Expression and significance of hypoxia inducible factor-1α and α-smooth-muscle actin in cervical cancer / 中国医师进修杂志

Objective@#To examine the expression levels of hypoxia inducible factor (HIF)-1α and α-smooth-muscle actin (SMA) in both cervical cancer tissues with concurrent chemoradiotherapy and non-cervical cancer tissues, and assess the clinical significance in cervical cancer.@*Methods@#The immune-histochemistry was used to detect HIF-1α and α-SMA in 68 cases of cervical cancer tissues and 56 cases of non-cervical cancer tissues (including normal cervical tissues, hysteromyoma and cervical intraepithelial neoplasias) from January 2013 to January 2014 in the First Affiliated Hospital of Hebei Northern University.@*Results@#The positive expression rates of HIF-1α and α-SMA in cervical cancer tissues and non-cervical cancer tissues were 58.8% (40/68), 39.3% (22/56) and 54.4% (37/68), 35.7% (20/56) respectively; the differences were significant statistically (P<0.05). The expression of HIF-1α was positively correlated with the expression of α-SMA protein in cervical cancer tissues (r = 0.376, P =0.001). The positive expression of HIF-1α was closely related to International Federation of Gynecology and Obstetrics (FIGO) stages (r = 0.371, P = 0.004), lymph node metastasis (r = 0.243, P = 0.039), abdominal aortic parathyroid lymph node metastasis (r = 0.286, P = 0.014) and serum squamous cell carcinoma antigen (SCC-Ag) (r = 0.271, P = 0.020), but it was not correlated with age and tumor size (P>0.05). The positive expression of α-SMA was associated with lymph node metastasis (r = 0.363, P = 0.001), abdominal aortic parathyroid lymph node metastasis (r = 0.271, P = 0.020) and SCC-Ag (r = 0.272, P = 0.020), but it was not correlated with age, FIGO stage and tumor size (P>0.05) . The short-term effect rates of concurrent chemoradiotherapy in cervical cancer tissues with positive and negative expression of HIF-1α and α-SMA were 27.5% (11/40), 21.6% (8/37), and 64.3% (18/28) and 48.4% (15/31), and there were statistical differences (P<0.05). The medial follow-up time of 68 cases was 60 months, and the 5-year survival rate was 52.9% (36/68). The 5-year survival rates of the patients with positive expression of HIF-1α and α-SMA were 42.5% (17/40) and 40.5% (15/31), the 5-year survival rates of the patients with negative expression of HIF-1α and α-SMA were 67.9% (19/28) and 67.7% (21/31), and the differences were statistically significant separately (χ2 = 4.091 and 4.573, P = 0.043 and 0.032).@*Conclusions@#The elevation of HIF-1α and α-SMA may be used to predict the development and prognosis of cervical cancer with concurrent chemoradiotherapy.

Expression and significance of hypoxia inducible factor-1α and α-smooth-muscle actin in cervical cancer / 中国医师进修杂志

Objective To examine the expression levels of hypoxia inducible factor (HIF)-1α and α-smooth-muscle actin (SMA) in both cervical cancer tissues with concurrent chemoradiotherapy and non-cervical cancer tissues,and assess the clinical significance in cervical cancer.Methods The immune-histochemistry was used to detect HIF-1α and α-SMA in 68 cases of cervical cancer tissues and 56 cases of non-cervical cancer tissues (including normal cervical tissues,hysteromyoma and cervical intraepithelial neoplasias) from January 2013 to January 2014 in the First Affiliated Hospital of Hebei Northern University.Results The positive expression rates of HIF-1α and α-SMA in cervical cancer tissues and non-cervical cancer tissues were 58.8％ (40/68),39.3％ (22/56) and 54.4％ (37/68),35.7％ (20/56) respectively;the differences were significant statistically (P＜ 0.05).The expression of HIF-1α was positively correlated with the expression of α-SMA protein in cervical cancer tissues (r =0.376,P =0.001).The positive expression of HIF-1α was closely related to International Federation of Gynecology and Obstetrics (FIGO) stages (r =0.371,P =0.004),lymph node metastasis (r =0.243,P =0.039),abdominal aortic parathyroid lymph node metastasis (r =0.286,P =0.014) and serum squamous cell carcinoma antigen (SCC-Ag) (r =0.271,P =0.020),but it was not correlated with age and tumor size (P ＞ 0.05).The positive expression of α-SMA was associated with lymph node metastasis (r =0.363,P =0.001),abdominal aortic parathyroid lymph node metastasis (r =0.271,P =0.020) and SCC-Ag (r =0.272,P =0.020),but it was not correlated with age,FIGO stage and tumor size (P ＞ 0.05).The shortterm effect rates of concurrent chemoradiotherapy in cervical cancer tissues with positive and negative expression of HIF-1α and α-SMA were 27.5％ (11/40),21.6％ (8/37),and 64.3％ (18/28) and 48.4％ (15/31),and there were statistical differences (P ＜ 0.05).The medial follow-up time of 68 cases was 60 months,and the 5-year survival rate was 52.9％ (36/68).The 5-year survival rates of the patients with positive expression of HIF-1α and α-SMA were 42.5％ (17/40) and 40.5％ (15/31),the 5-year survival rates of the patients with negative expression of HIF-1α and α-SMA were 67.9％ (19/28) and 67.7％ (21/31),and the differences were statistically significant separately (x2 =4.091 and 4.573,P =0.043 and 0.032).Conclusions The elevation of HIF-1α and α-SMA may be used to predict the development and prognosis of cervical cancer with concurrent chemoradiotherapy.

