Dab2 attenuates brain injury in APP/PS1 mice via targeting transforming growth factor-beta/SMAD signaling

Transforming growth factor-beta （TGF-β） type II receptor （TβRⅡ） levels are extremely low in the brain tissue of patients with Alzheimer＇s disease. This receptor inhibits TGF-β1/SMAD signaling and thereby aggravates amyolid-beta deposition and neuronal injury. Dab2, a specific adapter protein, protects T RII from degradation and ensures the effective conduction of TGF-β 1/SMAD signaling. In this study, we used an adenoviral vector to overexpress the Dab2 gene in the mouse hippocampus and investigated the regulatory effect of Dab2 protein on TGF-β1/SMAD signaling in a mouse model of Alzheimer＇s disease, and the potential neuroprotective effect. The results showed that the TβRⅡ level was lower.in APP/PS1 mouse hippocampus than in normal mouse hippocampus. After Dab2 expression, hippocampal TβRⅡ and p-SMAD2/3 levels were signifi- cantly increased, while amyloid-beta deposition, microglia activation, tumor necrosis factor- and interleulin-6 levels and neuronal loss were significantly attenuated in APP/PS1 mouse brain tissue. These results suggest that Dab2 can exhibit neuroprotective effects in Alzheimer＇s disease by regulating TGF-β1/SMAD signaling.

Total flavone of Abelmoschus manihot suppresses epithelial-mesenchymal transition via interfering transforming growth factor-β1 signaling in Crohn's disease intestinal fibrosis

AIM To explore the role and mechanism of total flavone of Abelmoschus manihot(TFA) on epithelial-mesenchymal transition(EMT) progress of Crohn's disease(CD) intestinal fibrosis.METHODS First,CCK-8 assay was performed to assess TFA on the viability of intestinal epithelial(IEC-6) cells and select the optimal concentrations of TFA for our further studies.Then cell morphology,wound healing and transwell assays were performed to examine the effect of TFA on morphology,migration and invasion of IEC-6 cells treated with TGF-β1.In addition,immunofluorescence,real-time PCR analysis(q RT-PCR) and western blotting assays were carried out to detect the impact of TFA on EMT progress.Moreover,western blotting assay was performed to evaluate the function of TFA on the Smad and MAPK signaling pathways.Further,the role of co-treatment of TFA and si-Smad or MAPK inhibitors has been examined by q RTPCR,western blotting,morphology,wound healing andtranswell assays.RESULTS In this study,TFA promoted transforming growth factor-β1(TGF-β1)-induced(IEC-6) morphological change,migration and invasion,and increased the expression of epithelial markers and reduced the levels of mesenchymal markers,along with the inactivation of Smad and MAPK signaling pathways.Moreover,we revealed that si-Smad and MAPK inhibitors effectively attenuated TGF-β1-induced EMT in IEC-6 cells.Importantly,co-treatment of TFA and si-Smad or MAPK inhibitors had better inhibitory effects on TGF-β1-induced EMT in IEC-6 cells than either one of them.CONCLUSION These findings could provide new insight into the molecular mechanisms of TFA on TGF-β1-induced EMT in IEC-6 cells and TFA is expected to advance as a new therapy to treat CD intestinal fibrosis.

