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Aim To investigate the effects of 2-dode-cyl-6-methoxycyclohexa-2 , 5-diene-l, 4-dione ( DM-DD) on resisting hepatic fibrosis induced by carbon tetrachloride ( CC14 ) in rats and the underlying mechanisms , with a specific focus on the TGF-pi/Smads signaling pathway. Methods The hepatic fibrosis model was replicated using 50% CC14. Various parameters, including levels of aspartate transferase ( AST) , ala-nine transferase ( ALT ) , albumin/globulin ( A/G ) , total protein (TP) , total bilirubin (T-BIL) , hyaluron-ic acid ( HA ) , laminin ( LN ) , collagen type Ж ( Col Ж) , and collagen type IV(ColIV) in the blood, were measured. Liver tissue lesions and fiber formation were observed using HE and Masson staining. The expression levels of a smooth muscle actin (a-SMA) , collagen type I ( Col I ) , transformed growth factor (TGF-pi), Smad2, and Smad7 proteins were assessed using immunohistochemistry. a-SMA, Coll, TGF-pi, and Smad7 mRNA levels in liver tissue were measured by RT-PCR. Additionally, the expression levels of TGF-pi, Smad4, and Smad7 proteins in liver tissue were determined by Western blot. Results In comparison to the normal control group, the model group exhibited significantly elevated levels of AST, ALT, TP, T-BIL, HA, LN, Col Ш and Col IV in serum. But A/G level notably decreased. Successful modeling was confirmed by the presence of extensive fiber formations observed through HE and Massonstaining in liver tissue. The DMDD administration group demonstrated a notable decrease levels of AST, ALT, TP, T-BIL, HA, LN, Col III, and CollV, but A/G was significantly elevated when compared to the model group. Furthermore, a-SMA, Coll, TGF-f31, Smad2 and Smad4 mRNA and protein levels in the DMDD administration group were significantly reduced, while Smad7 significantly declined. HE and Masson staining results reflected a marked reduction in fibrous hyper-plasia. Conclusion DMDD exhibits a protective effect against CCl4-induced hepatic fibrosis, and its mechanism appears to be associated with the TGF-fJl/ Smads signaling pathway.
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Objective To investigate the relationship between factors related to the transforming growth factor β(TGF-β)/Aerine-threonine kinase receptors(Smads)signaling pathway and cognitive dysfunction in peripheral blood of patients with aneurysmal subarachnoid hemorrhage(aSAH).Methods The clinical data of 100 patients with aSAH admitted to Chongzuo City People's Hospital from October 2018 to March 2022 were retrospectively selected and grouped according to the patients'Montreal Cognitive Assessment Scale(MoCA)scores,including 54 cases with cognitive dysfunction and 46 cases without cognitive dysfunction.The clinical data,peripheral blood TGF-β,Smad1,Smad3,and Smad7 mRNA expression levels of the two groups were compared.The relationship between pathway-related factors and cognitive dysfunction in patients with aSAH was analyzed in a multifactorial manner.The predictive value of pathway-related factors for cognitive dysfunction in aSAH patients was assessed using the receiver operating characteristic(ROC)curve.Results Peripheral blood TGF-β,Smad1,Smad3,and Smad7 mRNA expression levels were higher in the cognitively impaired group than in the group without cognitive impairment(P<0.05).Multifactorial showed that pathway-related factors were significantly associated with cognitive impairment in patients with aSAH(P<0.05).The ROC showed that the area under the curve(AUC)of pathway-related factors jointly predicted cognitive dysfunction in patients with aSAH was superior to that predicted alone(P<0.05).Conclusion The high expression of factors related to the TGF-β/Smads signaling pathway in the peripheral blood of aSAH patients suggests that this pathway may be associated with cognitive dysfunction in patients.
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Objective To observe the effect of sirtuin 1(SIRT1)on rat myocardial fibrosis induced by pressure overload and the proliferation of cardiac fibroblasts induced by angiotensin Ⅱ(Ang Ⅱ),and to explore the molecular mechanisms.Methods The pressure overload-induced myocardial fibrosis was established by abdominal aorta constriction(AAC)procedure in vi-vo.After treatment with SIRT1 activator,the myocardial interstitial fibrosis and the collagen volume fraction were evaluated by Masson's trichrome staining.The protein expressions of TGF-β1/Smads were determined by immunohistochemical analy-sis.After in vitro intervention of Ang Ⅱ or Ang Ⅱ with SIRT1 activator,the fibroblasts proliferation was detected by MTT as-say.The mRNA and protein expressions of collagen Ⅰ/Ⅲ(Col1α1/3α1),SIRT1 and TGF-β1/Smads in myocardial tissue and fi-broblasts were evaluated by qRT-PCR and Western blotting.Results Compared with the sham operation group,myocardial in-terstitial fibrosis was significantly observed in the pressure overload model group,myocardial collagen volume fraction was in-creased,expressions of Col1α1/3α1 and TGF-β1/Smads were significantly increased,and SIRT1 expression was decreased.After the intervention of SRT1720,SIRT1 activator could improve the myocardial interstitial fibrosis induced by pressure overload,downregulate the expressions of Col1α1/3α1 and TGF-β1/Smads,and upregulate the expression of SIRT1.Meanwhile,correla-tion analysis showed that the protein expression of SIRT1 was negatively correlated with the expression of TGF-β1.In addition,SRT1720 also inhibited Ang Ⅱ-induced fibroblast proliferation and increased expression of Col1α1/3α1 and TGF-β1.Conclusion Activation of SIRT1 inhibits pressure overload-induced myocardial fibrosis and Ang Ⅱ-induced fibroblasts proliferation via regu-lation of the TGF-β1/Smads signaling pathway.
