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

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

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

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

Molecular mechanism of benign biliary stricture inhibition by rosiglitazone-activated peroxisome proliferator-activated receptor gamma

SUMMARY OBJECTIVE: The aim of this study was to investigate whether rosiglitazone-activated peroxisome proliferator-activated receptor gamma can inhibit the occurrence of benign biliary stricture and further elucidate the relevant molecular signaling mechanism. METHODS: The primary cultured rat biliary fibroblasts following experiments were performed using within the fifth generation cells, which were separated from the bile ducts of Sprague-Dawley rats. The primary cultured rat biliary fibroblasts were co-cultured with 10 ng/mL transforming growth factor-beta 1 for stimulating collagen formation. Competent cells were transfected with siRNA that specifically target Smad3 or connective tissue growth factor to inhibit the expression of the corresponding proteins. The cells were incubated with 10 μmol/L rosiglitazone to activate peroxisome proliferator-activated receptor gamma. The cells were incubated with 10 μmol/L GW9662 in the pretreatment session to inactivate peroxisome proliferator-activated receptor gamma. ELISA was used to determine the levels of connective tissue growth factor and type I collagen in the cell supernatant. Western blotting was used to detect the levels of intracellular p-Smad3/t-Smad3. RESULTS: Rosiglitazone-activated peroxisome proliferator-activated receptor gamma inhibited the secretion of type I collagen induced by transforming growth factor-beta 1. Peroxisome proliferator-activated receptor gamma inhibitor GW9662 could significantly reverse the rosiglitazone-triggered inhibition of transforming growth factor-beta 1-induced type I collagen secretion by suppressing peroxisome proliferator-activated receptor gamma activation (p<0.01). Furthermore, we also found that the activation of peroxisome proliferator-activated receptor gamma was accompanied by the inhibition of transforming growth factor-beta 1-induced Smad3 phosphorylation (p<0.01), increased connective tissue growth factor expression (p<0.01), and production of type I collagen (p<0.01), all of which effects elicited by rosiglitazone could be reversed by peroxisome proliferator-activated receptor gamma inhibitor GW9662. CONCLUSION: Peroxisome proliferator-activated receptor gamma activated by rosiglitazone inhibits the transforming growth factor-beta1 -induced phosphorylation of Smad3 and the increased connective tissue growth factor expression as well as inhibits the secretion of type I collagen in biliary fibroblasts.

Research progress of endothelial-to-mesenchymal transition in ocular diseases / 国际眼科杂志(Guoji Yanke Zazhi)

@#Endothelial-to-mesenchymal transition(EndoMT)is a change in the transformation or differentiation of endothelial cells into mesenchymal cells under physiological or pathological conditions, accompanied by changes in phenotype and function, and is an important part of fiber repair. It is widely involved in the pathophysiological process of embryonic development, tumor invasion and a variety of fibrotic diseases. Research on the role of EndoMT in ocular diseases has also made some progress. This article will review the basic biological characteristics, mechanism and research results of EndoMT in ophthalmological diseases, intending to theoretically reveal its possibility as a treatment target and a key point of regenerative medicine technology in related diseases, provide a reference for clinical practice and scientific research.

Research progress on the role of transforming growth factor-β in tooth and craniofacial bone development and diseases / 口腔疾病防治

@#Dental and craniofacial bone development is a highly coordinated process that is tightly controlled by genetics and influenced by complex environments. The abnormal regulation of many development-related signaling molecules may lead to abnormal tooth development, severe craniofacial bone formation disorders, and developmental deformities. Transforming growth factor-β (TGF-β) is widely expressed in vivo and participates in many cellular biological processes, showing complex regulatory roles in mammalian craniofacial bone growth and tooth development. In tooth development, abnormal TGF-β signaling can lead to the failure of tooth germ formation, and its deletion mutation can directly affect odontoblast differentiation and enamel formation defects. However, the current research on TGF-β mainly focuses on the early stage of tooth development, and a comprehensive and systematic study of TGF-β-related tooth development is lacking. TGF-β signal transduction mainly controls the development of teeth and craniofacial bone by regulating the expression of development-related molecules via the classical Smad-dependent signaling pathway. In addition, the nonclassical mitogen-activated protein kinase (MAPK) pathway also participates in this process. Abnormal TGF-β signaling may cause jaw development disorders, temporomandibular joint dysplasia and inflammation, and cleft palate. Because the specific regulatory mechanism of TGF-β in craniofacial bone development has not been fully elucidated, its specific application in the treatment of related diseases is also greatly limited. This paper describes the new research progress of TGF-β in the development of teeth, jaws, temporomandibular joints and palate as well as related diseases.