TGF-β1/SMAD SIGNALING PATHWAY MEDIATES p53-DEPENDENT APOPTOSIS IN HEPATOMA CELL LINES

Objective To determine whether transforming growth factor betal (TGF-β1)/Smad signaling pathway mediates p53-dependent apoptosis in hepatoma cell lines.Methods Three human hepatic carcinoma cell lines, HepG2, Huh-7, and Hep3B, were used in this study.TGF-β1-induced apoptosis in hepatic carcinoma cell lines was analyzed using TUNEL assay.For identifying the mechanism of apoptosis induced by TGF-β1, cell lines were transfected with a TGF-β1-inducible luciferase reportor plasmid containing Smad4 binding elements.After transfection, cells were treated with TGF-β1, then assayed for luciferase activity.Results The apoptosis rate of HepG2 cell lines (48.51%± 8.21%) was significantly higher than control ( 12.72%±2.18%, P<0.05).But TGF-β1 was not able to induce apoptosis of Huh-7 and Hep3B cell lines.The relative luciferase activity of TGF-β1-treated HepG2 cell lines (4.38) was significantly higher than control (1.00, P< 0.05).But the relative luciferase activity of TGF-β1-treated Huh-7 and Hep3B cell lines less increased compared with control.Conclusions HepG2 cells seem to be highly susceptible to TGF-β1-induced apoptosis compared with Hep3B and Huh-7 cell lines.Smad4 is a central mediator of TGF-β1 signaling transdution pathway.TGF-β1/Smad signaling pathway might mediate p53-dependent apoptosis in hepatoma cell lines.

Regulation Mechanism of TFP on TGF-β1/STAT3 Signaling Pathway in Immune-mediated Liver Injury in Mice

[Objectives]To study the effect of total flavonoids extracted from Polygonum perfoliatum L.(TFP)on immune-mediated liver injury induced by bacillus Calmette-Guerin plus lipopolysaccharide(BCG+LPS)in mice,and to explore its action mechanism.[Methods]60 Kunming mice were divided into normal group,model group,control group(bifendate)and TFP low,medium and high dose groups according to random number table method,with 10 mice in each group.On the first day of modeling,mice were injected with 0.2 mL of BCG solution(12.5 mg/mL)through the tail vein,and on the eleventh day,0.2 mL of LPS(37.5μg/mL)were injected into the tail vein to prepare a mouse model of immune-mediated liver injury;from the first day of modeling,the normal group and the model group were administered intragastrically with the corresponding volume of distilled water,and the bifendate group and the TFP high,medium,and low dose groups were administered intragastrically with the corresponding doses once a day for 11 d.After the last time administration,fasting but giving water for 16 h,took blood from eyes,then collected the liver tissue.The levels of alanine transaminase(ALT)and aspartate transaminase(AST)in serum were detected by biochemical method;transforming growth factor-β1(TGF-β1),intercellular adhesion molecule-1(ICAM-1),interleukin-6(IL-6)and interleukin-1β(IL-1β)expression levels in liver tissue were detected by enzyme-linked immunosorbent assay(ELISA);phosphorylated protein tyrosine kinase JAK-2(p-JAK2),phosphorylated signal transducer and activator of transcription 3(p-STAT3)protein expression levels were detected by Western Blot method;the degree of liver tissue lesions was detected by HE staining.[Results]Compared with the model group,the levels of ALT and AST in the serum of mice in each dose group of TFP(high dose 600 mg/kg,medium dose 400 mg/kg,and low dose 200 mg/kg)were reduced,and the activities of T-SOD and GSH-Px were increased;the content or expression ofβ1,ICAM-1,IL-6,IL-1βdecreased,and the expression of p-JAK2 and p-STAT3 protein decreased;pathological sections showed that the degree of inflammatory necrosis and the degree of lesions in the liver tissues of each dose group of TFP were reduced by varying degrees.[Conclusions]TFP has a protective effect on BCG+LPS-induced immune-mediated liver injury in mice.The mechanism may be related to regulating the phosphorylation level of JAK2 and inhibiting the inflammatory reaction,thereby regulating the TGF-β1/STAT3 signaling pathway and improving the immune-mediated liver injury.

