an average of (4.0±1.8) years, there were 4 cases in gradeⅠ, 9 cases in gradeⅡand 4 cases in grade Ⅲ according to the Kellgren-Lawrence Grade, PRP 5 ml were injected into knee joints that the time and frequency were the same as those in the control group, and at the same time Bushen Chushi decoction orally were taken 1 dose per day with a total of 28 doses. All patients were treated for four weeks. Visual analogue scale(VAS) and Lequesne MG score before and after treatment were used to evaluate improvement of knee pain and joint function. The TGF-β1 and Smad-1 levels in serum were measured before and after treatment in two groups. The incidence of complications in two groups was observed.@*RESULTS@#All patients were followed up for 26 to 30 days with an average of (28.0±0.6) days. There was no significant difference in VAS and knee Lequesne MG scores between two groups before treatment(P>0.05). The scores of VAS and knee Lequesne MG on the first day after treatment in both groups were lower than those before treatment(P<0.05). The VAS and knee Lequesne MG scores in observation group were lower than those in control group(P<0.05) on the first day after treatment. The TGF-β1 level in serum after treatment were higher significantly than that before treatment in two groups(P<0.05). After treatment, TGF-β1 level in serum in observation group were lower than those in control group with statistically significant differences(P<0.05). The Smad-1 levels in serum after treatment in observation group were higher significantly than that in control group(P<0.05). The levels of Smad-1 were not statistically significant between before and after treatment(P>0.05). There was no significant difference in postopertaive complications between two groups (P>0.05).@*CONCLUSION@#The efficacy of Bushen Chushi decoction combined with PRP in treatment of early and middle KOA is better than that of PRP injection alone. The combined treatment could reduce TGF-β1 level and increase Smad-1 level in serum, which may be a mechanism to inhibit inflammation and alleviate cartilage degeneration to some extent.

Shenmai Injection Improves Hypertensive Heart Failure by Inhibiting Myocardial Fibrosis via TGF-β 1/Smad Pathway Regulation / 中国结合医学杂志

OBJECTIVE@#To study effects of Shenmai Injection on hypertensive heart failure and its mechanism for inhibiting myocardial fibrosis.@*METHODS@#Salt-sensitive (Dahl/SS) rats were fed with normal diet (0.3% NaCl) and the high-salt diet (8% NaCl) to observe the changes in blood pressure and heart function, as the control group and the model group. Salt-insensitive rats (SS-13BN) were fed with the high-salt diet (8% NaCl) as the negative control group. After modeling, the model rats were randomly divided into heart failure (HF) group, Shenmai Injection (SMI) group and pirfenidone (PFD) group by a random number table, with 6 rats in each group. They were given sterilized water, SMI and pirfenidone, respectively. Blood pressure, cardiac function, fibrosis and related molecular expression were detected by sphygmomanometer, echocardiogram, enzyme linked immunosorbent assay (ELISA), hematoxylin-eosin staining, Masson staining, immunofluorescence and qPCR analysis.@*RESULTS@#After high-salt feeding, compared with the control and negative control group, in the model group the blood pressure increased significantly, the left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS) were significantly reduced, and the serum NT-proBNP concentration increased significantly (all P<0.05); furthermore, the arrangement of myocardial cells was disordered, the edema was severe, and the degree of myocardial fibrosis was also significantly increased (P<0.05); the protein and mRNA expressions of collagen type I (Col I) were up-regulated (P<0.05), and the mRNA expressions of transforming growth factor β 1 (TGF- β 1), Smad2 and Smad3 were significantly up-regulated (P<0.05). Compared with HF group, after intervention of Shenmai Injection, LVEF and LVFS increased, myocardial morphology was improved, collagen volume fraction decreased significantly (P<0.05), and the mRNA expressions of Col I, TGF- β 1, Smad2 and Smad3, as well as Col I protein expression, were all significantly down-regulated (all P<0.05).@*CONCLUSION@#Myocardial fibrosis is the main pathological manifestation of hypertensive heart failure, and Shenmai Injection could inhibit myocardial fibrosis and effectively improve heart failure by regulating TGF-β 1/Smad signaling pathway.