Clinical significance of FOXD1 expression in esophageal squamous cell carcinoma and the mechanism of its effect on the malignant biological behaviors of TE1 cells / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：分析FOXD1在食管鳞状细胞癌（ESCC）组织中的表达及其与临床病理特征和患者预后的关系，探讨其对ESCC TE1细胞增殖、侵袭能力的影响及其对TGF-β1诱导TE1细胞EMT进程的影响。方法：采用qPCR和IHC法检测ESCC组织和细胞中FOXD1的表达，并分析其与临床病理特征和患者预后的关系；构建FOXD1敲减质粒并转染TE1细胞，检测其对TE1细胞增殖、侵袭能力的影响；用qPCR和WB法检测TGF-β1处理前后FOXD1及EMT相关基因和蛋白的表达变化及敲减FOXD1对EMT相关基因和蛋白表达的影响。结果：ESCC组织和细胞中FOXD1均呈高表达（均P<0.01），并与患者OS呈负相关；FOXD1表达水平、肿瘤TNM分期以及淋巴结转移均是影响ESCC患者预后的独立危险因素（均P<0.01）。TGF-β1可促进TE1细胞FOXD1的表达，并诱发其EMT进程（均P<0.05）；敲减FOXD1可抑制TE1细胞的增殖和侵袭能力，并可部分逆转由TGF-β1诱发的TE1细胞EMT进程。结论：FOXD1在ESCC组织及TE1细胞中呈高表达且是影响ESCC患者预后的独立危险因素，敲低FOXD1可显著抑制TE1细胞的增殖、侵袭及TGF-β1介导的EMT进程。

Ethanol Extract of Yacon Leaves (Smallanthus sonchifolius) Attenuates Fibroblast and Myofibroblast Expansion in Association With Downregulation of TGF β1 and Snail mRNA Expression in 5/6-Subtotal Nephrectomy Model in Mice

@#Introduction: Chronic kidney disease (CKD) is characterized by fibroblast activation, myofibroblast formation, and up-regulation of transforming growth factor-β1 (TGF-β1) that may activate Snail in fibroblast to myofibroblast transition. Ethanol extract of Yacon leaves is known to have a renoprotective effect on diabetic nephropathy but its effect in the CKD model is unknown. This experimental study aimed to elucidate the effect of ethanol extract from Yacon leaves in attenuating renal failure in a CKD mice model. Methods: Male Swiss-Webster mice (3 months, 30–40 grams, n=25) underwent 5/6 subtotal nephrectomy (SN) to induce CKD. The mice were divided into five groups: SN, SN mice with oral treatment of Yacon leaves ethanol extract with doses 0.735 μg/kg (SN+YK1), 1.47 μg/kg (SN+YK2), and 2.94 μg/kg (SN+YK3), and a Sham operation (SO) group with aquadest 0.1% supplementation. Mice were euthanized on day 14 after the operation and kidneys were harvested. Paraffin sections were used for histological analysis. Immunostaining was done for quantifying fibroblasts and myofibroblasts. We performed RT-PCR to measure TGF-β1 and Snail mRNA expressions. Results: The SN group had significantly higher fibroblast number, myofibroblast fraction area, TGF-β1 and Snail mRNA expressions compared to the SO. The fibroblasts number (p<0.001) and myofibroblast fraction areas (p<0.001) were significantly lower in Yacon treated-groups compared to the SN group. RT-PCR analysis showed lower mRNA expressions of TGF-β1 and Snail, but no significant differences were found among the various Yacon treated-groups. Conclusion: Ethanol extracts of Yacon leaves improved kidney damage in male mice with 5/6 subtotal nephrectomy model.