Mechanism of leflunomide in regulating pulmonary fibrosis by regulating miR-449a / 药学实践杂志

Objective To investigate the mechanism of leflunomide (LEF) in regulating pulmonary fibrosis by regulating microRNA (miR)-449a. Methods Human lung fibroblasts MRC-5 were divided into 6 groups: control group, LEF group, LEF+mimic group, mimic group, LEF+inhibitor group and inhibitor group. MiR-449a was overexpressed or silenced by plasmid transfection with miR-449a mimic or inhibitor and ncubate for 48 h at 5 mg / L LEF. The cell viability, cell proliferation ability and apoptotic rate of each group were measured by CCK-8 method, clone formation experiment and flow cytometry. Immunofluorescent staining was used to detect α smooth muscle actin (α-SMA) and collagen I (col I). The levels of miRNA and protein were detected using qPCR and Western blot, respectively. Results The miR-449a level in the mimic group was significantly higher than that in the control group (P<0.05). The level of miR-449a in LEF group and inhibitor group was significantly lower than that in control group (P<0.05). The expression level of miR-449a in LEF+mimic group was significantly higher than that in LEF group, and the level of miR-449a in LEF+inhibitor group was significantly lower than that in LEF group (P<0.05). The cell viability and cell proliferation ability of the LEF group and inhibitor group were significantly higher than those of the control group (P<0.05). The cell viability and cell proliferation ability of the mimic group were significantly lower than those of the control group (P<0.05). The cell viability and cell proliferation ability of the LEF+mimic group were significantly lower than those of the LEF group, while the cell viability of the LEF+inhibitor group was significantly higher than that of the LEF group (P<0.05). The apoptosis rate of LEF group and inhibitor group was lower than that of control group (P<0.05). The apoptosis rate of mimic group was significantly higher than that of control group (P<0.05). The apoptosis rate of LEF+mimic group was significantly higher than that of LEF group, while the apoptosis rate of LEF+inhibitor group was significantly lower than that of LEF group (P<0.05). The fluorescence intensity of α-SMA and Col I proteins in LEF group and inhibitor group were significantly higher than those in control group (P<0.05). The relative fluorescence intensity of mimic group was lower than that of control group (P<0.05). The relative fluorescence intensities of α-SMA and Col I proteins in LEF+mimic group were significantly lower than those in LEF group, while the relative fluorescence intensities of α-SMA and Col I protein in LEF+inhibitor group were significantly higher than those in LEF group (P<0.05). The levels of p-JNK / JNK in LEF group and inhibitor group were higher than those in control group (P<0.05). The p-JNK / JNK level in the mimic group was significantly lower than that in the control group (P<0.05). The level of p-JNK / JNK in LEF+mimic group was significantly lower than that in LEF group, while the level of p-JNK / JNK in LEF+inhibitor group was significantly higher than that in LEF group (P<0.05). Conclusion LEF may activate the JNK pathway by inhibiting the expression of miR-449a in lung fibroblasts, thereby inducing fibroblast activation and proliferation, inhibiting apoptosis, and causing pulmonary fibrosis.

The inhibition effect of miR-29c on lung fibroblasts transdifferentiation induced by SiO2 / 中华劳动卫生职业病杂志

Objective@#To investigate the regulatory effect of miR-29c on the trans-differentiation of pulmonary fibroblasts into myofibroblasts induced by silica dust.@*Methods@#Fibroblasts obtained from SD rat lung tissue and pulmonary macrophages (NR8383) were co-cultured to establish the silicosis cell model in vitro. And real time-quantitative polymerase chain reaction (RT-qPCR) and Western Blot assays were performed to detect the altered expression level of miR-29c and α-smooth muscle actin (α-SMA) . After that, the in vitro cell model was transfected with corresponding viruses to establish miR-29c overexpression and inhibition cell models, and the mRNA and protein expression levels of α-SMA were detected simultaneously.@*Results@#Compared with control group, the expression level of miR-29c in the silicosis cell model in vitro was down-regulated significantly after 12 or 18 h exposed to SiO2, and both of the mRNA and protein expression levels of α-SMA were up-regulated instead (P<0.05) . When transfected with corresponding viruses, the mRNA and protein expression levels of α-SMA in the pulmonary fibroblasts were significantly up-regulated in miR-29c inhibition group and down-regulated in miR-29c overexpression group (P<0.05) .@*Conclusion@#Based on the findings, it could be safely infered that the development of pulmonary fibrosis could be impeded by inhibiting transdifferentiation process of pulmonary fibroblasts into myofibroblasts regulated by miR-29c, miR-29c could be an potential therapeutic target to lung fibrosis induced by silica.

