Objective To investigate the effects and mechanism of Interleukin-33 (IL-33) mediated proliferation and differentiation of pulmonary myofibroblasts (MFbs) in pulmonary fibrosis (PF). Methods C57BL/6 mice were randomly divided into four groups: a control group, a bleomycin (BLM) group, a BLM combined with IL-33 group and a BLM combined with anti-IL-33 antibody group, 12 mice in each group. The PF model was induced by intratracheal injection of BLM (5000 U/kg). The degrees of fibrosis were examined using HE and Masson staining. ELISA was used to measure the plasma levels of IL-33. Immunohistochemical staining was used to measure the expression of alpha smooth muscle actin (α-SMA) in lung tissue. Primary pulmonary fibroblasts were isolated and cultured from lung tissues of mice. The cells were divided into four groups: a control group, an IL-33 group, an IL-33 combined with dimethyl sulfoxide (DMSO) group and an IL-33 combined with pyrrolidine dithiocarbamate (PDTC) group. The cells were treated with DMSO or PDTC for 1 hour and then with IL-33 for 48 hours. Cell proliferation was measured by 5-ethynyl-2'-deoxyuridine (EdU) assay and cell cycle was measured by flow cytometry. Transwell^TM assay was used to analyze cell migration. Real-time quantitative PCR was used to measure the expression of collagen type I (Col1), Col3 and α-SMA mRNA. The protein levels of IL-33, Col1, Col3, α-SMA, eukaryotic initiation factor 3a (eIF3a), phosphorylated IκBα (p-IκBα) (total lysate), p-NF-κB p65(total lysate) and NF-κB p65 (nucleus) were measured by Western blot analysis. Results In vivo, compared with the control group, the expressions of IL-33, p-IκBα (total lysate), p-NF-κB p65 (total lysate), NF-κB p65(nucleus), eIF3a, α-SMA, Col1 and Col3 in the BLM group significantly increased. Compared with the BLM group, the expressions of p-IκBα (total lysate), p-NF-κB p65 (total lysate), NF-κB p65 (nucleus), eIF3a, α-SMA, Col1 and Col3 in the IL-33 group increased further and the PF was further aggravated. But the effect of anti-IL-33 antibody was just opposite to that of IL-33. In vitro, IL-33 markedly induced the proliferation and migration of pulmonary fibroblasts, and significantly up-regulated the expression of p-IκBα (total lysate), p-NF-κB p65(total lysate), NF-κB p65 (nucleus), eIF3a, α-SMA, Col1 and Col3. But all these effects of IL-33 were reversed by pyrrolidine dithiocarbamate. Conclusion The results suggest that IL-33 may promote the expression of eIF3a by activating NF-κB signaling pathway, thus inducing the proliferation and differentiation of MFbs and promoting the occurrence and development of PF.
Animals ; Mice ; Bleomycin/metabolism* ; Cell Differentiation ; Cell Proliferation ; Dimethyl Sulfoxide/pharmacology* ; Fibroblasts ; Interleukin-33/pharmacology* ; Mice, Inbred C57BL ; Myofibroblasts/metabolism* ; NF-kappa B/metabolism* ; NF-KappaB Inhibitor alpha/metabolism* ; Pulmonary Fibrosis ; Signal Transduction

Objective:To explore the role of long non-coding RNA (LncRNA) AC000061.1 involved in the pathogenesis of nonobstructive azoospermia (NOA) by regulating the CFTR gene expression. Methods:Bioinformatics analysis was conducted with the use of gene microarray to screen the differentially expressed LncRNAs and corresponding mRNAs in the testicular tissue of patients in three groups including obstructive azoospermia (OA) group ( n=50), NOA group ( n=50), and control group ( n=50). The expression of apoptosis-related gene Bcl-2 in the testicular tissue of patients in these three groups was detected by qRT-PCR and Western blotting. Additionally, qRT-PCR was performed to determine the expression of LncRNA AC000061.1 and CFTR mRNA in the testicular tissue, serum, and seminal plasma of patients in the three groups, and enzyme-linked immunosorbent assay (ELISA) was conducted to detect the CFTR protein level in the serum and seminal plasma. LncRNA AC000061.1 overexpression (H-LncRNA group) and silencing vectors (Si-LncRNA group) and empty vectors were constructed and transfected into the testicular cancer cell line NTERA-2. Subsequently, the expression of LncRNA AC000061.1 and CFTR mRNA was determined by qRT-PCR, and CFTR protein expression was measured by Western blotting assay. Cell proliferation was assessed using CCK-8 and cell apoptosis was evaluated by flow cytometry and TUNEL. Results:Microarray analysis and qRT-PCR showed that the expression of LncRNA AC006100.1 and CFTR decreased in NOA group compared with control group ( P=0.033 and P=0.042). But there was no difference in the expression of LncRNA AC000061.1 between OA group and control group, while CFTR in OA group was lowly expressed compared with that in control group ( P=0.039). Bcl-2 expression was sequentially upregulated in OA and NOA groups relative to normal control group ( P=0.031 and P=0.008). No significant difference was noted in the serum levels of LncRNA AC000061.1 and CFTR mRNA and protein among the three groups ( P>0.05). The results also showed sequentially decreased levels of LncRNA AC000061.1 and CFTR mRNA and protein in seminal plasma in NOA and OA groups when compared with control group ( P=0.002, P=0.038 and P=0.006, P=0.026) and a positive correlation of LncRNA AC000061.1 expression with CFTR mRNA expression ( r=0.169, P=0.039). LncRNA AC000061.1 and CFTR mRNA and protein expression decreased in Si-LncRNA group ( P=0.005, P=0.003), but significantly increased in H-LncRNA group ( P=0.002, P=0.009). Furthermore, cell proliferation was significantly repressed while apoptosis rate was elevated in Si-LncRNA group ( P=0.003 and P=0.001). On the contrary, enhanced cell proliferation and inhibited apoptosis were observed in H-LncRNA group ( P=0.017 and P=0.017). Conclusion:LncRNA AC00006.1 in the testicular tissue of NOA patients induced abnormal cell proliferation and apoptosis via mediating CFTR gene expression, thereby participating in the pathogenesis of NOA.

