miR-34a mediates oxaliplatin resistance of colorectal cancer cells by inhibiting macroautophagy via transforming growth factor-β/Smad4 pathway

To investigate whether microRNA (miR)-34a mediates oxaliplatin (OXA) resistance of colorectal cancer (CRC) cells by inhibiting macroautophagy via the transforming growth factor (TGF)-β/Smad4 pathway.METHODSmiR-34a expression levels were detected in CRC tissues and CRC cell lines by quantitative real-time polymerase chain reaction. Computational search, functional luciferase assay and western blotting were used to demonstrate the downstream target of miR-34a in CRC cells. Cell viability was measured with Cell Counting Kit-8. Apoptosis and macroautophagy of CRC cells were analyzed by flow cytometry and transmission electron microscopy, and expression of beclin I and LC3-II was detected by western blotting.RESULTSExpression of miR-34a was significantly reduced while expression of TGF-β and Smad4 was increased in CRC patients treated with OXA-based chemotherapy. OXA treatment also resulted in decreased miR-34a levels and increased TGF-β and Smad4 levels in both parental cells and the OXA-resistant CRC cells. Activation of macroautophagy contributed to OXA resistance in CRC cells. Expression levels of Smad4 and miR-34a in CRC patients had a significant inverse correlation and overexpressing miR-34a inhibited macroautophagy activation by directly targeting Smad4 through the TGF-β/Smad4 pathway. OXA-induced downregulation of miR-34a and increased drug resistance by activating macroautophagy in CRC cells.CONCLUSIONmiR-34a mediates OXA resistance of CRC by inhibiting macroautophagy via the TGF-β/Smad4 pathway.

Interaction between insulin-like growth factor binding protein-related protein 1 and transforming growth factor beta 1 in primary hepatic stellate cells

BACKGROUND: We previously showed that insulin-like growth factor binding protein-related protein 1 (IGFBPrP1) is a novel mediator in liver fibrosis. Transforming growth factor beta 1 (TGF beta 1) is known as the strongest effector of liver fibrosis. Therefore, we aimed to investigate the detailed interaction between IGFBPrP1 and TGF beta 1 in primary hepatic stellate cells (HSCs). METHODS: We overexpressed TGF beta 1 or IGFBPrP1 and inhibited TGF beta 1 expression in primary HSCs for 6, 12, 24, 48, 72, and 96 hours to investigate their interaction and observe the accompanying expressions of a-smooth muscle actin (alpha-SMA), collagen I, fibronectin, and phosphorylated-mothers against decapentaplegic homolog 2/3 (p-Smad2/3). RESULTS: We found that the adenovirus vector encoding the TGF beta 1 gene (AdTGF beta 1) induced IGFBPrP1 expression while that of alpha-SMA, collagen I, fibronectin, and TGF beta 1 increased gradually. Concomitantly, AdIGFBPrP1 upregulated TGF beta 1, alpha-SMA, collagen I, fibronectin, and p-Smad2/3 in a time-dependent manner while IGFBPrP1 expression was decreased at 96 hours. Inhibition of TGF beta 1 expression reduced the IGFBPrP1-stimulated expression of alpha-SMA, collagen I, fibronectin, and p-Smad2/3. CONCLUSIONS: These findings for the first time suggest the existence of a possible mutually regulation between IGFBPrP1 and TGF beta 1, which likely accelerates liver fibrosis progression. Furthermore, IGFBPrP1 likely participates in liver fibrosis in a TGF beta 1-depedent manner, and may act as an upstream regulatory factor of TGF beta 1 in the Smad pathway.

THE ROLE OF CONNECTIVE TISSUE GROWTH FACTOR, TRANSFORMING GROWTH FACTOR Β1 AND SMAD SIGNALING PATHWAY IN CORNEA WOUND HEALING

The cornea is a highly specialized and unique organ in the human body. Its main function is to project light from the external environment onto the retina, and it has a specific transparency to perform its function properly. The transparency and integrity of the cornea is of vital importance. The corneal wound, especially laceration deep to Bowman＇s membrane and stroma, which will inevitably cause scar formation, may cause the degeneration or even loss of sight.