Cannabinoid receptor-1 antagonism: a new perspective on treating a murine schistosomal liver fibrosis model

BACKGROUND Formation of schistosomal granulomata surrounding the ova can result in schistosomiasis-associated liver fibrosis (SSLF). The current standard of treatment is praziquantel (PZQ), which cannot effectively reverse SSLF. The role of the cannabinoid (CB) receptor family in liver fibrosis has recently been highlighted. OBJECTIVES This study aimed to assess the therapeutic effect of CB1 receptor antagonism in reversing SSLF in a murine model of Schistosoma mansoni infection. METHODS One hundred male Swiss albino mice were divided equally into five groups: healthy uninfected control (group I), infected control (group II), PZQ treated (group III), rimonabant (RIM) (SR141716, a CB1 receptor antagonist)-treated (group IV) and group V was treated with combined PZQ and RIM. Liver sections were obtained for histopathological examination, alpha-1 smooth muscle actin (α-SMA) immunostaining and assessment of CB1 receptor expression using real-time polymerase chain reaction (RT-PCR). FINDINGS The most effective reduction in fibrotic marker levels and granuloma load was achieved by combined treatment with PZQ+RIM (group V): CB1 receptor expression (H = 26.612, p < 0.001), number of α-SMA-positive cells (F = 57.086, p < 0.001), % hepatic portal fibrosis (F = 42.849, p < 0.001) and number of granulomata (F = 69.088, p < 0.001). MAIN CONCLUSIONS Combining PZQ with CB1 receptor antagonists yielded the best results in reversing SSLF. To our knowledge, this is the first study to test this regimen in S. mansoni infection.

Effect of electroacupuncture on renal fibrosis in spontaneously hypertension rats and its related mechanisms / 针刺研究

OBJECTIVE: To observe the effect of electroacupuncture (EA) on blood pressure, renal fibrosis and expression of tissue inhibitors of metalloproteinase-1 (TIMP-1), plasminogen activator inhibitor 1 (PAI-1), and alpha smooth muscle actin (α-SMA) in spontaneous hypertension rats (SHR), so as to explore its mechanisms underlying improving hypertensive renal damage. METHODS: Forty male SHR (15 weeks in age) were randomly divided into 5 groups: model, medication (Losartan), Shenshu, Geshu, and Shenshu＋Geshu groups(n=8 rats in each group), and the same age-old male 8 Wistar-Kyoto (WKY) rats were used as the normal control group. Rats of the medication group were treated by gavage of Losartan potassium solution (3 mg/mL, 30 mg·kg-1·d-1, once a day for 12 weeks), and those of the 3 EA groups treated by EA stimulation of bilateral "Shenshu" (BL23), "Geshu"(BL17) or both BL23 and BL17 (2 Hz/100 Hz, 1 mA, 15 min each time, once every other day for 12 weeks). The systolic blood pressure of the tail artery was measured before, and 4, 8 and 12 weeks after the intervention. The expression of TIMP-1, PAI-1 and α-SMA proteins of the right kidney tissue was measured by immunohistochemistry. Histopathological changes of the right renal tissue were observed under light microscope after H.E. stain. RESULTS: The blood pressure was significantly higher in the mo-del group than those in the normal control group (P<0.01), and considerably decreased at the 4th , 8th, and 12th week of the interventions in the medication and 3 EA groups (P<0.01). The expression levels of renal TIMP-1, PAI-1 and α-SMA proteins were notably higher in the model group than those in the normal control group and considerably decreased at the 12th week of the interventions in the medication and 3 EA groups than in the model group (P<0.01). H.E. staining of the renal tissue showed disordered arrangement of the renal cells, congestion and dilation of capillaries with thickened vascular wall, renal tubule atrophy and lumen stenosis with some necrosis of renal tubules, protein tubule and cell tubules, increase of some glomerular mesangial matrix and hyperplasia of fibrous tissue in the model group, which was re-latively milder in the medication and 3 EA groups. CONCLUSION: EA of BL23 and BL17 can reduce the blood pressure in SHR, which may be related to its function in down-regulating expression of TIMP-1, PAI-1 and α-SMA proteins.

Expression of ?-smooth muscle actin in benign and malignant salivary gland tumors: An immunohistochemical study