Yangfei Kongliu Formula, a compound Chinese herbal medicine, combined with cisplatin, inhibits growth of lung cancer cells through transforming growth factor-β1 signaling pathway

OBJECTIVE： To investigate the tumor inhibition effect of Yangfei Kongliu Formula （YKF）, a compound Chinese herbal medicine, combined with cisplatin （DDP） and its action mechanisms. METHODS： C57BL/6 mice with Lewis lung carcinoma were divided into six groups： control group （C）, DDP group （2 mg/kg, DDP）, low-dose YKF group （2.43 g/kg, L）, high-dose YKF group （24.3 g/kg, H）, low- dose YKF combined with DDP group （L ＋ DDP） and high-dose YKF combined with DDP group （H ＋ DDP）. Transforming growth factor-β1 （TGF-β1）, mothers against decapentaplegic homolog 3 （Smad3） and Smad7 levels were measured with quantitative real-time polymerase chain reaction （qPCR）, Western blotting and immunohistochemistry. An enzyme-linked immunosorbent assay was used to analyze the expressions of interleukin-2 （IL-2） and tumor necrosis factor-α （TNF-α）. RESULTS： YKF combined with DDP significantly inhibited the growth and metastasis of tumors relative to the control group, and YKF groups （P 〈 0.05）. There was no significant difference between high-dose YKF group and low-dose YKF group （P 〉 0.05）. We also found that the expression levels of TGF-β1 and Smad3 were both significantly decreased by YKF relative to the control group （P 〈 0.05）. Furthermore, after treatment with YKF combined with DDP, the expression levels of TGF-β1 and Smad3 were decreased but the expression level of Smad7 was increased relative to the DDP group （P 〈 0.05）. Compared to the DDP group, the combination of YKF and DDP enhanced the effect of tumor inhibition （P 〈 0.05）, showing obvious synergy between YKF and DDP. Treatment with DDP or YKF decreased serum levels of IL-2 and TNF-α relative to the control group （P 〈 0.05）. Furthermore, the expression levels of IL-2 and TNF-α were significantly decreased when treated with YKF in combination with DDP. Co-treatment with YKF and DDP significantly inhibited tumor growth, decreased the expressions of TGF-β1, Smad3, IL-2 and TNF-α and increased the expression of Smad7; these differences were significant relative to both YKF groups and the control group （P 〈 0.05）. CONCLUSION： YKF can inhibit tumor growth synergistically with DDP, mainly through the TGF-β1 signaling pathway.

Role of TGF-β1/Smads pathway in carotid artery remodeling in renovascular hypertensive rats and prevention by Enalapril and Amlodipine

Objective To investigate the role of transforming growth factor-β1 （TGF-β1）, Smad2/3 and Smad7 expressions in carotid artery remodeling in renovascular hypertensive rats, and also the therapeutic effect of Enalapril and Amlodipine. Methods The renovascular hypertensive rat （RHR） models with ＂two-kidney and one-clip＂ were established, including model group （n = 6）, sham-operated group （n = 6）, Enalapril group （10 mg/kg per day, n = 6）, Amlodipine group （5 mg/kg per day, n = 6） and combination group （Amlodipine 2.5 mg/kg per day ＋ Enalapril 5mg/kg per day, n = 6）. The medication were continuous administrated for six weeks. Carotid artery morphological and structural changes in the media were observed by HE staining, Masson staining and immuno histochemical staining. Media thickness （MT）, MT and lumen diameter ratio （MT/LD）, and the expression levels of media a-smooth muscle actin （α-actin）, proliferating cell nuclear antigen （PCNA）, TGF-β1, phosphorylated Smad2/3 （p-Smad2/3） and Smad7 in carotid arteries were measured. Results The media of carotid arteries in RHR model group was significantly thickened, the volume of smooth muscle cell was increased, and the array was in disorder; MT, MT/LD, the proliferation index of smooth muscle cell and collagen fiber area percentage of carotid arteries in the model group were significantly higher than those in the sham-operated group （P 〈 0.01）. Compared to sham-operated group, the model group had significantly higher expressions of TGF-β1 and p-Smad2/3 （P 〈 0.05） and lower Smad7 expression. Both Enalapril and Amlodipine improved smooth muscle hypertrophy and collagen deposition, reduced RHR carotid MT, MT/LD, proliferation index of smooth muscle cell, collagen fiber area percentage and the expressions of TGF-β1 and p-Smad2/3 （P 〈 0.05）, increased Smad7 expression （P 〈 0.05）. Moreover, the combination treatment of Enalapril and Amlodipine had significantly better effects than single Amlodipine group （P 〈 0.05）, but not single Enalapril group. Conclusions TGF-β1/Smads pathway may participate in the mechanism of carotid artery remodeling in RHR; the role of Amlodipine and Enalapril in inversing carotid artery remodeling may be related to the change of TGF-β1/Smads pathway, the combination treatment of Amlodipine and Enalapril had better effects than single administration of Amlodipine.