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Objective To investigate the mechanism of Gentianopsis paludosa xanthone(GPX)combined with probiotics in the intervention of colon inflammation-tumor transformation in rats by regulating TGF-β1/Smads pathway and inflammatory factors.Methods Ninety rats were divided into the normal group,the model group[drinking sodium dextran sulfate(DSS)for 3 days]and the intervention group by random number table method.The model group was subdivided into the inflammatory stage group,the pre-inflammatory cancer group(DMH injection for 4 weeks),the intermediate inflammatory cancer group(DMH injection for 13 weeks)and the advanced inflammatory cancer group(DMH injection for 21 weeks).The administration group was subdivided into the groups(after the first day of drinking DSS,drugs for each group were given by gavage once a day for 8 weeks)on the basis of the advanced inflammatory cancer group,including the GPX group(GPX 69.3 mg/kg),the probiotic group,the combined group(GPX+probiotics 400 mg/kg)and the thalidomide group(thalidomide 13.5 mg/kg).The disease activity index(DAI),colon length and wet mass index were compared between all groups.Characteristics of colon tumors were observed,and pathological changes of colon were observed by HE staining.The expression levels of transforming growth factor(TGF)-β1,Smad4,Smad7,interleukin(IL)-6 and tumor necrosis factor(TNF)-α were detected by Western blot assay and enzyme-linked immunosorbent assay,respectively.Results Compared with the advanced inflammatory cancer group,the administration groups showed an increase in colon length,the expression levels of TGF-β1 and Smad4 protein,a decrease in colon wall thickness,wet mass index,maximum tumor diameter,the levels of Smad7,IL-6,TNF-α,and DAI score decreased in the GPX group and the combined group(P＜0.05).The structure and morphology of intestinal mucosa were improved in the GPX group,the probiotic group and the combination group,and the structure of colonic crypt and goblet cell number were increased.Compared with the probiotic group and the GPX group,the colon wall thickness,colon wet mass index and tumor number were decreased,the protein expression levels of TGF-β1 and Smad4 were increased,and levels of IL-6 and TNF-α were decreased in the combination group(P＜0.05).Conclusion GPX combined with probiotics could inhibit the transformation of colon inflammation-tumor,and the mechanism may be related to the regulation of TGF-β1/Smads pathway and the inhibition of pro-inflammatory factors of IL-6 and TNF-α.
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Aim To explore the effect of ZLY18 on angiotensin II-induced cardiac fibrosis and the underlying mechanism. Methods Ang II was used to induce cardiac fibrosis in vitro and in vivo. Cardiac fibroblasts were divided into blank control group, model group and medicine group. The medicine group was subdivided into ZLY18(L)group, ZLY18(M)group and ZLY18(H)group. Compound ZLY18 was given 1, 2, 5 μmol·L-1 respectively. C57BL/6 mice were randomly divided into control group, model group and medicine group. The medicine group were subdivided into ZLY18(L)group, ZLY18(M)group and ZLY18(H)group. Compound ZLY18 was given 10,20 and 50 mg·kg-1 respectively. Both the model group and the medicine group were given with Ang II to induce cardiac fibrosis. The changes of protein levels were detected by Western blot and immunofluorescence. The changes of cardiac function indexes in C57BL/6 mice were detected by small animal echocardiography. The morphology, cell arrangement and collagen fibers of cardiac fibroblasts were observed by tissue section staining and other methods. Results The model of Ang II-induced myocardial fibrosis was successfully established at the cell and animal levels, and ZLY18 treatment improved the elevated fibrosis-related protein caused by Ang II and abnormal cardiac function in mice. Moreover, ZLY18 was able to inhibit the increased phosphorylation of TGF-1 and Smad3 caused by Ang II and increased Smad2/3 nuclear entry, suggesting that the antifibrotic effect of ZLY18 might be related to the activation of TGF-1/Smads signaling pathway. Conclusions ZLY18 has a protective effect on Ang II-induced cardiac fibrosis. ZLY18 may inhibit TGF-β/Smads signaling pathway activation to exert anti-fibrotic effects.
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Liver fibrosis is a wound healing response that occurs in the setting of chronic liver injury and is caused by imbalance in the synthesis and degradation of extracellular matrix （ECM）. If left untreated， it can progress to liver cirrhosis and hepatocellular carcinoma. The activation of hepatic stellate cell （HSC） is now well established as a central driver of liver fibrosis. The activated HSC will transform into myofibroblasts that produce ECM protein. Transforming growth factor-β1 （TGF-β1） can induce the activation of hepatic stellate cell （HSC）， and TGF-β1/Smads signaling pathway is one of the important pathways to promote liver fibrosis. Non-coding RNA （ncRNA） does not encode proteins during the transcription but plays an important regulatory role in the post-transcriptional process of genes. Accumulating evidence shows that the occurrence of liver fibrosis is closely related to the abnormal expression of ncRNA which participates in the activation of HSC by regulating TGF-β1 signal transduction and then affects the process of liver fibrosis. MiRNA-mediated TGF-β1/Smads signaling pathway can not only promote liver fibrosis but also play a role in anti-fibrosis. Long non-coding RNA （lncRNA） not only promotes the development of liver fibrosis by binding to target genes but also enhances TGF-β1 signal transduction by acting as competitive endogenous RNA. circular RNA （circRNA） acts as a ''sponge'' to regulate TGF-β1/Smads pathway， thereby inhibiting HSC activation and exerting the anti-liver fibrosis effect. Chinese medicinal plays an essential part in the prevention and treatment of liver fibrosis， and the active components can inhibit TGF-β1/Smads pathway by regulating the expression of miRNA， thus alleviating liver fibrosis. This article reviews the role and mechanism of miRNA-， lncRNA- and circRNA-mediated TGF-β1/Smads signaling pathway in liver fibrosis and summarizes the anti-liver fibrosis effect of active components of Chinese medicinals by regulating miRNA-mediated TGF-β1/Smads signaling pathway， which can serve as a reference for clinical treatment of liver fibrosis and the development of new drugs.