Background and Objectives: Myoepithelial cells (ME) are known to contribute in the patterning of salivary gland neoplasms (SGN) and possess cytoplasmic smooth muscle actin (SMA) revealed by alpha SMA (?-SMA). The present study aimed to assess the expression of ?-SMA in selected benign and malignant SGN (pleomorphic adenoma printarticle.asp?issn=0377-4929;year=2018;volume=61;issue=4;spage=479;epage=484;aulast=Ravi, mucoepidermoid carcinoma (MEC), adenoid cystic carcinoma (ACC), and polymorphous low-grade adenocarcinoma (PLGA). Materials and Methods: The intensity and pattern of expression of ?-SMA were studied in 25 cases of SGN's ACC (n = 7), MEC (n = 8), PA (n = 8), and PLGA (n = 2), and correlated with the histological patterns. Results: Maximum expression of ?-SMA in the epithelial compartment was seen in ACC, followed by PA, whereas MEC and PLGA showed completely negative staining. The connective tissue expression was mild in ACC and MEC. The myxoid stroma of PA with “melting” pattern was weakly positive for ?-SMA. The stroma in PLGA showed complete negativity. In ACC, ?-SMA-positive cells were lining the cribriform spaces, small islands, and dispersed within large islands. Small nests showed complete positivity for ?-SMA. Interpretation and Conclusion: In ACC, ?-SMA expression supports the involvement of ME in epithelial organization explaining the histological patterns seen. In PA, the expression correlates with the predominantly secretory nature of ME. The absence of epithelial positivity in MEC and PLGA suggest that ME has less role to play in their histogenesis. The weak stromal positivity observed in MEC and ACC may be attributed to the positive immunoreactivity of myofibroblasts playing a role in modulating the course of SGN's.

Effects of uranium dust on collagen synthesis in lung fibroblasts / 中国职业医学

OBJECTIVE: To observe the effects and mechanism of uranium dust on collagen synthesis in fibroblastic cells.METHODS: i) RAW264. 7 macrophages were divided into solvent-control group and dust-affected group,and then treated with 0 and 120 mg / L( final concentration) uranium dust suspension for 0,2,4,8,16,24 and 32 hours. The supernatants of cells from these two groups were collected,and transforming growth factor beta 1( TGF-β1) levels were measured by enzyme-linked immunosorbent assay( ELISA). The best time phase was defined,and the supernatant of RAW264. 7 macrophages culture were separated as the conditioned medium( CM) for subsequent experiments. ii) Normal mouse lung fibroblasts L929 cells were divided into control group and uranium dust group,and treated with the CM of solvent-control group and dust-affected group respectively. After cultivated for 0,6,12,18,24,30 and 36 hours,the expression of α-smooth muscle actin( α-SMA) in L929 cells was detected by immunocytochemistry. The levels of extrac ellular collagen type Ⅰ( Col Ⅰ),collagen type Ⅲ( Col Ⅲ) and hydroxyproline( HYP) were detected by ELISA.RESULTS: i) The level of TGF-β1 in RAW264. 7 macrophages supernatant of the dust-affected group was higher than that of the solvent-control group after treatment with uranium dust for 4 hours,and showed an increasing trend with increasing time during 4-24 hours( P < 0. 05). The peak value was in 24 hours. The best time phase was 24 hours and used for CM of the two groups in subsequent experiments. ii) Compared with the control group at the same time points, the α-SMA expressions of L929 cells in uranium dust group increased after treatment with CM for 18-36 hours( P < 0. 05),the levels of Col Ⅰ,Col Ⅲ and HYP in uranium dust group increased after treatment with CM for 12-36 hours( P < 0. 05); both the expression of α-SMA and levels of Col Ⅲ and HYP increased with increasing time,showing a time-effect relationship( P <0. 05). CONCLUSION: Uranium dust can induce macrophages to secret TGF-β1 and affect fibroblast,increase α-SMAexpression,and increase the Col Ⅰ,Col Ⅲ and HYP synthesis. These might be the important mechanisms of lung fibrosis.

Effects of DNA methylation of thymocyte differentiation antigen-1 in beryllium sulfate induced human fetal lung fibroblast fibrosis / 中国职业医学

OBJECTIVE: The change of DNA methylation of thymocyte differentiation antigen-1( Thy-1) was observed in beryllium sulfate( Be SO4) stimulated human fetal lung fibroblast( MRC-5 cell) to explore the effects of Thy-1 in Be SO4 induced lung fibrosis. METHODS: MRC-5 cell culture in vitro model was used. The final concentrations of Be SO4were1. 0,10. 0 and 100. 0 μmol / L( low-,medium- and high-dose groups). The control was untreated. Other 2 intervention groups were the 5-azacytidine( AZC) intervention group( 10. 0 μmol / L of AZC and 10. 0 μmol / L Be SO4) and the trichostatin A( TSA) intervention group( 0. 5 μmol / L of TSA and 10. 0 μmol / L Be SO4). The cells were collected 24,48 and 72 hours after exposure. Real-time quantitative polymerase chain reaction( PCR) was used to determine the relative expression of collagen typeⅠ( Col Ⅰ),collagen type Ⅲ( Col Ⅲ),α-smooth muscle actin( α-SMA) and Thy-1 mRNA.The nested landed methylation specific PCR was used to detect the Thy-1 DNA methylation level. RESULTS: At 24 hours,the relative expression level of Col Ⅲ mRNA in MRC-5 cells showed an increasing trend with increasing dose( P < 0. 05);at 48 and 72 hours,the relative expression levels of Col Ⅰ,Col Ⅲ and α-SMA mRNA in MRC-5 cells increased with the increasing dose( P < 0. 05). All these 3 indicators in MRC-5 cells of 3 dose groups increased with the increase of expose time( P < 0. 05). The relative expression level of Thy-1 mRNA in MRC-5 cells of all 3 dose groups were lower than that in control( P < 0. 05). The relative expression level of Thy-1 mRNA of the high-dose group was lower than that of the lowdose group( P < 0. 05). The Thy-1 DNA methylation levels in the medium- and high-dose groups were both higher than that of the control( P < 0. 05). The Thy-1 DNA methylation levels of the 3 dose groups increased with the increasing dose( P < 0. 05). The Thy-1 DNA methylation levels of MRC-5 cells in the 2 intervention groups were higher than that of the control( P < 0. 05),but there was no significant difference when compared with the medium-dose group( P > 0. 05).CONCLUSION: Be SO4 stimulation can induce the fibrosis of MRC-5 cells. In this process,the Thy-1 DNA methylation level increases,while the Thy-1 mRNA expression level decrease. Thy-1 DNA methylation might be one of the important mechanisms of lung fibrosis induced by Be SO4.