lncRNA-Z38 promotes occurrence and progression of gastric cancer though regulating TGF-β signaling pathway / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨lncRNA-Z38对胃癌细胞恶性生物学行为的影响及其可能的调控机制。方法：构建lncRNA-Z38稳定过表达的胃癌细胞系（AGS、MKN74细胞），通过细胞成球实验检测过表达lncRNA-Z38对胃癌细胞肿瘤特征的影响。对过表达lncRNA-Z38的AGS细胞及对照细胞进行高通量测序，对差异表达基因进行KEGG富集分析，筛选出可能参与调控胃癌发生发展的信号通路，采用WB等技术验证lncRNA-Z38通过调控该信号通路参与提高胃癌细胞的干性。结果：成功构建lncRNA-Z38过表达细胞株，细胞成球实验提示lncRNA-Z38过表达细胞的成球能力显著高于对照细胞（P<0.05）。通过测序筛选出lncRNA-Z38过表达胃癌细胞中的1 999个差异表达基因，其中上调基因1 238个、下调基因761个；经KEGG富集分析得到其中变化最显著的通路为TGF-β信号通路（P<0.05）。WB实验结果提示，lncRNA-Z38高表达胃癌细胞中TGF-β信号通路组成基因编码蛋白ID3、BMP6显著上调（均P<0.05）、GDF-5、WNT8B显著下调（P<0.05）。结论：在胃癌中高度表达的lncRNA-Z38通过调控TGF-β信号通路提高胃癌细胞的干性。

Differential expression of TGF-β1 receptors ALK1/ALK5 in pterygium and normal conjunctiva tissues / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To investigate the expression of different kinds of transforming growth factors beta- 1(TGF- β1)and changes of activin receptor-like kinase(ALK)in pterygium and normal conjunctiva tissues. <p>METHODS: A total of 40 cases(40 eyes)of pterygium patients who underwent surgical treatment in our hospital were selected. In the same period, 40 cases(40 eyes)of normal conjunctiva tissues removed from the eye due to cataract surgery were selected. The expression of TGF-β1 receptors(ALK1/ALK5)in pterygium and normal conjunctiva tissues was detected by immunohistochemistry, with the proportion of positive staining cells counted. The expression of ALK1 and ALK5 mRNA and their proteins were quantified by reverse transcription polymerase chain reaction(RT-PCR)and Western Blot, respectively.<p>RESULTS: According to immunohistochemistry results, the ALK1 expression level was increased more distinct in pterygium group, compared to the normal conjunctiva group, and it was detected throughout the full-thickness pterygium epithelial cells, while only in the basal layer of epithelial cells in normal conjunctiva tissues; the ALK5 was detected in the basal layer of epithelial cells in both groups, while its level was decreased in the pterygium group compared to normal conjunctiva group. There was significant difference in the proportion of ALK1 and ALK5 positive cells between the two groups(all <i>P</i><0.05). The expression of the ALK1 mRNA and its protein in the pterygium tissues were significantly elevated, while the ALK5 mRNA level and its protein was significantly decreased, compared with the normal conjunctival group(<i>P</i><0.05).<p>CONCLUSION: Compared with the normal conjunctiva tissues, the expression of ALK1 and ALK5 in pterygium tissues was increased and decreased, respectively. This indicated different activation status of TGF-β signaling pathway, providing experimental evidence for further study on the pathogenesis of pterygium.

Impact of Allium sativum ethanol extract on immuno-regulatory T cells and anti-inflammatory cytokine profile in murine schistosomiasis

@#Parasite immune response against schistosomal antigens involves both the innate and adaptive immune response. Tregs have a suppressive effect and play a role on the parasite’s immune evasion. This study aimed to evaluate active compounds of Allium sativum (AS) ethanol extract and the impact of AS extract alone or in combination with praziquantel on Tregs and anti-inflammatory cytokines TGF-β and IL-10 in mice infected with S. mansoni. Phytochemical screening of AS bulbs for various active constituents and qualitative and quantitative analysis of the flavonoids and phenolic acids were done using HPLC. Measurement of splenocytes Treg cell phenotypes and anti-inflammatory cytokines TGF-β and IL-10 was done by flow cytometric analysis. The data are expressed as mean ± SD. Statistical significance was determined by one-way ANOVA utilizing the statistical package (SPSS version 17.0). HPLC of AS ethanol extract revealed presence of 22 and 18 compounds of flavonoids and phenolic acids, respectively. S. mansoni infection upregulated the Treg cells subsets (CD4, CD25, Foxp3) frequencies and the levels of TGF-β and IL-10 anti-inflammatory cytokines when compared to healthy control. AS ethanol extract alone or combined with PZQ decreases the production of Treg cells from spleen in addition to the reduction in antiinflammatory cytokines IL-10 and TGF-β. This study recommends that the combination of AS ethanol extract and PZQ may play a role in maintaining the homeostasis of the immune system during schistosomiasis by decreasing Treg cells and anti-inflammatory cytokines IL10 and TGF-β production.