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Objective:To investigate the effect of gallic acid on the morphology, proliferation and cell cycle of keloid fibroblasts, as well as on collagen contraction and the transforming growth factor-β (TGF-β) /Sma- and Mad-related proteins (Smads) signaling pathway, and to explore the role and mechanisms of action of gallic acid in the treatment of keloids.Methods:From August to December 2022, 3 keloid tissue samples were collected from 3 patients with clinically and pathologically confirmed keloids after surgery in the Department of Dermatologic Surgery, Wuhan No.1 Hospital. Primary fibroblasts were isolated and cultured by using the tissue culture method, and 3- to 8-passage fibroblasts were used for subsequent experiments. Cultured keloid fibroblasts were divided into 4 groups: low-, medium- and high-dose gallic acid groups treated with 0.025, 0.05 and 0.1 mg/ml gallic acid respectively, and a control group cultured with Dulbecco′s modified Eagle′s medium (DMEM) containing 10% fetal calf serum. After 24-, 48-, and 72-hour treatment, cellular proliferative activity was evaluated by cell counting kit 8 (CCK8) assay, and collagen contraction by using a three-dimensional culture method. After 24-hour treatment in the above groups, pictures were taken using a differential interference inverted fluorescence microscope, and changes in the cell cycle were analyzed by flow cytometry. Some keloid fibroblasts were divided into 2 groups: an experimental group (high-dose gallic acid group) treated with 0.1 mg/ml gallic acid, and a control group cultured with DMEM containing 10% fetal calf serum. After 24-hour treatment, enzyme-linked immunosorbent assay (ELISA) was performed to determine the changes in supernatant concentrations of TGF-β1, TGF-β2, and TGF-β3 in the two groups, real-time fluorescence-based quantitative PCR to detect the relative mRNA expression levels of TGF-β1, TGF-β2, TGF-β3, Smad2, Smad3, Smad4, and α-smooth muscle actin (α-SMA). Statistical analysis was carried out using t test, one-way analysis of variance and two-way analysis of variance, and least significant difference (LSD) - t test was used for multiple comparisons. Results:Compared with the control group, the gallic acid groups showed gradual changes in the shape of keloid fibroblasts under the microscope as the dose of gallic acid increased, including gradually shrinking cell bodies, enlarged intercellular spaces, cell atrophy, increased number of apoptotic cells, etc. CCK8 assay showed that the cellular proliferative activity changed significantly as the dose of gallic acid increased and the treatment time was prolonged ( Fgroup = 78.31, P < 0.001; Ftime = 4.17, P = 0.037), and the proliferative activity of keloid fibroblasts was significantly lower in the high-dose gallic acid group than in the control group at 24, 48, and 72 hours (all P < 0.05). The three-dimensional culture showed that different degrees of collagen contraction occurred in all groups over time, marked collagen contraction was observed in the control group, and a lower degree of collagen contraction in the gallic acid groups; the collagen contraction indices were significantly lower in the medium- and high-dose gallic acid groups than in the control group at 24, 48, and 72 hours (all P < 0.05). Flow cytometry showed that the cell apoptosis rates were significantly higher in the low-, medium- and high-dose gallic acid groups (38.68% ± 3.05%, 41.82% ± 2.19%, 43.56% ± 3.58%, respectively) than in the control group (12.58% ± 1.56%, all P < 0.001) after 24-hour treatment; compared with the control group, the medium- and high-dose gallic acid groups showed significantly decreased proportions of cells in the G0/G1 phase (both P < 0.01), but significantly increased proportions of cells in the S phase and G2/M phase (all P < 0.05). ELISA revealed that the TGF-β1 concentration was significantly lower in the high-dose gallic acid group (758.58 ± 31.42 pg/ml) than in the control group (1 081.30 ± 44.72 pg/ml, t = 11.81, P<0.001), there was no significant difference in the TGF-β2 concentration between the high-dose gallic acid group (71.05 ± 7.40 pg/ml) and the control group (76.43 ± 6.51 pg/ml, t = 1.09, P = 0.317), while the TGF-β3 concentration was significantly higher in the high-dose gallic acid group (5.70 ± 3.87 pg/ml) than in the control group (0.00 ± 0.00 pg/ml, t = 2.94, P = 0.026). As real-time fluorescence-based quantitative PCR revealed, the high-dose gallic acid group showed significantly decreased mRNA expression levels of TGF-β1, Smad2, Smad3, Smad4, and α-SMA (all P < 0.05), but significantly increased mRNA expression level of TGF-β3 ( t = 6.78, P = 0.002) compared with the control group; however, there was no significant difference in the TGF-β2 mRNA expression level between the above two groups ( t = 0.05, P = 0.962) . Conclusion:Gallic acid could change the cell cycle, inhibit the proliferative activity, promote apoptosis and change the shape of keloid fibroblasts, and thus inhibit scar formation and contraction, which may be related to the inhibition of TGF-β/Smads signaling pathway.
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Diabetic kidney disease is one of the complications of diabetes,which can progress to end-stage renal disease.In recent years,it has been found that miRNAs have become a research hotspot,with miRNA-21 regulating transforming growth factor β1(TGF-β1)/Smads,phosphoinositide 3-kinase(PI3K)/protein kinase B(AKT),Wnt/β-catenin and other signaling pathways to promote the progress of diabetic kidney disease.Studies have showed that traditional Chinese medicine has a regulatory effect on the expression of miRNA-21 and can target miRNA-21 to regulate TGF-β1/Smads,phosphatase and tensin homolog/PI3K/AKT/mammalian target of rapamycin(mTOR),peroxisome proliferator activated receptors and other signal transduction pathways to trigger signal cascade reactions,which intervene in pathological processes such as fibrosis,inflammation,oxidative stress and autophagy.In this article,the role of miRNA-21 in diabetic kidney disease and the intervention of traditional Chinese medicine were summarized,in order to provide some reference for the treatment of diabetic kidney disease and the development of new drugs.