Smoking-Related Renal Histologic Injury in IgA Nephropathy Patients

PURPOSE: Smoking reportedly exerts deleterious effects on renal function; however, its effects on histology have not been clarified in patients with IgA nephropathy (IgAN). MATERIALS AND METHODS: Renal histology was evaluated in a cohort of 397 patients diagnosed with IgAN according to smoking status and dose in relation to renal function. RESULTS: Among the study cohort, which was predominantly male (88.5%), 52 patients (13%) were current smokers. These current smokers demonstrated more frequent hypertension and higher serum creatinine levels than non/ex-smokers at the time of diagnosis, which was apparent with increased smoking dose. The percentages of global glomerulosclerosis and arteriolar hyalinosis increased with increased smoking dose, whereas tubulointerstitial fibrosis or arterial intimal thickening did not. Glomerular mesangial alpha-smooth muscle actin expression were similar between current and non/ex-smokers matched for age, gender, hypertension, and histologic severity, although the number of glomerular CD68+ cells was significantly fewer in smokers. Initial serum creatinine level, estimated glomerular filtration rate (eGFR), and global glomerulosclerosis were found to be risk factors of serum creatinine doubling in both smokers and non/ex-smokers by univariate analysis during a mean follow-up of 3.8 years. CONCLUSION: In addition to dose dependent renal functional decline and hypertension, smoking contributes to renal disease progression by eliciting microvascular injury in IgAN patients.

Antioxidative effect of total ginsenosides on exprimental pulmonary fibrosis of mice / 中国药理学与毒理学杂志

OBJECTIVE To observe the protective effect and underlying mechanism of total ginsenosides (TG) on bleomycin-induced pulmonary fibrosis. METHODS Intratracheal instillation of bleomycin 5 mg · kg-1 was conducted to establish a pulmonary fibrosis mouse model. Kunming mice(1/2 males and 1/2 females)were randomly divided into sham-operation(Sham),model,total ginsen?osides 40,80 and 160 mg·kg-1 and prednisone acetate(5 mg·kg-1) groups. After 28 d administration,the histopathological changes in the lung were analyzed by hematoxylin eosin(HE)and Masson staining. The exprssion of alpha smooth muscle actin(α-SMA)in the lung was detected by real-time PCR. The content of hydroxyproline(HYP)and glutathione(GSH),level of total antioxidant capacity(T-AOC)and hydroxy radical(·OH),activity of myeloperoxidase(MPO)and nitric oxide synthase(NOS)in the lung were detected by corresponding kits. RESULTS Compared with the sham group,the pulmonary indexes in model group were significantly increased(P<0.01),alveolitis and pulmonary fibrosis were obvious. The mRNA expression ofα-SMA,content of HYP and · OH,activity of MPO and NOS were increased(P<0.05),but the content of GSH and T-AOC in model group was decreased(P<0.05). Compared with model group,the pulmonary indexes in TG 80 and 160 mg · kg-1 and prednisone acetate 5 mg · kg-1 groups were reduced(P<0.05),and the degree of alveolitis and pulmonary fibrosis was mitigated. The mRNA expression ofα-SMA,content of HYP and · OH, the activity of MPO and NO were decreased (P<0.05),while the content of GSH and T-AOC was increased(P<0.05). CONCLUSION TG can improve the degree of mice pulmonary fibrosis induced by bleomycin. The mechanism may be related to the increased antioxidant capacity of organisms.

The anti-fibrosis effect of ghrelin in human hepatic stellate cells via PI3K-AKT pathway / 中国综合临床