Results of the bone healing performance of Osteo-calcium 5 on based preparation tested by internal fixation surgery patients / Шинэ санаа Шинэ нээлт

Background@#Even though there are lots of good treatment methods, medicines and biomaterials in use for broken bones due to international scientific researches on the ossifying process and its mechanism, which are being studied on high level and bringing new solutions, there is still a lack of inexpensive but good quality medicines that have low side effects and specifically targets on the enhancement of osteoblasts. </br> Despite of the level of fractures, therapeutic effects and multiple bone fractures, only certain drugs and medicines such as solution of baragshun and rhodiola, kitchen grime, chili-san and calcium are used for ossifying broken bones and fractures in Mongolia which indicates that there are certain needs of new medicine and drugs.@*Methods@#Patients who had a intramedullary osteosynthesis surgery to deal with tibia bone fractures were divided randomly into two groups; 1.5 gram Osteo calcium-5 is given to the experimental group while the comparison group got Calcium nycomed D3 for 60 days. </br> X-ray pictures were taken at the 56th and the 84th day after the fractures were diagnosed and analyzed by Warden method (et al 2009). TGF-β1, BMP-2 cytokines and alkaline phosphatase were evaluated on the 3rd, the 14th and the 42nd day of the study.@*Results@#Compared to the comparison group, which used Calcium Nycomed D3, the experimental group patient, who used osteocalcium-5, had a similar TGF-β1 level at the 3rd day (p=1.0), a higher level at the 14th day (p=0.0001) and a lower level at the 42nd day (p=0.015).Analyzing fracture ossifying process with the Warden et al method shows that the ossifying points of the Osteo calcium-5 group were 1.73 on 53rd day and 2.79 on 84th day while the ossifying points of the comparison group was 1.41 on 56th day and 2.35 on 84th day (p=0.034),( p=0.04).@*Conclusions@#According to the study, Osteo calcium-5 has proven to have better effects on decreasing inflammation process and accelerating bone ossify, cartilage and bone formation with the analysis of cytokine in serum and structure.

The Potential of Red Dragon Fruit (Hylocereuspolyrhizus) Rind Extract Accelerates Wound Healing Process Post Tooth Extraction

@#Introduction: Biological process of wound healing in soft tissue injury which include some phase that its highly organized cascade, such as haemostasis, inflammation, proliferation and remodeling. Healing process will be impaired when there is an interference with one of the four of the phase not going well. This study is aimed to proving the role of dragon fruit peel extract towards the interpretation of TGF- β, fibroblast cell and formation of new blood vessels during wound healing process. Methods: 20 rattus norwegicus wistar strain rats are separated into four groups. The one lower incisor is extracted, afterward the wound post extraction of the treatment group was treated with dragon fruit peel extract gel with the concentration of 15%, 30% and 60%, meanwhile, for the control groups, they are treated with polyethylene glycol gel. On the fourth day, the rats were sacrificed and preparation of immunohistochemical and histopathology are made. Observe under the light microscope with 400x magnification. Results: This study shows that the 30% concentration increase the expression of TGF- β (16.6±2.07), fibroblast proliferation (77.6±5.27) and new blood vessels (33.8±2.28) compare to the control group, 15% and 60% concentration in a meaningful way (p=0.00, p=0.00 and p=0.00). Conclusions: The dragon fruit peel extract gel may affect the wound healing process of extraction by increasing the expression of TGF- β, number of fibroblast and formation of new blood vessels.

Effect of lncRNA PCGEM1 on malignant biological behavior of lung cancer A549 cells and its mechanism / 中国肿瘤生物治疗杂志