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Objective To investigate the expression and significance of BMP7/Smads signaling pathway in hepatic fibrosis tissues induced by common bile duct ligation in rats.Methods Male Wistar rats were simple randomizationally divided into sham operation group,common bile duct ligation group for 7days group,14days group,28days group and 42days group.Common bile duct ligation meth-od was used to establish the cholestatic hepatic fibrosis animal model.hematoxylin-eosin staining and Masson staining were used to ob-serve the hepatic tissues morphology and collagen deposition of the rats in each group.Real-time quantitative polymerase chain reaction(RT-qPCR)technology was used to detect the mRNA expression of hepatic fibrosis indicators,such as α-smooth muscle actin(α-SMA)and fibronectin(FN)in each group.Immunohistochemistry was used to determine the deposition of collagen Ⅰ in the hepatic tis-sues,Western blot was used to detect the proteins expression of FN,collagen Ⅰ,collagen Ⅲ,α-SMA and tissue inhibitor of matrix metalloproteinase(TIMP1)in the hepatic tissues.The expression and localization of BMP-7 and p-Smad1/5/8 proteins in the hepatic tissues were detected by Western blot and Immunohistochemistry.Results Compared with the sham operation group,with the extension of common bile duct ligation time,the degree of hepatic fibrosis gradually increased,the intrahepatic collagen hyperplasia was obvious,and thick fibrous septum were obviously formed in the hepatic issues of BDL groups.The expression of FN,α-SMA,collagen Ⅰ,colla-gen Ⅲ and TIMP1 proteins in the BDL hepatic tissues were significantly increased,the difference were statistically significant(P<0.05).In the hepatic fibrosis tissue,the expression of BMP-7 and p-Smad1/5/8 proteins were significantly increased in the early stage of hepatic fibrosis,while which were decreased with the aggravation of hepatic fibrosis.Compare with the sham operation group,the difference was statistically significant(P<0.05).Conclusion The BMP-7/Smads signaling pathway is significantly activated in the early stage of hepatic fibrosis in rats,which exerted protective effect on bile duct ligation-induced hepatic fibrosis.However,the protec-tive effect of activation of BMP-7/Smads signaling pathway was inhibited with the progression of hepatic fibrotic severity.
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ObjectiveTraditional Chinese medicine， namely Dahuang Zhechongwan （DHZCW） was used to treat myocardial fibrosis in model rats， observe its effect on myocardial fibrosis in rats， and explore its action mechanism. MethodThirty-six SPF male Kunming rats were divided into blank group， model group， low-， medium-， high-dose groups of DHZCW （0.056， 0.084， 0.168 g·kg-1）， captopril group （10 mg·kg-1）， with six rats in each group. Except for the blank group， the other groups were intraperitoneally injected isoproterenol solution of 5 mg·kg-1 for 15 consecutive days to replicate the myocardial fibrosis model. At the beginning of modeling， the rats in each group took drugs， and they were sacrificed 28 days after administration. Serum and heart tissue were collected for the corresponding detection. Hematoxylin-eosin （HE） staining and Masson staining were used to observe tissue inflammation， cellular degeneration， necrosis， and fibrosis. The contents of hydroxyproline （HYP）， tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， interleukin-6 （IL-6）， hyaluronic acid （HA）， laminin （LN）， type-Ⅲ procollagen （PC Ⅲ） in serum of rats and rats were determined by enzyme-related immunosorbent assay （ELISA）. The expression levels of key pathway proteins transforming growth factor-β1 （TGF-β1）， α-smooth muscle actin （α-SMA）， Smad2， Smad3， and Smad7 were detected by Western blot. The expression levels of key pathway genes TGF-β1， α-SMA， Smad2， Smad3， Smad7， miR-29a-5p， miR-29b-2-5p， and miR-29c-5p were detected by Real-time quantitative polymerase chain reaction （Real-time PCR）. ResultCompared with the blank group， the pathological changes of fibrosis in the model group were obvious， the contents of serum HYP， TNF-α， IL-1β， IL-6， HA， LN， and PCⅢ were increased （P<0.01）， the protein expression levels of TGF-β1， α-SMA， Smad2， and Smad3 were increased； the protein expression level of Smad7 was decreased （P<0.01）. The mRNA expression levels of TGF-β1， α-SMA， Smad2， and Smad3 were increased （P<0.05， P<0.01）， while those of Smad7， miR-29a-5p， miR-29b-2-5p， and miR-29c-5p were decreased （P<0.01）. Compared with the model group， after 28 days of administration， serum HYP， TNF-α， IL-1β， IL-6， HA， LN， and PCⅢ in high-， medium-， and low-dose groups of DHZCW and captopril groups were decreased （P<0.01）. Except for the low-dose group， the protein contents of TGF-β1， α-SMA， Smad2， and Smad3 were decreased， while the protein content of Smad7 was increased （P<0.01）. The mRNA expression levels of TGF-β1， Smad2， α-SMA， and Smad3 in high-dose group of DHZCW were decreased （P<0.05，P<0.01）， while those of Smad7， miR-29a-5p， miR-29b-2-5p， and miR-29c-5p were increased （P<0.05）. The mRNA expressions of TGF-β1， Smad2， and Smad3 in the medium-dose group of DHZCW were decreased （P<0.05， P<0.01）， while mRNA expression of Smad7 was increased （P<0.01）. The mRNA levels of TGF-β1 and Smad2 in the low-dose group of DHZCW were decreased （P<0.01）. ConclusionDHZCW can improve myocardial fibrosis in rats， and its action mechanism may be related to the regulation of the TGF-β1/Smads/miR-29 pathway. In addition， there is dose dependence in the range of 0.056-0.168 g·kg-1， and the effect of the high-dose group is more stable.
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AIM: To explore the protective effect of fluorofenidone (AKF-PD) on diethylnitrosamine-induced liver injury in rats and its inhibition of the TGF-β1/Smads pathway in hepatocytes. METHODS: Fifty-five male Sprague Dawley (SD) rats were randomly divided into three groups: model group (DEN group, n=20) were gavaged with DEN (10 mg/kg), 5 times for 14 weeks; control group (n=20) were gavaged with saline with the same volume of the model group; treatment group (DEN+AKF-PD Group, n=15), after 4 weeks of modeling, they were gavaged with AKF-PD (500 mg/kg) daily, and stopped at 14 weeks. At the end of experiment, the rats were killed by anesthesia and spinal dislocation. Masson staining was used to observe collagen deposition; primary hepatocytes were extracted and identified, and the levels of α-smooth muscle actin (α-SMA), TGF-β1, Smad3, and Smad7 mRNA, and the expression of Smad3 and Smad7 proteins in hepatocytes were detected. RESULTS: Compared with the control group, Masson staining showed that collagen deposition increased in the DEN group; AKF-PD treatment could significantly improve liver pathological damage and reduce collagen deposition. In addition, compared with the DEN group, the α-SMA, TGF-β1, and Smad3 mRNA levels of the AKF-PD group were significantly reduced, and the Smad7 mRNA level was increased. Moreover, AKF-PD treatment could dependably reduce the expression of Smad3 and increase Smad7. CONCLUSION: AKF-PD can significantly improve liver injury and fibrosis in rats caused by DEN. This effect may be related to the down-regulation of α-SMA, TGF-β1, and Smad3 mRNA levels in hepatocytes and the increase of Smad7 mRNA levels.