Astragaloside IV alleviates lung inflammation in Klebsiella pneumonia rats by suppressing TGF-β1/Smad pathway

Astragaloside IV is a biologically active substance derived from the traditional Chinese medicine Astragalus mambranaceus Bunge, and has antioxidant, anti-inflammatory, and anti-apoptotic properties. In this study, we aimed to investigate the effects of astragaloside IV on Klebsiella pneumonia rats and the underlying mechanisms. Klebsiella pneumoniae (K. pneumoniae) rats were treated with different dosages of astragaloside IV (5, 10, and 20 mg/kg) by intragastric administration. The levels of pro-inflammatory cytokines interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α in bronchoalveolar lavage fluid (BALF) were determined. Pathological changes of lung tissue were inspected by HE staining. The expression of transforming growth factor (TGF)-β1 in lung tissue was determined with immunohistochemistry, and the expression levels of TGF-β1, p-Smad2/Smad2, p-Smad3/Smad3, IκBα/p-IκBα, and p65/p-p65 in lung tissue were determined by western blot. The mechanism was further investigated with TGF-β1 inhibitor SB-431542. Astragaloside IV reduced the elevated levels of pro-inflammatory cytokines caused by K. pneumoniae and improved lung tissue damage in a dose-dependent manner. Astragaloside IV also decreased the expression of TGF-β1/Smad signaling pathway-related proteins and decreased the protein levels of inflammation-related p-IκBα and p65 in lung tissues induced by K. pneumoniae. Additionally, it was found that the effects of 20 mg/kg astragaloside IV were similar to SB-431542, which could improve pulmonary fibrosis induced by K. pneumoniae, decrease the levels of TGF-β1/Smad signaling pathway-related proteins in lung, and reduce inflammation at the same time. Astragaloside IV could alleviate the inflammation of rat pneumonia induced by K. pneumoniae through suppressing the TGF-β1/Smad pathway.

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).

Progress on the roles of TGF-βSmad signaling pathway in HIV pathogenesis / 中国热带医学

@#Abstract: TGF-β/Smad signaling pathway has a wide range of biological activities and plays an important roles in regulating cell growth, adhesion, differentiation, cell dynamic balance, and immune responses. The higher activity of TGF-β/Smad signaling pathway may promote scar formation, organ fibrosis, immunosuppression, and late-stage cancer progression, while low activity may lead to inflammation, dysplasia, poor healing and oncogenesis. The function of the TGF-β/Smad signaling pathway is extremely complex and can exhibit inhibitory or enhancing effects on immunity and inflammation under different circumstances, but immunosuppressive and anti-inflammatory effects are dominant. During HIV infection, the TGF-β/Smad signaling pathway interacts with HIV in a complex manner as HIV proteins tat and gp120 can induce TGF-β expression. Meanwhile, this signaling pathway may also play a role in HIV infection and replication, latent virus reservoir, host immune deficiency and HIV-related inflammation. It is worth noting that even though TGF-β, which mainly exhibits anti-inflammatory effects, is induced by HIV, high levels of TGF-β do not seem to inhibit HIV-related inflammation. So far, the relationship between TGF-β/Smad signaling pathway and HIV infection has not been elucidated, and its role and mechanism in HIV infection and related illnesses need further exploration and validation. This review summarizes the relevant research progress on the TGF-β/Smad signaling pathway and HIV infection, and provides a reference for further understanding of HIV pathogenesis and exploring strategies of AIDS treatment.

Bacteroides fragilis ATCC25285 relieves colitis by inducing Treg cells differentiation via the TGF-β/Smad3 signaling pathway / 中国药科大学学报

@#The changes in intestinal flora are usually associated with different gastrointestinal diseases, and intestinal flora homeostasis can enhance immune tolerance and regulate intestinal immune balance.Previous studies have found that the increase of the relative abundance of Bacteroides fragilis (B.fragilis) in Bacteroides intestinalis can significantly enhance the expression of intestinal regulatory T cells (Treg) and anti-inflammatory cytokines, thus alleviating intestinal inflammation.However, the mechanism of B.fragilis regulating intestinal immunity is still unclear.In this study, an acute colitis model was constructed by giving 3% DSS in drinking water solution to SPF-grade C57BL/6 mice for 7 days, and exogenous supplementation B.fragilis was given to mice by gastric gavage to study its regulatory effect on intestinal immunity and its mechanism of action.The results showed that B.fragilis could improve the intestinal flora disorder in mice with colitis and increase the content of short-chain fatty acids (SCFAs), the main metabolite of the intestinal flora.By extracting mouse tissue lymphocytes, naive CD4+ T cells, and liposome-modified siRNA knockdown mouse Smad3, it was further discovered by flow cytometry that B.fragilis induced the expression of intestinal Treg cells and related cytokines through the TGF-β/Smad3 signaling pathway, which enhanced intestinal regulatory immunity and alleviated colitis.It was also found that B.fragilis activated TGF-β by increasing the expression of reactive oxygen species (ROS), thus inducing Treg cell differentiation and playing an immunomodulatory role.