@#[Abstract] Objective ： ：To investigate the long-chain noncoding RNA (Lnc RNA) PCGEM1 regulating the lung cancer (LC) cell invasion and metastasis through the TGF-β/Smad signaling pathways. Methods： ：From March 2016 to May 2018, total 62 cases of LC patients receiving surgical treatment in our hospital were collected, including cancer tissues and normal tissues more than 2 cm away from the cancer tissues. qRT-PCR was used to detect the expression of lncRNA PCGEM1 and miR-148a in LC, corresponding para-cancer tissues and different LC cell strains. LncRNA PCGEM1 silenced cell line A549-siPCGEM1 and negative control A549-NC were constructed, and A549 was used as blank control. MTT and plate cloning assay were used to detect the effect of PCGEM1 on the proliferation of A549 cells. Transwell and scratch assay were used to detect the effect of PCGEM1 on the invasion and migration of A549 cells. The bioinformatics website StarBase was used to predict the complementary binding miRNAof PCGEM1. Furthermore, according to the website Targetscan, the genes that the corresponding miRNAs could target and bind were predicted. Results： ：qRT-PCR results showed that the expression of PCGEM1 in LC tissues and lung cancer cell lines was higher than that in normal tissues, and the expression level of miR-148a was lower than that in normal tissues (all P<0.05). The expression level of PCGEM1 in A549 cells was the highest, and the difference was statistically significant compared with other cell lines (P<0.05). After successful construction of PCGEM1 silenced cells, compared with the blank control group and A549-NC group, the cell OD492nm value of A549-siPCGEM1 group was significantly decreased, the number of cell clones and the number of matrigel matrix gels was significantly reduced, the cell migration rate was significantly reduced, the differences were statistically significant (P<0.05). According to the prediction results of StarBase website, PCGEM1 could be complementary to miR-148a, and the prediction analysis on microRNA.org website shows that miR-148a had a targeted binding site with TGF-β2. qRT-PCR and Western blotting results showed that the expression of miR-148a was significantly increased in the A549-siPCGEM1 group compared with the blank control group and A549-NC group, and the expression of TGF-β2 and p-Smad 2 was significantly decreased (P<0.05), while the expression of the above indicators in the blank control group and A549-NC group was not statistically significant (P>0.05). Conclusion： ：Lnc RNA PCGEM1 is highly expressed in lung cancer. High expression of PCGEM1 may enhance the TGF-β2/Smad2 signaling pathway by downregulation of miR-148a, thus promoting the development of LC and the malignant biological behavior.

Autophagy regulates high glucose-induced epithelial-mesenchymal transition in human lens epithelial cells / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To investigate the regulation of autophagy on high glucose-induced epithelial-mesenchymal transition(EMT)in human lens epithelial cells.<p>METHODS: In order to investigate the changes of EMT and autophagy induced by high glucose, HLE-B3 cells were divided into two groups. In NC group, cells were cultured in DMEM with 5.5mmol/L glucose, and in HG group, cells were treated with DMEM in addition with 30mmol/L glucose for 12h, 24h, and 48h. Western blot was used to detect the expression of EMT-marker proteins(E-cadherin and α-SMA)and autophagy-marker proteins(LC3, Beclin 1 and SQSTM1/p62). Wound healing assay was conducted to observe the migration ability. To investigate the regulation of autophagy on EMT, we employed rapamycin, an agonist of autophagy. HLE-B3 cells were divided into 4 groups. Two of them were mentioned as above, and the other two groups were treated with high glucose combined with DMSO(DMSO)and high glucose combined with 200nmol/L rapamycin(RAPA), respectively. Migration ability of cells was evaluated by Transwell assay. Expressions of proteins, such as EMT marker proteins, molecules in TGF-β signaling pathway(TGF-β2, Smad2/3, p-Smad2/3, Snail), and autophagy markers were detected by Western blot. The intracellular co-localization of SQSTM1/p62 and Smad2/3 was observed by immunofluorescence staining, and their interaction was confirmed by co-immunoprecipitation assay. <p>RESULTS: The expression of E-cadherin, LC3 Ⅱ/Ⅰ, and Beclin 1 in HLE-B3 cells of HG group gradually decreased(<i>F</i>=67.52, 163, 206; all <i>P</i><0.0001), the expressions of α-SMA, SQSTM1/p62 increased with time(<i>F</i>=53.37, 302.1; all <i>P</i><0.0001), and cell migration also increased compared with the cells in NC group(all <i>P</i><0.001), indicating that high glucose stimulated EMT and suppressed autophagy. After treatment with rapamycin, the expressions of LC3 Ⅱ/Ⅰ and E-cadherin increased, the expressions of α-SMA, p-Smad2/Smad2, p-Smad3/Smad3 and Snail decreased(all <i>P</i><0.05), and the expressions of TGF-β2 did not change(all <i>P</i>>0.05)in RAPA group compared with HG group and DMSO group, cell migration was also suppressed(all <i>P</i><0.001), indicating that Rapamycin down regulated the expressions of molecules in TGF-βsignaling pathway after activation of autophagy, which resulted in inhibiting EMT. Immunofluorescence staining showed co-localization of SQSTM1/p62 and Smad2/3 in cytoplasm. Co-immunoprecipitation confirmed the combination between SQSTM1/p62 and Smad2/3.<p>CONCLUSION: High glucose stimulates the process of EMT and suppresses the autophagy in HLE-B3 cells. Autophagy regulates EMT by interacting with Smad2/3 via SQSTM1/p62, altering the amount of Smad2/3 which works in the TGF-β signaling pathway.