Objective:To explore the role of long non-coding RNA (LncRNA) AC000061.1 involved in the pathogenesis of nonobstructive azoospermia (NOA) by regulating the CFTR gene expression. Methods:Bioinformatics analysis was conducted with the use of gene microarray to screen the differentially expressed LncRNAs and corresponding mRNAs in the testicular tissue of patients in three groups including obstructive azoospermia (OA) group ( n=50), NOA group ( n=50), and control group ( n=50). The expression of apoptosis-related gene Bcl-2 in the testicular tissue of patients in these three groups was detected by qRT-PCR and Western blotting. Additionally, qRT-PCR was performed to determine the expression of LncRNA AC000061.1 and CFTR mRNA in the testicular tissue, serum, and seminal plasma of patients in the three groups, and enzyme-linked immunosorbent assay (ELISA) was conducted to detect the CFTR protein level in the serum and seminal plasma. LncRNA AC000061.1 overexpression (H-LncRNA group) and silencing vectors (Si-LncRNA group) and empty vectors were constructed and transfected into the testicular cancer cell line NTERA-2. Subsequently, the expression of LncRNA AC000061.1 and CFTR mRNA was determined by qRT-PCR, and CFTR protein expression was measured by Western blotting assay. Cell proliferation was assessed using CCK-8 and cell apoptosis was evaluated by flow cytometry and TUNEL. Results:Microarray analysis and qRT-PCR showed that the expression of LncRNA AC006100.1 and CFTR decreased in NOA group compared with control group ( P=0.033 and P=0.042). But there was no difference in the expression of LncRNA AC000061.1 between OA group and control group, while CFTR in OA group was lowly expressed compared with that in control group ( P=0.039). Bcl-2 expression was sequentially upregulated in OA and NOA groups relative to normal control group ( P=0.031 and P=0.008). No significant difference was noted in the serum levels of LncRNA AC000061.1 and CFTR mRNA and protein among the three groups ( P>0.05). The results also showed sequentially decreased levels of LncRNA AC000061.1 and CFTR mRNA and protein in seminal plasma in NOA and OA groups when compared with control group ( P=0.002, P=0.038 and P=0.006, P=0.026) and a positive correlation of LncRNA AC000061.1 expression with CFTR mRNA expression ( r=0.169, P=0.039). LncRNA AC000061.1 and CFTR mRNA and protein expression decreased in Si-LncRNA group ( P=0.005, P=0.003), but significantly increased in H-LncRNA group ( P=0.002, P=0.009). Furthermore, cell proliferation was significantly repressed while apoptosis rate was elevated in Si-LncRNA group ( P=0.003 and P=0.001). On the contrary, enhanced cell proliferation and inhibited apoptosis were observed in H-LncRNA group ( P=0.017 and P=0.017). Conclusion:LncRNA AC00006.1 in the testicular tissue of NOA patients induced abnormal cell proliferation and apoptosis via mediating CFTR gene expression, thereby participating in the pathogenesis of NOA.

ObjectiveTo evaluate the daily vitamin intakes in diets of diabetic patients.MethodsUsing a food questionnaire,the contents of 63 type Ⅱ diabetics(group A) and 64 non diabetics(group B) were recorded and analysised by computer.The daily vitamin intakes were assessed and compared with the Recommend Dietary Allowance(RDA).ResultsThe daily intakes of vitamin B1 and vitamin B2 were both obviously deficient,less than 50% of the RDA.The daily intakes of vitamin E and A were significantly different between group A and group B.ConclusionsThe vitamin intakes of the senile diabetics in diets were inadequate.