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OBJECTIVE To s tudy the impr ovement effects of tilianin on the atherosclerosis （AS）model mice and its potential mechanism. METHODS Eight C 57BL/6J mice were taken as the normal group. Forty ApoE－/－ mice were randomly divided into model group ，tilianin low-dose ，medium-dose and high-dose groups [ 2.1，3.5，7.0 mg/（kg·d）] and simvastatin group [positive control drug ，3.5 mg/（kg·d）]，with 8 mice in each group. Normal group was given normal diet ，and other groups were given high-lipid diet to induce AS model. At the same time ，normal group and model group were given normal saline intragastrically ， administration groups were given relevant drug intragastrically ，once a day ，for 12 consecutive weeks. The levels of TC ，TG， LDL-C，HDL-C，Ox-LDL，IL-1β，IL-6，MCP-1 and TNF-α in plasma were determined. The pathomorphological changes of the aorta in mice were observed. The positive rate of ICAM- 1，VCAM-1 and PCNA in the aorta were determined. mRNA expressions of MMP- 2，MMP-9，TGF-β1，Smad2 and Smad 3 as well as protein expressions of TGF-β1，Smad2/3 and p-Smad 2/3 were also determined in aorta of mice. RESULTS Compared with normal group ，the plasma levels of TC ，TG，LDL-C，Ox-LDL，IL-1β， IL-6，MCP-1 and TNF-α in model group were increased significantly（P＜0.01），while HDL-C level was significantly reduced （P＜0.01）. Lipid plaques were formed in the aorta ，and the plaque area was large and caused severe stenosis of the lumen. mRNA expressions of MMP- 2，MMP-9，TGF-β1，Smad2 and Smad 3 as well as positive rate of ICAM- 1，VCAM-1，PCNA and protein expression TGF-β1，Smad2/3，and p-Smad 2/3 in the aorta were significantly increased （P＜0.01）. Compared with model group ， most of above indexes of medication groups were improved significantly （P＜0.05 or P＜0.01）. CONCLUSIONS Tilianin can inhibit the activation of TGF-β1/Smads signaling pathway and then inhibit the proliferation of vascular smooth muscle cells ，reduce ， inflammation and regulate lipid metabolism to inhibit the No.81960766） formation of AS.
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OBJECTIVE To investigate th e intervention effect of Jinkui shenqi pills on renal fibrosis （RF）model rats and its mechanism based on transforming growth factor β1/Smads（TGF-β1/Smads）and TGF-β1/extracellular signal regulated kinase （ERK） signaling pathway. METHODS Male SD rats were given adenine suspension （250 mg/kg）to induce RF model. After modeling ， they were randomly divided into model group ，Colchicine tablet group （positive control ，0.45 mg/kg）and Jinkui shenqi pills low-dose，medium-dose and high-dose groups （0.5，1，2 g/kg），with 10 rats in each group. Other 10 healthy rats were selected as normal group. The rats in administration groups were given the corresponding drugs intragastrically ；normal group and model group were given 0.1％ sodium carboxymethyl cellulose solution ，once a day ，for consecutive 30 d. After last medication ，the serum levels of creatinine （Cr）and blood urea nitrogen （BUN），renal weight and body weight were detected. The ratio of BUN/Cr and renal coefficient were calculated. The pathological morphology of renal tissue in rats were observed. The protein and mRNA expressions of TGF-β1，Smad2，Smad3，ERK1 and ERK 2 were detected. RESULTS Compared with normal group ，serum levels of Cr and BUN and renal coefficient were all increased significantly in model group （P＜0.05），while the ratio of BUN/Cr was decreased significantly （P＜0.05）. The volume of the kidney was significantly increased ，and the surface was seriously granulated. Mesangial hyperplasia ，dilation or atrophy of renal tubules ，accompanied by large-area collagen deposition，could be found. Protein and mRNA expressions of TGF-β 1，Smad2，Smad3，ERK1 and ERK 2 were increased significantly in renal tissue （P＜0.05）. Compared with model group ，above indexes of Jinkui shenqi pills groups were all reversed significantly （P＜0.05）；dilation or atrophy of renal tubules was relieved ，and collagen deposition was reduced to different extents. CONCLUSIONS Jinkui shenqi pills can improve renal function of RF model rats ，the mechanism of which may be associated with inhibiting TGF-β1/Smads and TGF-β1/ERK signaling pathway.
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Objective To construct the clustered regularly interspaced short palindromic repeats / associated protein 9 (CRISPR/ Cas9) plasmid targeting forkhead box J2 (FOXJ2) gene and investigate the effects of FOXJ2 interference on the expression of transforming growth factor-β(TGF-β) / Smads and proliferation in hepatocellular carcinoma cells of mouse. Methods Small guide RNA(sgRNA) sequence of FOXJ2 was designed, linked with PX458 vector and transfected into competent E. coli for proliferation. The recombinant plasmids were sent for sequencing to confirm the accuracy of the sgRNA sequence. The PX458-FOXJ2-sgRNAs plasmids were transfected into Hepa1-6 cells by liposome transfection, respectively. The empty vectors of PX458 were transfected as control group. After 48 hours, the expression of FOXJ2, TGF-β and Smads were obtained by RT-PCR and agarose gel electrophoresis, respectively. The cell proliferation was detected by methylthio tetrazole (MTT) method . Results The CRISPR/ Cas9 plasmids of PX458-FOXJ2-sgRNAs were successfully constructed. The recombinant plasmid of PX458-FOXJ2-sgRNA2 could effectively inhibit FOXJ2 gene expression which induced increasing expression of TGF-β, Smad2 and Smad4 in Hepa1-6 cells comparing to the control group transfected with PX458 only. And the proliferation of Hepa1-6 was promoted in PX458-FOXJ2-sgRNA2 interference group. Conclusion In hepatocellular carcinoma cells of mouse, FOXJ2 gene inhibits the expression of TGF-β, Smad2, Smad4 and cell proliferation partially, which indicates the relationship between FOXJ2 and TGF-β signal pathway. The result provides the target molecule of FOXJ2 for the prevention and treatment of hepatocellular carcinoma.