C-phycocyanin inhibits epithelial-mesenchymal transformation ofTGF-β1-induced cervical cancer Caski cells / 中国肿瘤生物治疗杂志

@# Objective: To investigate the effect of C-phycocyanin (C-PC) on the epithelial-mesenchymal transition (EMT) of cervical cancer Caski cells induced by transforming growth factor beta1 (TGF-β1). Methods: According to different treatment methods, Caski cells were divided into three groups: 10 ng/ml TGF-β1 treatment group, 10 ng/ml TGF-β1+300 μg/ml C-PC co-treatment group and control group (untreated). After 24 h of treatment, the morphological changes of Caski cells were observed, and the effects of TGF-β1 and C-PC on the migration and invasion of Caski cells were detected by Scratch test and Transwell test, respectively. Western blotting was used to detect the effect of C-PC on the expression of epithelial phenotypic marker protein E-cadherin and stromal phenotypic marker protein N-cadherin in TGF-β1-induced Caski cells, and qPCR was used to detect the mRNA expressions of EMT related factors Snail, Zeb1 and Twist. Results: Caski cells in the TGF-β1 treatment group lost the characteristics of the original epithelial phenotype, while the cells in the TGF-β1+C-PC co-treatment group maintained the characteristics of normal epithelial phenotype; the migration rate ([60.0±1.4]% vs [33.5±2.2]%, [40.0±2.8]%, both P<0.05) and the number of invasive transmembrane cells ([108.2±6.2] vs [25.2±3.1], [39.8±5.4], both P<0.01]) of Caski cells in the TGF- β1 treatment group were significantly higher than those in the co-treatment group and the control group. Compared with the control group, the expression of E-cadherin in Caski cells treated with TGF-β1 decreased significantly (P<0.05), while the mRNA expressions of Twist, Snail and Zeb1 increased significantly (all P<0.05); However, co-treatment with C-PC reversed above changes (P<0.05 or P<0.01), and significantly decreased the protein expression level of N-cadherin (P< 0.05). Conclusion: C-PC treatment can inhibit the invasion and metastasis ability of Caski cells induced by TGF-β1 and further affects the EMT process. The mechanism may be related to the decrease of mRNAexpressions of Twist, Snail and Zeb1 by C-PC treatment. ·

Silencing GFAP gene regulates the proliferation and apoptosis of high glucose-induced retinal microvascular endothelial cells / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM:To investigate the effect of silencing glial fibrillary acidic protein gene(GFAP)on proliferation and apoptosis of high glucose-induced human retinal microvascular endothelial cells(hRMECs)and its mechanism.<p>METHODS: Expression of GFAP in hRMECs treated with high sugar(30mmol/mL)and low sugar(5mmol/mL)was detected by qRT-PCR. The high glucose-induced hRMECs cells of silencing GFAP gene was established by lentiviral-mediated method. High glucose-induced hRMECs cells were treated with SRI-011381(TGF-β signaling pathway activator)and dimethyl sulfoxide(DMSO); Expression of GFAP, transforming growth factor-1(TGF-β1), activating transcription factor2(Smad2), Smad3 proteins were measured by Western blot, and cell proliferation and apoptosis were detected by CCK-8 and flow cytometry, respectively.<p>RESULTS: Expression of GFAP was significantly increased in high glucose treated hRMECs. The high glucose induced hRMECs cell model of GFAP gene silencing was successfully constructed by lentivirus mediation, and the cell proliferation ability was significantly improved, the apoptosis rate is significantly inhibited, and expression of TGF-1, Smad2 and Smad3 proteins in the TGF-β signaling pathway was significantly inhibited after silencing GFAP, while activation of TGF-β signaling pathway could reverse the inhibitory effect of silencing GFAP on the proliferation and apoptosis in high glucose hRMECs.<p>CONCLUSION: Silencing GFAP gene can promote the proliferation of high glucoseinducedhuman retinal microvascular endothelial cells and inhibit cell apoptosis,the mechanism may be related to the inactivation of TGF-β signaling pathway.