Objective To observe the effect of ghrelin on the expression of procollagen type Ⅰ and alpha smooth muscle actin (α-SMA) synthesis in vitro cultured human hepatic stellate cell (HSC-LX2) stimulated by Platelet-derived growth factor-BB (PDGF-BB).Besides,the effect of PI3K-AKT pathway was studied.Methods Cultured LX2 were intervented and jointing intervented according to the different ghrelin concentration by ghrelin and PDGF:control group,0.1 μmol/L Ghrelin group,10 μg/L PDGF group,0.05 μmol/L Ghrelin +10 μg/L PDGF group,0.1 μmol/L Ghrelin + 10 μg/L PDGF group,0.15 μmol/L Ghrelin + 10 μg/L PDGF group.Culture HSC-LX2 in vitro,joint intervention cells with different concentrations.Procollagen Ⅰ mRNA expression were detected by Polymerase chain reaction (PCR),besides,α-SMA and AKT expression were detected by Western blot in each groups.After treatment by PI3K specific inhibitor LY294002 in LX2,three groups were divided into PDGF,Ghrelin + PDGF and LY294002 + Ghrelin +PDGF.Procollagen Ⅰ mRNA expression were detected by PCR,and α-SMA was detected by Western blot.Results PCR results showed that procollagen Ⅰ expression in PDGF treated group was significantly higher than the control group ((6.91 ± 0.46) vs.(1.00 ± 0.08),P ＜ 0.05),so PDGF can promote the expression of procollagen type Ⅰ.Procollagen Ⅰ mRNA expression between ghrelin group(0.60±0.13) and blank control group had no significant change(P＞0.05).Procollagen Ⅰ mRNA expression between different concentrations of Ghrelin and PDGF (3.11 ± 0.28,2.03 ±0.23,0.70 ± 0.06) was significantly reduced than PDGF group.The difference was statistically significant (P ＜0.05),and with the increase of the ghrelin concentration of the inhibition,the effect was more obvious in a concentration dependent manner.Western blot showed that α-SMA expression was lower in Ghrelin +PDGF group than PDGF group.AKT expression was higher in Ghrelin +PDGF group than PDGF group,indicating that PI3K-AKT may participate in the anti-fibrosis effect of ghrelin in LX2.After treatment of PI3K specific inhibitor,procollagen Ⅰ expression in LY294002+Ghrelin +PDGF group was significantly higher than Ghrelin +PDGF group((4.13±0.21) vs.(2.34±0.25),P＜0.05).Western blot also showed that α-SMA expression was higher in LY294002 + Ghrelin + PDGF group than Ghrelin + PDGF group.It was suggested that after inhibitation of PI3K,the anti-fibrosis effect of ghrelin in LX2 was attenuated.Conclusion After stimulated by PDGF in hepatic stellate cell,ghrelin can inhibit procollagen type Ⅰ and alpha-SMA synthesis in the process of hepatic fibrosis via PI3K-AKT pathway,thus,ghrelin may become one of the new ways of prevention and treatment of liver fibrosis.

EZH2 plays a role in HSC-T6 cell proliferation and activation affecting MAPK/ERK and PI3 K/AKT pathway / 中国药理学通报

Aim To investigate the effects of cell pro-liferation and activation in HSC-T6 cells by inhibiting the expression of EZH2 , and its partial relevant mech-anism. Methods By introducing the inhibitor DZNep in activated HSC-T6 cells stimulated by TGF-β1 , the protein expression levels of EZH2, p-ERK, p-AKT andα-SMA were detected by Western blot. The siRNA targeting EZH2 was designed and synthesized according to its nucleotide sequence, and their corresponding ex-pression vectors were constructed and transfected into HSC-T6 cells with LipofectamineTM 2000. The prolifer-ation of HSC-T6 cells was determined by MTT. And the protein expression levels of EZH2, p-ERK, p-AKT and α-SMA were measured by Western blot. Results By introducing the inhibitor DZNep in activated HSC-T6 cells stimulated by TGF-β1 , it effectively de-creased the protein levels of EZH2 and also the protein levels of p-ERK, p-AKT and α-SMA. By introducing EZH2-siRNA in activated HSC-T6 cells, it effectively inhibited the cell proliferation, and also the protein levels of EZH2, p-ERK, p-AKT andα-SMA. Conclu-sion Silencing EZH2 expression inhibits HSC-T6 cell proliferation and activation, and EZH2 may be a poten-tial therapeutic target gene for hepatic fibrosis.

Dynamic expression of desmin, α-SMA and TGF-β1 during hepatic fibrogenesis induced by selective bile duct ligation in young rats

We previously described a selective bile duct ligation model to elucidate the process of hepatic fibrogenesis in children with biliary atresia or intrahepatic biliary stenosis. Using this model, we identified changes in the expression of alpha smooth muscle actin (α-SMA) both in the obstructed parenchyma and in the hepatic parenchyma adjacent to the obstruction. However, the expression profiles of desmin and TGF-β1, molecules known to be involved in hepatic fibrogenesis, were unchanged when analyzed by semiquantitative polymerase chain reaction (RT-PCR). Thus, the molecular mechanisms involved in the modulation of liver fibrosis in this experimental model are not fully understood. This study aimed to evaluate the molecular changes in an experimental model of selective bile duct ligation and to compare the gene expression changes observed in RT-PCR and in real-time quantitative PCR (qRT‐PCR). Twenty-eight Wistar rats of both sexes and weaning age (21-23 days old) were used. The rats were separated into groups that were assessed 7 or 60 days after selective biliary duct ligation. The expression of desmin, α-SMA and TGF-β1 was examined in tissue from hepatic parenchyma with biliary obstruction (BO) and in hepatic parenchyma without biliary obstruction (WBO), using RT-PCR and qRT‐PCR. The results obtained in this study using these two methods were significantly different. The BO parenchyma had a more severe fibrogenic reaction, with increased α-SMA and TGF-β1 expression after 7 days. The WBO parenchyma presented a later, fibrotic response, with increased desmin expression 7 days after surgery and increased α-SMA 60 days after surgery. The qRT‐PCR technique was more sensitive to expression changes than the semiquantitative method.