Effect of ursodeoxycholic acid on TGF betal/Smad signaling pathway in rat hepatic stellate cells

Background Hepatic fibrosis is the key stage of the pathological progress from hepatic injury to cirrhosis. Ursodeoxycholic acid （UDCA） has been known as having significant clinical therapeutic effects on chronic liver diseases. Our research aimed to study the effect of UDCA on the signaling pathway of transforming growth factor beta1 （TGFβ1）/Smad and discuss its possible molecular mechanisms of inhibiting hepatic fibrosis. Methods Rat hepatic stellate cells were cultured in vitro and randomly assigned to 4 groups. Group A was control group with only DMEM culture medium applied, and groups B, C, D were experimental groups, with different doses of UDCA （1.0 mmol/L, 0.5 mmol/L and 0.25 mmol/L respectively） added into their DMEM culture medium for further culture of 24 hours and 48 hours. The protein expressions of TGFβ1, TGF type 1 receptor, Smad3, Smad4 and Smad7 were measured by Western blotting, as well as the expressions of TGFβ1, Smad3, Smad7 and cAMP response element （CREB） binding protein （CBP） mRNA by real-time PCR. SPSS 11.5 statistical package was adopted for data analyses. Results Compared with control group, the mRNA expressions of TGFβ1 in the high and middle UDCA dose groups for 24 hours and 48 hours significantly decreased （P 〈0.05）, the protein expressions of TGFβ1 in the two above groups for 48 hours and in the high dose group for 24 hours significantly decreased （P 〈0.05）. The protein and mRNA expressions of Smad3 in each UDCA dose group for 24 hours and 48 hours significantly decreased, with significant difference among different UDCA dose groups and between that of 24 hours and 48 hours observed （P 〈0.05）. The protein and mRNA expressions of Smad7 in the high and middle UDCA dose groups for 24 hours and 48 hours significantly increased. The CBP mRNA expression in each UDCA dose group for 24 hours and 48 hours significantly decreased （P 〈0.05）, with significant difference among different UDCA dose groups observed （P 〈0.05）. Conclusion UDCA could curb the development of hepatic fibrosis through affecting the signaling pathway of TGFβ1/Smad by inhibiting the expressions of TGFβ1, Smad3 and CBP and increasing the expression of Smad7.

Qingxuan Jiangya Decoction(清眩降压汤) Prevents Blood Pressure Elevation and Ameliorates Vascular Structural Remodeling via Modulating TGF-β 1/Smad Pathway in Spontaneously Hypertensive Rats

Objective:To elevate the effects of Qingxuan Jiangya Decoction(清眩降压汤,QXJYD)on hypertension and vascular structural remodeling(VSR)in spontaneously hypertensive rats(SHRs),and investigate the underlying mechanisms.Methods:SHRs(n=8)were given intra-gastric administration with 60 mg/kg of QXJYD or saline,daily for 8 weeks,while rats in SHR-control(n=8)and WKY(n=8)groups were received equal volumes of saline solution.Systolic blood pressures(SBP),diastolic blood pressures(DBP)and mean blood pressures(MBP)were measured once a week.The levels of angiotensinⅡ(AngⅡ),endothelin 1(ET-1)and plasma renin activity(PRA)were tested by enzyme-linked immunosorbent assay(ELISA)and radioimmunoassay,respectively.The effect of QXJYD on VSR was determined by examining the media thickness and the ex vivo contractility of thoracic aortic.The proliferation and fibrosis of vascular smooth muscle cells(VSMCs)were examined via immunohistochemical(IHC)staining for proliferating cell nuclear antigen(PCNA),collagen Ⅰ and collagen Ⅲ,respectively.The mRNA and protein expressions of transforming growth factor β1(TGF-β1),Smad3 and phosphorylation of Smad3 in thoracic aorta tissues were determined by real-time polymerase chain reaction(PCR)and Western blot assay,respectively.Results:QXJYD treatment led to a significant decrease of the elevation of blood pressure in SHRs and reduced the levels of AngⅡ,ET-1 and PRA in the serum(P<0.05).In addition,QXJYD treatment remarkably ameliorated VSR and vascular function in SHRs.Moreover,QXJYD inhibited VSMC proliferation and fibrosis by suppressing the expression of PCNA,collagen Ⅰ and collagen Ⅲ in thoracic aortic.Furthermore,QXJYD inhibited the expression of TGF-β1,Smad3 and the phosphorylation of Smad3,respectively(P<0.05).Conclusion:QXJYD reversed VSR by inhibiting VSMC proliferation and collagen deposition via regulation of TGF-β1/Smad signaling pathway,which may,in part,illuminate its anti-hypertensive activities.

Gardenia jasminoides attenuates hepatocellular injury and fibrosis in bile duct-ligated rats and human hepatic stellate cells