基于TGF-β_(1)/Smad3信号通路探讨参附注射液对缺血性脑卒中大鼠的神经保护作用

目的 基于转化生长因子β_(1)(TGF-β_(1))/Smad家族成员3(Smad3)信号通路探讨参附注射液对缺血性脑卒中(IS)大鼠的神经保护作用机制。方法 将48只无特定病原体(SPF)级SD雄性大鼠随机分为Sham组、Vehicle组、参附注射液组、TGF-β_(1)组。采用改良Longa线栓法构建Vehicle组、参附注射液组、TGF-β_(1)组大鼠永久性中动脉栓塞模型,分别腹腔注射相应剂量的生理盐水、参附注射液和TGF-β_(1),Sham组只插入线栓不结扎其余操作同Vehicle组。建模14 d后进行神经功能缺损评分;采用激光散斑成像观察大鼠大脑皮层血流变化;伊文思蓝染色检测血脑屏障通透性;2,3,5-三苯基氯化四氮唑(TTC)染色检测脑梗死面积;尼氏染色观察脑组织神经元损伤程度;原位末端凋亡检测(TUNEL)法检测细胞凋亡;检测白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、超氧化物歧化酶(SOD)、丙二醛(MDA)水平;蛋白免疫印迹(Western Blot)法检测TGF-β_(1)、p-Smad3、B细胞淋巴瘤-2基因(Bcl-2)、Bcl-2相关X蛋白(Bax)表达。结果 与Sham组比较,Vehicle组大鼠大脑皮层血流灌注量、SOD水平、Bcl-2蛋白水平减少,神经功能损伤评分、脑梗死面积、血脑屏障通透性、细胞凋亡、神经元损伤、炎性因子IL-1β、IL-6、TNF-α含量、MDA水平及TGF-β_(1)、p-Smad3水平、Bax蛋白水平增加(P<0.05);与Vehicle组比较,参附注射液组和TGF-β_(1)组大鼠大脑皮层血流灌注量、SOD水平、TGF-β_(1)、p-Smad3水平、Bcl-2蛋白水平增加,神经功能损伤评分、脑梗死面积、血脑屏障通透性、细胞凋亡、神经元损伤、炎性因子IL-1β、IL-6、TNF-α表达、MDA水平、Bax蛋白水平降低(P<0.05)。结论 参附注射液可改善脑缺血大鼠血流灌注,缩小脑梗死体积,抑制机体氧化应激、炎症反应及细胞凋亡,可能通过激活TGF-β_(1)/Smad3信号通路发挥对IS大鼠神经细胞的保护作用。

Targeting key signalling pathways in oesophageal adenocarcinoma:A reality for personalised medicine?

Cancer treatments are rapidly changing.Curative treatment for oesophageal adenocarcinoma currently involves surgery and cytotoxic chemotherapy or chemoradiotherapy.Outcomes for both regimes are generally poor as a result of tumor recurrence.We have reviewed the key signalling pathways associated with oesophageal adenocarcinomas and discussed the recent trials of novel agents that attempt to target these pathways.There are many trials underway with the aim of improving survival in oesophageal cancer.Currently,phase 2 and 3 trials are focused on MAP kinase inhibition,either through inhibition of growth factor receptors or signal transducer proteins.In order to avoid tumor resistance,it appears to be clear that targeted therapy will be needed to combat the multiple signalling pathways that are in operation in oesophageal adenocarcinomas.This may be achievable in the future with the advent of gene signatures and a combinatorial approach.