Role of Caffeic Acid on Collagen Production in Nasal Polyp-Derived Fibroblasts

OBJECTIVES: Caffeic acids are known to have anti-oxidant, anti-inflammatory, immunomodulatory, and tissue reparative effects. The purposes of this study were to determine the effect of caffeic acid on transforming growth factor (TGF) beta1-induced myofibroblast differentiation and collagen production, and to determine whether caffeic acid is involved in the antioxidant effect in nasal polyp-derived fibroblasts (NPDFs). METHODS: NPDFs were pretreated with caffeic acid (1-10 microM) for 2 hours and stimulated with TGF-beta1 (5 ng/mL) for 24 hours. The expression of alpha-smooth muscle actin (SMA), collagen types I and III, and Nox4 mRNA was determined by a reverse transcription-polymerase chain reaction, and the expression of alpha-SMA protein was determined by actin ned by immunofluorescence microscopy. The amount of total soluble collagen production was analyzed by the Sircol collagen dye-binding assay. The reactive oxygen species (ROS) generated by NPDFs were determined using 2',7'-dichlorfluorescein-diacetate. siNox4 was used to determine the effect of Nox4. RESULTS: The expression of alpha-SMA and production of collagen were significantly increased following TGF-beta1 treatment. In contrast, the level of expression of alpha-SMA and the level of production of collagen were decreased by pretreatment with caffeic acid. The activation of Nox4 and the subsequent production of ROS were also reduced by pretreatment with caffeic acid. The expression of alpha-SMA was prevented by inhibition of ROS generation with siNox4. CONCLUSION: Caffeic acid may inhibit TGF-beta1-induced differentiation of fibroblasts into myofibroblasts and collagen production by regulating ROS.

Primary leiomyoma of the ovaries in a dog / 동물의과학연구지

We would like to report a case of leiomyoma of the ovaries in a dog. Leiomyoma is commonly seen in the vagina in dogs. However, it is a very rare neoplasm in the ovaries. As there have only been a few reported cases, this report provides valuable information on veterinary medicine and pathology. Masses found in the ovaries need to be differentiated from other ovarian tumors. Therefore, we describe the gross, histopathological, and immunohistochemical features of a case of ovarian leiomyoma in a 10-year-old female Yorkshire Terrier dog. The mass on the right of the uterus was found accidentally by pelvic ultrasonography. Laparatomy revealed a large multi-nodulated ovarian mass. Grossly, cut surfaces of the mass showed multiple firm whitish nodules in the cortex and bloody loose connective tissue in the medulla. Histopathologically, the cortex of the mass was composed of spindle cells forming interlacing fascicles. The cells had elongated, blunt-ended nuclei and eosinophilic cytoplasm as detected by hematoxylin and eosin staining. Immunohistochemical stained sections were immunoreactive for alpha-smooth muscle actin and desmin but negative for vimentin and S-100. Therefore, differential diagnosis confirmed leiomyoma based on morphology and positive staining for alpha-smooth muscle actin and desmin.

Effects of silibinin on expression of integrin linked kinase, transforming growth factor β1 and α-smooth muscle actin in rat peritoneal mesothelial cells induced by high glucose / 中华肾脏病杂志

Objective To observe the effect of silibinin on the expression of integrin linked kinase (ILK),transforming growth factor β1 (TGF-β1) and α-smooth muscle actin (α-SMA) in rat peritoneal mesothelial cells (RPMCs) induced by high glucose.Methods RPMCs were isolated,cultured and passaged by trypsin,then identified.The second generation of cultured RPMCs were used in the experiment.RPMCs were divided into normal control group,high glucose(1.5％,2.5％,4.25％)for 24 hours,high glucose (2.5％) for 12,24,48,72 hours,high glucose (2.5％) for 24 hours after silibinin (5,10,20 mg/L) preincubate for 2 hours.ILK and α-SMA mRNA were detected by real-time PCR.ILK protein was detected by Western blotting.TGF-β1 protein in supernatants was detected by ELISA.Results Compared with the control group,the expresssion of ILK,TGF-β1 and α-SAM was significantly increased in groups stimulated by high glucose (all P ＜ 0.05).Silibinin could significantly decrease the expression of ILK,TGF-β1 and α-SMA induced by high glucose (all P ＜ 0.05).Conclusions High glucose can up-regulate the expression of ILK,TGF-β1 and α-SMA.Silibinin can reverse these changes.