Medicated Serum of Bupleuri Radix Regulates HFL1 Apoptosis and Fibroblast-myofibroblast Transition via Smad3/Rheb Axis / 中国实验方剂学杂志

ObjectiveTransforming growth factor-β1 （TGF-β1） was used to stimulate human fetal lung fibroblast 1 （HFL1） for simulating the pathological process of idiopathic pulmonary fibrosis （IPF） and thereby the effects and mechanism of medicated serum of Bupleuri Radix against IPF were investigated. MethodTGF-β1 （10 μg·L-1） was employed to stimulate HFL1， and cells were treated with medicated serum of Bupleuri Radix （5%， 10%， 15%， 20%） for 24 h. Then cell proliferation rate was determined with cell counting kit-8 （CCK-8）. Subsequently， cells were classified into the control group （20% blank serum）， TGF-β1 group （20% blank serum and 10 μg·L-1 TGF-β1）， TGF-β1 + medicated serum of Bupleuri Radix group （5% blank serum， 15% medicated serum， and 10 μg·L-1 TGF-β1）， and TGF-β1 + SIS3 group （3 μmol·L-1 SIS3， 20% blank serum， 10 μg·L-1 TGF-β1）. Based on in situ end labeling （TUNEL） staining， the apoptosis rate was examined， and mRNA expression of apoptosis-related proteins B-cell lymphoma 2 （Bcl-2）， Bcl-2 associated X protein （Bax） and myofibroblast marker α-smooth muscle actin （α-SMA） was detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. The protein expression of α-SMA， Ras homolog enriched in brain （Rheb）， and phosphorylated （p）-Smad3 was determined by immunofluorescence. Expression of Rheb， p-Smad3， and Smad3 was examined by Western blot. ResultThe cell proliferation rate of TGF-β1 group increased compared with that of the control group （P<0.05）. The cell proliferation rate of TGF+15% medicated serum of Bupleuri Radix group and TGF+20% medicated serum of Bupleuri Radix group decreased compared with that of the TGF-β1 group （P<0.01）. Compared with the control group， TGF-β1 group showed decrease in apoptosis rate， increase in mRNA expression of Bcl-2 and α-SMA， reduction in Bax mRNA expression， and rise of α-SMA and Rheb protein expression and p-Smad3 level （P<0.05）. Compared with TGF-β1 group， TGF-β1 + medicated serum of Bupleuri Radix group and TGF-β1 + SIS3 group demonstrated high apoptosis rate， low Bcl-2 and α-SMA mRNA expression， high Bax mRNA expression， and low α-SMA and Rheb protein expression and p-Smad3 level （P<0.05）. ConclusionMedicated serum of Bupleuri Radix can inhibit TGF-β1-induced HFL1 proliferation and fibroblast-myofibroblast transition and promote fibroblast apoptosis by regulating the Smad3/Rheb axis.

Low-dose paclitaxel modulates the cross talk between the JNK and Smad signaling in primary biliary fibroblasts

SUMMARY OBJECTIVE: The objective of this study was to explore the molecular mechanism underlying the occurrence of benign bile duct stricture and the target of low-dose paclitaxel in the prevention of benign bile duct stricture. METHODS: Under the stimulation of transforming growth factor beta 1, the expression of collagen type I and connective tissue growth factor were detected on isolated primary fibroblasts. The phosphorylation levels of JNK and Smad2L were detected using Western blot. The effect of low-dose paclitaxel on the transforming growth factor beta 1-induced inhibition of type I collagen and connective tissue growth factor expression and JNK and Smad2L phosphorylation was also observed. RESULTS: Transforming growth factor beta 1 induced the secretion of type I collagen and connective tissue growth factor as well as JNK phosphorylation in biliary fibroblasts. The JNK inhibitor or siRNA-Smad2 inhibited the transforming growth factor beta 1-induced secretion of type I collagen and connective tissue growth factor. Low-dose paclitaxel inhibited the expression of type I collagen induced by transforming growth factor beta 1 and may inhibit the secretion of collagen in biliary fibroblasts. CONCLUSION: The activation of JNK/Smad2L induced by transforming growth factor beta 1 is involved in the occurrence of benign bile duct stricture that is mediated by the overexpression of type I collagen and connective tissue growth factor, and low-dose paclitaxel may inhibit the phosphorylation of JNK/Smad2L.

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