AIM:To investigate the anti-hepatofibrotic effects of Gardenia jasminoides in liver fibrosis.METHODS:Male Sprague-Dawley rats underwent common bile duct ligation(BDL) for 14 d and were treated with Gardenia jasminoides by gavage.The ef-fects of Gardenia jasminoides on liver fibrosis and the detailed molecular mechanisms were also assessed in human hepatic stellate cells(LX-2) in vitro.RESULTS:Treatment with Gardenia jasminoides decreased serum alanine aminotransferase(BDL vs BDL + 100 mg/kg Gardenia jasminoides,146.6 ± 15 U/L vs 77 ± 6.5 U/L,P = 0.0007) and aspartate aminotransferase(BDL vs BDL + 100 mg/kg Gardenia jasminoides,188 ± 35.2 U/L vs 128 ± 19 U/L,P = 0.005) as well as hydroxyproline(BDL vs BDL + 100 mg/kg Gardenia jasminoides,438 ± 40.2 μg/g vs 228 ± 10.3 μg/g liver tissue,P = 0.004) after BDL.Furthermore,Gardenia jasminoides significantly reduced liver mRNA and/or protein expression of transforming growth factor β1(TGF-β1),collagen type?Ⅰ?(Col?Ⅰ) and α-smooth muscle actin(α-SMA).Gardenia jasminoides significantly suppressed the upregulation of TGF-β1,Col?Ⅰand α-SMA in LX-2 exposed to recombinant TGF-β1.Moreover,Gardenia jasminoides inhibited TGF-β1-induced Smad2 phosphorylation in LX-2 cells.CONCLUSION:Gardenia jasminoides exerts antifibrotic effects in the liver fibrosis and may represent a novel antifibrotic agent.

Association of overexpression of TIF1γ with colorectal carcinogenesis and advanced colorectal adenocarcinoma

AIM:To determine the expression and clinical significance of transcriptional intermediary factor 1 gamma (TIF1γ),Smad4 and transforming growth factor-beta (TGFβR) across a spectrum representing colorectal cancer (CRC) development.METHODS:Tissue microarrays were prepared from archival paraffin embedded tissue,including 51 colorectal carcinomas,25 tubular adenomas (TA) and 26 HPs,each with matched normal colonic epithelium.Immunohistochemistry was performed using antibodies against TIF1γ,Smad4 and TGFβ RⅡ.The levels of expression were scored semi-quantitatively (score 0-3 or loss and retention for Smad4).RESULTS:Overexpression of TIF1γ was detected in 5/26 (19%) HP;however,it was seen in a significantly higher proportion of neoplasms,15/25 (60%) TAs and 24/51 (47%) CRCs (P<0.05).Normal colonic mucosa,HP,and TAs showed strong Smad4 expression,while its expression was absent in 22/51 (43%) CRCs.Over-expression of TGFβ RⅡ was more commonly seen in neoplasms,13/25 (52%) TAs and 29/51 (57%) CRCs compared to 9/26 (35%) HP (P<0.05).Furthermore,there was a correlation between TIF1γ overexpression and Smad4 loss in CRC (Kendall tau rank correlation value=0.35,P<0.05).The levels of TIF1γ overexpression were significantly higher in stage Ⅲ than in stage Ⅰ and Ⅱ CRC (P<0.05).CONCLUSION:The findings suggest that over-expression of TIF1γ occurs in early stages of colorectal carcinogenesis,is inversely related with Smad4 loss,and may be a prognostic indicator for poor outcome.

Acupuncture and Moxibustion Inhibited Intestinal Epithelial-Mesenchymal Transition in Patients with Crohn's Disease Induced by TGF-β1/Smad3/Snail Pathway:A Clinical Trial Study

Objective:To explore whether acupuncture combined with moxibustion could inhibit epithelialmesenchymal transition in Crohn’s disease by affecting the transforming growth factorβ1(TGF-β1)/Smad3/Snail pathway.Methods:Sixty-three patients with Crohn’s disease were randomly divided into an observation group(31 cases)receiving moxibustion at 43℃ combined with acupuncture,and a control group(32 cases)receiving moxibustion at 37℃combined with sham acupuncture using a random number table.Patients were treated for12 weeks.Crohn’s Disease Activity Index(CDAI)was used to evaluate disease activity.Hematoxylin-eosin staining and transmission electron microscopy were utilized to observe the morphological and ultrastructural changes.Immunohistochemistry was used to detect the expression of transforming growth factorβ1(TGF-β1),TβR1,TβR2,Smad3,Snail,E-cadherin and fibronectin in intestinal mucosal tissues.Results:The decrease of the CDAI score,morphological and ultrastructural changes were more significant in observation group.The expression levels of TGF-β1,TβR2,Smad3,and Snail in the observation group were significantly lower than those before the treatment(P<0.05 or P<0.01).After treatment,the expression levels of TGF-β1,TβR2,and Snail in the observation group were significantly lower than those in the control group(all P<0.05);compared with the control group,the expression of fibronectin in the observation group was significantly decreased,and the expression of E-cadherin was significantly increased(all P<0.05).Conclusions:Moxibustion at 43℃combined with acupuncture may suppress TGF-β1/Smad3/Snail pathway-mediated epithelial-mesenchymal transition of intestinal epithelial cells in Crohn’s disease patients by inhibiting the expression levels of TGF-β1,TβR2,Smad3,and Snail.(Registration No.ChiCTR-IIR-16007751).

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

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