Association Between Epithelial Cytokines and Clinical Phenotypes of Elderly Asthma

PURPOSE: Asthma in the elderly has different clinical features including more severe phenotypes with higher comorbidities. Epithelial cells are known to initiate innate/adaptive immune responses in asthmatic airways. We investigated clinical features and epithelial derived cytokine levels in elderly asthmatics compared to non-elderly asthmatics in a cross-sectional cohort of adult asthmatics in order to further understand its pathogenic mechanisms. METHODS: A total of 1,452 adult asthmatics were enrolled from a single tertiary hospital and were classified into 2 groups: 234 elderly (≥ 60 years at initial diagnosis) and 1,218 non-elderly (< 60 years at initial diagnosis) asthmatics. Asthma-related clinical parameters were compared between the 2 groups. Serum levels of epithelial cell-derived cytokines including interleukin (IL)-31, IL-33, IL-8, eotaxin-2, transforming growth factor beta 1 (TGF-β1) and periostin were measured by enzyme-linked immunosorbent assay. RESULTS: Significantly higher prevalence rates of late-onset asthma (onset age ≥ 40 years) and severe asthma, as well as the lower rate of atopy, blood/sputum eosinophil counts, total immunoglobulin E and eosinophil cationic protein levels were noted in elderly asthmatics compared to non-elderly asthmatics (P < 0.05, respectively). The forced expiratory volume in 1 second (FEV1, % predicted) level tended to be lower in elderly asthmatics (P = 0.07). In addition, serum IL-33 and IL-31 levels were significantly lower in elderly asthmatics, while no differences were found in the serum level of IL-8, eotaxin-2, TGF-β1 or periostin. Among elderly asthmatics, subjects with severe asthma had lower FEV1 (% predicted) value, but showed significantly higher serum levels of eotaxin-2 and TGF-β1, than those with non-severe asthma (P < 0.05 for each). CONCLUSIONS: These findings suggest that age-related changes of epithelial cell-derived cytokines may affect clinical phenotypes and severity of elderly asthma: decreased levels of IL-33 and IL-31 may contribute to less Th2 phenotype, while increased levels of eotaxin-2 and TGF-β1 may contribute to severity.

Ipilimumab affectsTlymphocytesandBcl-2mRNAexpression in xenograft tissues of lung cancer-bearing mice by inhibiting TGF-β1/ERK signaling pathway / 中国肿瘤生物治疗杂志

@# Objective: To study the effect of ipilimumab on T lymphocytes and Bcl-2 mRNA expression in lung cancer-bearing mice by inhibiting TGF-β1/ERK signaling pathway. Methods: Forty-five C57 mices inoculated with Lewis lung cancer cells were randomly divided into control group, low dose ipilimumab group and high dose ipilimumab group with 15 mice in each. The low and high dose groups were given 3 mg/kg and 5 mg/kg ipilimumab respectively, while the control group was given 0.9% sodium chloride solution with the same volume. The effects of ipilimumab on TGF-β1/ERK signaling pathway, Bcl-2 mRNA expression, immune function improvement and tumor inhibition in three groups were detected by WB and qPCR. Results: After administration of ipilimumab, the tumor weight and volume of mice in low-dose and high-dose groups were significantly lower than that of the control group, and the tumor inhibition rate increased in a dose-dependent manner (P<0.05). The thymus index and spleen index of mice were significantly higher than that of control group, which also increased in a dose-dependent manner (P<0.05). The levels of CD3+, CD4+, CD4+/CD8+ cells in the high and low dose groups were significantly higher than those in the control group, with significantly higher levels in high dose group compared with the low dose group (P<0.05). The levels of serum inflammatory factors were significantly lower than those in control group, and the levels of serum TNF-α, IL-6 and IL-3 in the high dose group were significantly lower than those in the low dose group (P<0.05). The expressions of TGF-β1, ERK1/2, p-ERK1/2 and MEK in tumor tissues of both high and low dose groups significantly decreased, with more lower levels in high dose group than in low dose groups (all P<0.05), and the positive rate of TGF-β1 expression in high dose group was the lowest. The mRNAexpression of Bcl-2 in tumor tissues of high and low dose groups decreased significantly after drug administration, with a significantly lower level in high does group than that in low dose group (P<0.05). Conclusion: Ipilimumab can effectively inhibit TGF-β1/ERK signaling pathway, improve immune function and down-regulate the expression of Bcl-2, thus inhibit the growth of Lewis lung cancer cells and play an antitumor role in mice.