Podoplanin, alpha-Smooth Muscle Actin or S100A4 Expressing Cancer-Associated Fibroblasts Are Associated with Different Prognosis in Colorectal Cancers

The interactions between the tumor microenvironment and tumor cells determine the behavior of the primary tumors. Whether cancer-associated fibroblasts (CAF) have a tumor progressive or a protective role likely depends on the type of tumor cells and the CAF subpopulation. In the present study, we analyzed the prognostic significance of CAF subpopulations in colorectal cancer (CRC). CAF phenotypes were analyzed in 302 CRC patients by using antibodies against podoplanin (PDPN), alpha-smooth muscle actin (alpha-SMA), and S100A4. The relationship between the CAF phenotypes and 11 clinicopathological parameters were evaluated and their prognostic significance was analyzed from the disease-free and overall survival times. We observed that at the tumor invasive front, PDPN CAFs were present in 40% of the cases, and S100A4 or alpha-SMA CAFs were detected in all the cases. PDPN/S100A4 and alpha-SMA/S100A4 dual-stained CAFs were observed in 10% and 40% of the cases, respectively. The PDPN+ CAFs were associated with 6 favorable clinicopathological parameters and prolonged disease-free survival time. The PDPN-/alpha-SMA(high) CAFs were associated with 6 aggressive clinicopathological parameters and tended to exhibit shorter disease-free survival time. On the other hand, the PDPN-/S100A4(high) CAFs were associated with 2 tumor progression parameters, but not with disease prognosis. The PDPN+ CAF phenotype is distinct from the alpha-SMA or S100A4 CAFs in that it is associated with less aggressive tumors and a favorable prognosis, whereas the PDPN-/alpha-SMA(high) or PDPN-/S100A4(high) CAFs are associated with tumor progression in CRC. These findings suggest that CAFs can be a useful prognostic biomarker or potential targets of anti-cancer therapy in CRC.

Effects of valsartan on renal interstitium fibrosis in rats after unilateral ureteral obstruction / 第二军医大学学报

Objective To study the effect of valsartan, an angiotensin II type I receptor antagonist AT1RA), on renal interstitium fibrosis(RIF)in rats with unilateral ureteral obstruction (UUO), and to discuss the possible mechanisms. Methods Thirty-five Sprague-Dawley rats were randomly divided into sham-operation, model and valsartan groups. The rat UUO model was established. From the day after operation, the rats in sham-operation and model groups received intragastric valsartan and sodium chloride in tales doses. The serum creatinine (SCr), blood urea nitrogen (BUN), angiotensin- II (Ang II ) in blood plasma, N-acetyl-β-D-glucosaminidase(NAG)and 24 h urine β2-microglobulin(β2-MG)were examined 4 weeks after operation. The renal tissues of the obstructed sides were harvested; H-E staining and Masson staining were used to observe the tubulointerstitial lesions; and immunohistochemistry staining was used for semiquantitative analysis of alpha-smooth muscle actin(α-SMA), fibronectin(FN), plasminogen activator inhibitor-1 (PAI-1), transforming growth factor-beta 1 (TGF-β1), and hepatocyte growth factor(HGF). Results Compared with those in the sham-operation group, SCr, BUN, Ang II, NAG and (β2- MG levels, and the expression of α-SMA, FN, PAI-l, and TGF-β1 in model group were significantly higher(P0. 05). The expression levels of orSMA, FN, PAI-l, and TGF-β1 in valsartan group were significantly lower than and the expression of HGF was significantly higher than those in the model group(P<0. 01). Conclusion Valsartan does not improve the tubular and glomerular functions, but it can inhibit production of Ang-II. Valsartan may inhibit renal interstitial fibrosis by inhibiting renal tubule epithelial mesenchymal transdifferentiation and reducing extracellular matrix deposition through blocking up Ang Q, inhibiting overexpression of α-SMA, FN, PAI-l, and TGF-β1, and inducing the HGF expression.

Synovial fluid of patients with rheumatoid arthritis induces alpha-smooth muscle actin in human adipose tissue-derived mesenchymal stem cells through a TGF-beta1-dependent mechanism

Rheumatoid arthritis (RA) is a chronic, inflammatory autoimmune disorder that causes the immune system to attack the joints. Transforming growth factor-beta1 (TGF-beta1) is a secreted protein that promotes differentiation of synovial fibroblasts to alpha-smooth muscle actin (alpha-SMA)-positive myofibroblasts to repair the damaged joints. Synovial fluid from patients with RA (RA-SF) induced expression of alpha-SMA in human adipose tissue-derived mesenchymal stem cells (hASCs). RA-SF-induced alpha-SMA expression was abrogated by immunodepletion of TGF-beta1 from RA-SF with anti-TGF-beta1 antibody. Furthermore, pretreatment of hASCs with the TGF-beta type I receptor inhibitor SB431542 or lentiviral small hairpin RNA-mediated silencing of TGF-beta type I receptor expression in hASCs blocked RA-SF-induced alpha-SMA expression. Small interfering RNA-mediated silencing of Smad2 or adenoviral overexpression of Smad7 (an inhibitory Smad isoform) completely inhibited RA-SF-stimulated alpha-SMA expression. These results suggest that TGF-beta1 plays a pivotal role in RA-SF-induced differentiation of hASCs to alpha-SMA-positive cells.