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).

Mechanism of ELL-associated factor 2 and vasohibin 1 regulating invasion,migration,and angiogenesis in colorectal cancer

BACKGROUND As a novel endogenous anti-angiogenic molecule, vasohibin 1(VASH1) is not only expressed in tumor stroma, but also in tumor tissue. Moreover, studies have shown that VASH1 may be a prognostic marker in colorectal cancer(CRC). Knockdown of VASH1 enhanced transforming growth factor-β1(TGF-β1)/Smad3 pathway activity and type Ⅰ/Ⅲ collagen production. Our previous findings suggest that ELL-associated factor 2(EAF2) may play a tumor suppressor and protective role in the development and progression of CRC by regulating signal transducer and activator of transcription 3(STAT3)/TGF-β1 signaling pathway. However, the functional role and mechanism of VASH1-mediated TGF-β1 related pathway in CRC has not been elucidated.AIM To investigate the expression of VASH1 in CRC and its correlation with the expression of EAF2. Furthermore, we studied the functional role and mechanism of VASH1 involved in the regulation and protection of EAF2 in CRC cells in vitro.METHODS We collected colorectal adenocarcinoma and corresponding adjacent tissues to investigate the clinical expression of EAF2 protein and VASH1 protein in patients with advanced CRC. Following, we investigated the effect and mechanism of EAF2 and VASH1 on the invasion, migration and angiogenesis of CRC cells in vitro using plasmid transfection.RESULTS Our findings indicated that EAF2 was down-regulated and VASH1 was upregulated in advanced CRC tissue compared to normal colorectal tissue. KaplanMeier survival analysis showed that the higher EAF2 Level group and the lower VASH1 Level group had a higher survival rate. Overexpression of EAF2 might inhibit the activity of STAT3/TGF-β1 pathway by up-regulating the expression of VASH1, and then weaken the invasion, migration and angiogenesis of CRC cells.CONCLUSION This study suggests that EAF2 and VASH1 may serve as new diagnostic and prognostic markers for CRC, and provide a clinical basis for exploring new biomarkers for CRC. This study complements the mechanism of EAF2 in CRC cells, enriches the role and mechanism of CRC cellderived VASH1, and provides a new possible subtype of CRC as a therapeutic target of STAT3/TGF-β1 pathway.

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.

Overexpression of ELL-associated factor 2 suppresses invasion,migration,and angiogenesis in colorectal cancer

BACKGROUND The androgen responsive gene,ELL-associated factor 2(EAF2),expressed in benign prostate tissues,has been shown to play an important role in tumor suppression in a variety of malignant tumors.In addition,some scholars found that EAF2 frameshift mutations are associated with intratumor heterogeneity in colorectal cancer(CRC)and inactivation of EAF2 in microsatellite instability-high CRC.However,the molecular mechanism by which EAF2 is involved in CRC invasion and metastasis remains unclear.AIM To determine the clinical value of expression of EAF2 protein in CRC,and to study the effects of EAF2 on the invasion,migration,and angiogenesis of CRC cells in vitro.METHODS In this study,we collected colorectal adenocarcinoma and corresponding adjacent tissues to investigate the clinical expression of EAF2 protein in patients with advanced CRC.Subsequently,we investigated the effect of EAF2 on the invasion,migration,and angiogenesis of CRC cells in vitro using plasmid transfection.RESULTS EAF2 protein was lowly expressed in cancer tissues of patients with advanced CRC.Kaplan-Meier survival analysis showed that the survival rate of the high EAF2 level group was higher than that of the low EAF2 level group.CONCLUSION Our results demonstrated that EAF2,as a tumor suppressor,may inhibit the invasion,metastasis,and angiogenesis of CRC cells by regulating the signal transducer and activator of transcription 3/transforming growth factor-β1 crosstalk pathway,and play a cancer suppressive and protective role in the occurrence and development of CRC.Our findings are of great significance to provide a new idea and theoretical basis for the targeted diagnosis and treatment of CRC.