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Objective:To establish a rat model of neurogenic bladder and analyze the changes in kidney morphology and function and the expression of proteins in AngiotensinⅡ(AngⅡ)/transforming growth factor β1 (TGF-β1)/Smads pathway.Methods:Sprague-Dawley rats were randomly divided into experimental group (spinal nerve amputation, n=36) and control group (sham operation, n=12). At 6, 12, and 24 weeks, the bladder compliance was measured by cystometry, the kidney morphology was detected by B-ultrasound, blood urea nitrogen (BUN) and serum creatinine (Scr) in blood samples were examined, the kidney pathological changes were detected by Masson and HE staining, the distribution of AngⅡ/TGF-β1/Smads pathway proteins was analyzed by immunohistochemisty, and the protein expressions in kidney were detected by Western blotting. Results:Urodynamics showed that the basic bladder pressure in experimental group was higher than that in control group. B-ultrasound showed that compared with the control group, the diameter of the renal pelvis of the rats with nerve dissection gradually increased ( P<0.05), and the hydronephrosis was gradually obvious. Compared with the control group, the BUN and Scr in experimental group gradually increased (both P<0.01). Masson and HE staining showed that compared with the control group, the collagen expression and renal tubulointerstitial scores in experimental group were gradually increased (both P<0.01). Immunohistochemisty showed that compared with the control group, in experimental group the expression of angiotensinⅡ receptor type 1 (AT1), TGF-β receptor 1(TGF-βR1), phosphorylated Smad2 gradually increased (all P<0.01), the pathway inhibitor Smad6 gradually decreased ( P<0.01), and the distribution of each protein in kidney was consistent. Western blotting showed a corresponding expression trend with immunohistochemisty. Conclusions:In neurogenic bladder caused by bilateral spinal nerve amputation, due to bladder dysfunction, increased bladder pressure induces hydronephrosis, destruction of the nephron structure, activation of AngⅡ/TGF-β1/Smads pathway, and renal fibrosis. This method is effective and has clinical similarities, laying a foundation for exploring neurogenic bladder treatment.
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Objective:To observe the improving effect of Danggui Shaoyaosan on diminished ovarian reserve （DOR） in rats triggered by Tripterygia wilfordii polyglycoside tablet combined with stress， and to explore the role of transforming growth factor-<italic>β</italic><sub>1 </sub>（TGF-<italic>β</italic><sub>1</sub>）/Smads signaling pathway in such improvement. Method:Forty-eight female SD rats with normal sexual cycle were selected and randomly divided into a normal group （<italic>n</italic>=8） and a modeling group （<italic>n</italic>=40）， and the ones in the modeling group were given Tripterygium wilfordii polyglycoside tablets （50 mg·kg<sup>-1</sup>） combined with random stress for 15 d. After successful modeling， the rats were randomized into the model group， low-， medium-， and high-dose （3.96， 7.92， 15.84 g·kg<sup>-1</sup>） Danggui Shaoyaosan groups， and estradiol valerate group （0.09 mg·kg<sup>-1</sup>）， with eight in each group. Under the premise of stress exposure， they were separately gavaged with the normal saline， low-， medium- and high-dose Danggui Shaoyaosan， and estradiol valerate for 15 successive days. The estrous cycle of rats in each group was observed daily. After intervention， the rats were sacrificed and the ovarian visceral index was calculated. The pathological changes in ovarian tissues were observed by hematoxylin eosin （HE） staining. The protein expression levels of TGF-<italic>β</italic><sub>1</sub> and TGF-<italic>β</italic><sub>1 </sub>receptor （TGF-<italic>β</italic><sub>1</sub>R） in the ovarian tissues of rats were measured by immunohistochemistry （IHC）， and the mRNA expression levels of Smad2， Smad3， and Smad7 in the ovarian tissues by real-time polymerase chain reaction （Real-time PCR）. Result:Compared with the normal group， the model group exhibited disordered estrus cycle （<italic>P</italic><0.05）， reduced visceral index （<italic>P</italic><0.01）， and down-regulated TGF-<italic>β</italic><sub>1</sub> and TGF-<italic>β</italic><sub>1</sub>R protein and Smad2 and Smad3 mRNA expression in the ovarian tissues （<italic>P</italic><0.01）， and up-regulated Smad7 mRNA expression （<italic>P</italic><0.01）. Compared with the model group， Danggui Shaoyaosan at the low， medium， and high doses and estradiol valerate improved the estrus cycle of rats to varying degrees （<italic>P</italic><0.05） and increased the visceral index， with better effects observed in the medium-group and high-dose Danggui Shaoyaosan groups （<italic>P</italic><0.05，<italic>P</italic><0.01）. Besides， the protein expression levels of TGF-<italic>β</italic><sub>1</sub> and TGF-<italic>β</italic><sub>1</sub>R and the mRNA expression levels of Smad2 and Smad3 in the ovarian tissues were elevated to varying degrees （<italic>P</italic><0.01）， and the Smad7 mRNA expression declined （<italic>P</italic><0.01）. The improvements in TGF-<italic>β</italic><sub>1</sub> and TGF-<italic>β</italic><sub>1</sub>R protein expression of the medium-dose Danggui Shaoyaosan group and estradiol valerate group were more obvious. Conclusion:Danggui Shaoyaosan significantly improves ovarian reserve in DOR rats， which is closely related to the regulation of TGF-<italic>β</italic><sub>1</sub>/Smads signaling pathway.