Mechanisms of Retinoic Acid-AP-1 Signaling Pathway Modulating Secretion of TGF-β2 by Retinal Pigment Epithelial Cells / 中山大学学报(医学科学版)

@#【Objective】To investigate the effects of up-regulating RA signal and inhibiting AP-1 transcriptional activity on TGF-β2 secretion by RPEs and its possible pathways.【Methods】① To investigate the effects of ATRA treat⁃ ment，human retinal pigment epithelial cell line ARPE-19 cells were divided into 5 groups：control group and 4 interven⁃ tion groups（6 h，12 h，24 h and 48 h after RA treatment）. Western blot，RT-qPCR and immunofluorescence staining were carried out to analyze RARβ and c-Fos expression. ②To investigate the effects of RARβ inhibitor LE540 treatment on expression of RARβ and c-Fos that were induced by ATRA，ARPE-19 cells were divided into 4 groups：control group，ATRA group，LE540 group and ATRA+LE540 group. RARβ and c-Fos expression was assessed by western blot and RT-qPCR. ③ To investigate the effects of AP-1 inhibitor T-5224 treatment，ARPE-19 cells were divided into 4 groups：control group and treatment groups（12 h，24 h and 48 h after T-5224 treatment）. EMSA was carried out to ana⁃ lyze the AP-1 DNA binding activity. ④To investigate the effects of LE540 and T-5224 administration on ATRA- induced TGF-β2 secretion，ARPE-19 cells were divided into 4 groups：control group，ATRA group，ATRA+LE540 group and ATRA+LE540 group. Western blot and ELISA were carried out to analyze TGF-β2 secretion in ARPE-19 cells.【Results】 RARβ level in ARPE-19 cells was significantly higher in treatment group than in control group after being treated with ATRA for 24 and 48 hours（P＜0.05）. C-Fos level was first up-regulated and then decreased. After treatment with ATRA for 6 and 12 hours，c-Fos expression were significantly upregulated（P＜0.01），but at 48 h after treatment，their expression were significantly decreased to the level which had no statistical difference compared with the control group （P＞0.05）. The AP-1 DNA binding activity was significantly decreased in ARPE-19 cells after being treated with T-5224 for 24 and 48 hours（P＜0.01）. Compared with ATRA group，TGF-β2 secretion was statistically down-regulated after being treated with LE540 and T-5224 for 48 hours（P＜0.05）.【Conclusion】ATRA can induce TGF-β2 secretion in RPE cells through affecting RARβ expression and AP-1 transcriptional activity.

Effect of miR-141-3p targeting TGF-β2 on malignant biological behaviors of human prostatic cancer C4-2B cells / 中国肿瘤生物治疗杂志

@#Objective: To investigate the relationship between miR-141-3p and transforming growth factorβ2 (TGF-β2), and its effects on the malignant biological behaviors of human prostate cancer cell line C4-2B. Methods:After the transfection of miR-141-3p mimic, the mRNAexpression of miR-141-3p and TGF-β2 in C4-2B cells was detected by qRT-PCR. Bioinformatics method validated the relationship between miR-141-3p and TGF-β2. miR-141-3p mimic alone or with TGF-β2 over-expression vector was transfected into C42B cells, and then Western blotting was used to detect the expression of TGF-β2 protein in C4-2B cells, Hochest33258 staining was used to detect cell apoptosis, and Transwell assay was used to detect the invasion ability of cells in each group. Results:After the transfection of C4-2B cells with miR-141-3p mimic, the level of miR-141-3p increased significantly, and the level of TGF-β2 mRNA decreased significantly (all P<0.01). The activity of luciferase was significantly reduced after the co-transfection with miR-141-3p mimic and wild type report plasmid (P<0.01); However, the activity of luciferase was not obviously changed after co-transfection with miR141-3p mimic and mutant type report plasmid (P>0.05).After co-transfection with miR-141-3p mimic and pc-TGF-β2, the proliferation of C4-2B cells decreased significantly, the number of apoptotic cells increased significantly, and the cell invasion ability decreased significantly (all P<0.01). Conclusion: miR-141-3p inhibits the proliferation and invasion of human prostate cancer C4-2B cells and induces cell apoptosis by targeting TGF-β2.