左归丸治疗骨质疏松症相关机制

左归丸是中医经典方剂之一，为“阳中求阴”的代表方，具有滋肾补阴的功效和纯补无泻的特点。骨质疏松症是一种以骨量低，骨组织结构破坏，导致骨脆性增加，骨强度下降及骨折风险增加，易发生骨折为特征的全身性骨病，它已成为威胁人类健康的常见疾病之一。中医学有“肾藏精，主骨生髓”的理论，左归丸在治疗骨质疏松症方面有着较好的临床疗效，同时也具有较少的副作用。近年来，针对左归丸治疗骨质疏松症的实验研究数量与日俱增，相关分子以及信号通路的机制研究亦取得一定拓展，特别是左归丸治疗骨质疏松症的相关机制研究陆续涌现。本研究通过整理左归丸治疗骨质疏松症的文献，对其作用机制及信号通路，包括环腺苷酸（cAMP）／蛋白激酶（PKA）／cAMP应答元件结合蛋白（CREB）通路，Notch信号通路，转化生长因子巾，（TGF-卢，）／Smad信号通路，Wnt／卢一链蛋白（卢一catenin）信号通路和丝裂原活化蛋白激酶（MAPK）信号通路等方面做一综述。这些研究表明左归丸治疗骨质疏松症具有多靶点、多途径的干预特点。虽然左归丸治疗骨质疏松症的疗效明确，且对于一些基本的机制研究亦取得一定进展，但仍存在一定的局限性，尚需结合现代最新研究方法和技术对其机制进行更全面更深入的探索。

Effect and Mechanism of Ganoderma lucidum Polysaccharides on Human Fibroblasts and Skin Wound Healing in Mice

Objective: To investigate the effects of Ganoderma lucidum polysaccharides(GL-PS) on human fibroblasts and skin wound healing in Kunming male mice and to explore the putative molecular mechanism. Methods: Primary human skin fibroblasts were cultured. The viability of fibroblasts treated with 0, 10, 20, 40, 80, and 160 μg/mL of GL-PS, respectively were detected by 3-4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2-Htetrazolium bromide(MTT). The migration ability of fibroblasts treated with 0, 10, 20, and 40 μg/mL of GL-PS were measured by transwell assay. The secretion of the C-terminal peptide of procollagen type Ⅰ(CICP) and transforming growth factor-β1(TGF-β1) in the cell supernatant was tested by enzyme-linked immunosorbent assay. The expression of β-catenin was detected by Western blot. Furthermore, the Kunming mouse model with full-layer skin resection trauma was established, and was treated with 10, 20, and 40 mg/mL of GL-PS, respectively as external use. The size of the wound was measured daily, complete healing time in each group was recorded and the percentage of wound contraction was calculated. Results: Compared with the control group, 10, 20, and 40 μg/mL of GL-PS significantly increased the viability of fibroblasts, promoted the migration ability of fibroblasts, and up-regulated the expressions of CICP and TGF-β1 in fibroblasts(P<0.05 or P<0.01). The expression of β-catenin in fibroblasts treated with 20 and 40 μg/mL of GL-PS was significantly higher than that of the control group(P<0.01). Furthermore, after external use of 10, 20, and 40 mg/mL of GL-PS, the rates of wound healing in mice were significantly higher and the wound healing time was significantly less than the control group(P<0.05 or P<0.01). Conclusion: A certain concentration of GL-PS may promote wound healing via activation of the Wnt/β-catenin signaling pathway and up-regulation of TGF-β1, which might serve as a promising source of skin wound healing.

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.