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@#Periodontitis is the inflammation of periodontal tissue caused by dental plaque, which absorbs the alveolar bone and cementum. The immune response triggered by CD4+T cells is the key factor for the aggravation of periodontitis. The activation of dendritic cells and the receptor activator of the NF-κB ligand (RANKL) pathway is an important link in the alveolar bone resorption of periodontal tissue. Pro-inflammatory factors such as interferon-γ (IFN-γ), tumor necrosis factor (TNF-α) and interleukin-1β (IL-1β) also play important roles in the development of periodontitis. Interleukin-37(IL-37), which is a newly discovered cytokine in the IL-1 family, has five shear variants from a to e, among which the clover β-structure encoded by exon 4 plays an important role in the binding of cytokines and the corresponding receptors. IL-37 has strong anti-inflammatory and inhibition of autoimmunity, can enter the nucleus with the help of caspase-1 and bind with Smad proteins to regulate the transcription of pro-inflammatory genes. Extracellular IL-37 can bind to IL-18 binding protein and inhibit the production of pro-inflammatory factors. IL-37 can inhibit the progression of periodontitis by inhibiting the RANKL signaling pathway, inhibiting the proliferation and differentiation of dendritic cells and CD4+T cells, binding to Smad proteins, and releasing pro-inflammatory factors such as IFN-γ and TNF-α. The IL-37 concentration in periodontal tissue can indicate the progression of periodontitis. Few studies have described the interaction between the anti-inflammatory factor IL-37 and periodontitis. Thus, in this paper, the structure and function of IL-37 and the related factors between IL-37 and periodontitis will be reviewed.
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<b>Objective::To observe the effect of Guizhitang with different proportions of Cinnamomi Ramulus and Paeoniae Alba Radix on the expressions of transforming growth factor-<italic>β</italic><sub>1</sub>(TGF-<italic>β</italic><sub>1</sub>)/Smads signaling pathway and interleukin-10(IL-10), IL-6 and tumour necrosis factor-<italic>α</italic>(TNF-<italic>α</italic>)related inflammatory cytokines in salt-sensitive hypertensive rats, in order to explore the mechanism of Guizhitang in improving myocardial fibrosis in salt-sensitive hypertensive rats. <b>Method::Totally 40 male 6-week-old salt-sensitive rats were randomly divided into 5 groups: the normal control group, the model group, the 1∶1(RC/peony)Guishao group, the 1∶2 Guishao group, and the 2∶1 Guishao group, with 8 in each group. The normal control group was fed with normal salt diet, while the other four groups were fed with high-salt diet. After 4 weeks of feeding, the rats were given intragastric administration, the normal control group and the model group were given the same amount of normal saline, and the 1∶1 Guishao group, the 1∶2 Guishao group and the 2∶1 Guishao group were given 4.0, 5.5, 5.5 g·kg<sup>-1</sup> of Guizhitang by gavage for 4 weeks. Blood pressure was measured once a week, left ventricular end systolic diameter(LVESD), left ventricular end diastolic diameter (LVEDD), left ventricular ejection fraction (LVEF) and left ventricular short axis shortening fraction (LVFS) were detected by using echocardiogram. The pathological changes of myocardial morphology were observed by htoxylin eosin(HE)and Masson staining. The expressions of type Ⅰ and Ⅲ collagen in myocardial tissue of each group was detected by immunohistochemistry. The mRNA expression levels of IL-10, IL-6 and TNF-<italic>α</italic> in myocardial tissue of each group were detected by quantitative real-time fluorescence polymerase chain reaction(Real-time PCR). The protein expression levels of TGF-<italic>β</italic><sub>1</sub>, <italic>α</italic>-smooth muscle actin(<italic>α</italic>-SMA), Smad2, Smad3 and Smad7 in myocardial tissue of each group were detected by Western blot. <b>Result::Compared with the normal control group, the blood pressure was increased in the model group at 8-15 weeks, LVESD, LVEDD were increased in the model group, while LVFS, LVEF were decreased in the model group. The collagen volume fraction was increased, immunohistochemistry showed the expression levels of type Ⅰ and Ⅲ collagen were increased, mRNA expression levels of IL-10, IL-6 and TNF-<italic>α</italic> were increased, the protein expression levels of TGF-<italic>β</italic><sub>1</sub>, Smad2, Smad3 and <italic>α</italic>-SMA were increased, whereas the protein expression of Smad7 was decreased (<italic>P</italic><0.01). Compared with the model group, the blood pressure rise of each group of Guizhitang was delayed in 12-15 weeks, LVESD and LVEDD were decreased in Guizhitang group (<italic>P</italic><0.01), LVFS, LVEF were increased in Guizhitang group (<italic>P</italic><0.01), the collagen volume fraction was decreased in Guizhitang group (<italic>P</italic><0.01), and the expressions of type Ⅰ and Ⅲ collagen were decreased in Guizhitang group (<italic>P</italic><0.01). At the same time, the mRNA expression of IL-10 was increased in Guizhitang group (<italic>P</italic><0.05, <italic>P</italic><0.01), the mRNA expressions of IL-6 and TNF-<italic>α</italic> were decreased in Guizhitang group (<italic>P</italic><0.01), and the protein expressions of TGF-<italic>β</italic><sub>1</sub>, Smad2, Smad3 and <italic>α</italic>-SMA were decreased in Guizhitang group (<italic>P</italic><0.05, <italic>P</italic><0.01), and the protein expression of Smad7 was increased in Guizhitang group (<italic>P</italic><0.01). Compared with the 2∶1 Guishao group, the effect of the 1∶1 Guishao group in improving the above indicators was more obvious (<italic>P</italic><0.05, <italic>P</italic><0.01). <b>Conclusion::Guizhitang with different proportions of Ramulus Cinnamomi and Poeny can alleviate the degree of myocardial fibrosis in salt-sensitive hypertensive rats. The mechanism may be related to the regulation of TGF-<italic>β</italic><sub>1</sub>/Smads signaling pathway and the reduction of inflammatory response. Besides, the 1∶1 Guishao group showed the optimal effect in reducing inflammation and improving myocardial fibrosis.
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Objective:To explore the effect of Qizhu Zhenwutang on renal interstitial fibrosis in rats ligated with unilateral ureter， transforming growth factor-β1 （TGF-β1）/Smads and oxidative stress. Method:A total of 30 male SD rats were randomly divided into sham operation group， model group， high-dose group， low-dose group and irbesartan group （n=6）. The left ureter ligation was performed in the model group and the treatment group. In the sham operation group， the ureter was not ligated， only the ureter was separated， and the abdominal cavity was closed. Rats in each group were given drugs by gavage on the next day after operation. Sham operation group and model group were given aseptic distilled water 10 mL·kg-1 by gavage， high-dose Qizhizhenwu Tang group was given 22.2 g·kg-1 by gavage， low-dose group was given 11.1 g·kg-1 by gavage， and irbesartan group was given 0.02 g·kg-1 by gavage. Rats in each group were sacrificed on the 14th day after operation， 24-hour urine was collected before sampling， and the total amount of 24 hour urine protein （24 h-Upr） was detected. Blood samples were collected from the abdominal aorta to detect serum creatinine（SCr） and blood urea nitrogen （BUN）. The tissues were stained with htoxylin eosin （HE） and Masson， and the pathological changes were observed under light microscope， immunohistochemical method was used to detect α-SMA， FN and Col-Ⅰ expressions. Western blot method was used to detect the expressions of TGF-β1， Smad3， p-Smad3 and NOX4. Result:Compared with sham group， SCr， BUN and collagen volume fraction （CVF），24 h-Upr in model group were all increased （P<0.05， P<0.01）. The expressions of α-SMA， Col-Ⅰ， FN， TGF-β1， p-Smad3， NOX4 were higher （P<0.05）. Compared with the model group， SCr， BUN and CVF were lower in high-dose group and irbesartan group （P<0.05）. 24 h-Upr was lower in high-dose group （P<0.05）， the expressions of α-SMA， Col-Ⅰ， FN， TGF-β1， Smad3， p-Smad3， NOX4 in traditional Chinese medicine treatment group were less （P<0.05）. Conclusion:Qizhi Zhenwutang can reduce the urinary protein of UUO rats， protect the renal function， and inhibit the occurrence and development of renal interstitial fibrosis， the mechanism may be related to the inhibition of TGF-β1/Smads signaling pathway and oxidative stress response.
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Objective: To observe the effect of neuroopilin-1 (NRP1) gene on the process of radiation-induced pulmonary fibrosis (RIPF), and to explore its roles in the occurrence and development of epithelial-mesenchymal transition (EMT) mediated by Wnt/fi-catenin pathway tail identification was performed in and TGF-β1/Smads pathway, and extracellular matrix (ECM) deposition. Methods: The Cre-LoxP recombinase system was used to construct the transgenic C57BL/6J mice with NRP1 gene specific knockout in alveolar type II epithelial cells (AT-II) and the mice. A total of 160 mice were randomly divided into 4-week group, 8-week group, 16-week group and 24-week group. In each group, the mice were randomly divided into wild type (Con) group, wild type+irradiation (IR) group, NRP1 gene-specific knockout (KO-Con) group, NRP1 gene-specific knockout+irradiation (KO+IR) group according to the method of random number table; there were 10 mice per group. In KO-Con and KO + IR groups, the NRP1 gene was specifically knocked out in the AT-II cells by intraperitoneal injection of tamoxifen, and the mouse models of RTPF were established by 20 Gy total thoracic irradiation in IR group and KO+IR group. After the models were constructed, HE staining and Masson staining were used to verify whether the models were successfully constructed. Immunohistochemistry (IHC) method was used to detect the type I collagen (Col I) and crsmooth muscle actin α-SMA) protein expression levels; Western blotting method was performed to detect the NRP1, β-catenin, TGF-β1, and Smad2 protein expression levels in the lung tissue of the mice; Quantitative fluorensence real-time polymerase chain reaction (qRT-PCR) method was used to detect the expression levels of NRP1, Col I, α-SMA, β-catenin, TGF-βl, Smad2, E-cadherin, N-cadherin, and Vimentin mRNA in the lung tissue of the mice. Results: The results of HE and Masson staining showed the RTPF models were successfully established, and the lung tissue of the mice in IR group mainly showed the pathomorphology of radiation pneumonitis. Compared with Con group, the protein and mRNA expression levels of NRP1 in the lung tissue of the mice in IR group were gradually increased with the prolongation of time (P<0.05), and reached the highest at 24 weeks (P<0.01). Compared with Con group, the expression levels of Col I, α-SMA, β-catenin, TGF-β1, and Smad2 proteins and mRNA in the lung tissue of the mice in IR group and KO+IR group were increased gradually with the prolongation of time (P<0.05 or P<0.01). Compared with IR group, the expression levels of Col I, α-SMA, β-catenin, TGF-β1, and Smad2 protein and mRNA in the lung tissue of the mice in KO+IR group were significantly decreased (P<0.05 or P<0.01), but they were higher than those in Con group (P<0.05 or P<0.01). Compared with Con group, the expression levels of the epithelial cell marker E-Cadherin mRNA in the lung tissue of the mice in IR group and KO+IR group were gradually decreased with the prolongation of time (P< 0.01), and the expression levels of the interstitial cell markers N-Cadherin and Vimentin were increased (P<0.05 or P<0.01), but the expression levels of E-cadhern mRNA in the lung tissue of the mice in KO-IR group were significantly higher than those in IR group (P<0.05 or P<0.01), and the expression levels of N-Cadherin and Vimentin mRNA in the lung tissue of the mice in KO + IR group at each time point were lower than those in IR group (P<0.05 or P<0.01). Conclusion: Knockout of NRP1 gene can inhibit the occurrence and development of RTPF, and its mechanism may be involved in regulating the expressions of Wnt/fi-catenin and TGF-β1/Smads signaling pathways in the lung tissue and inhibiting the EMT process in the mice.