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<p><b>Objective</b>To study the effect of Icariin on rat Leydig cells with TGF-β1-induced injury.</p><p><b>METHODS</b>We determined the optimal concentration of Icariin for protecting primarily cultured Leydig cells against TGF-β1-induced injury by methyl thiazolyl tetrazolium assay. We detected the effects of Icariin on the secretion of estradiol (E2) and activity of aromatase in the injured Leydig cells by radioimmunoassay and Tritium water release experiment and its effect on the gap junctional intercellular communication (GJIC) between the Leydig cells by fluorescence distribution after photobleaching.</p><p><b>RESULTS</b>Different concentrations of Icariin showed different degrees of protective effect on the TGF-β1-treated Leydig cells, the effect observed at 20 μg/ml and at its optimum at 160 μg/ml. After treatment of the injured Leydig cells with Icariin at 160 μg/ml, significant improvement was observed in the E2 secretion and aromatase activity (P<0.01) as well as in the GJIC between the Leydig cells (P<0.01).</p><p><b>CONCLUSIONS</b>Icariin can effectively protect rat Leydig cells against TGF-β1-induced injury, which is largely attributed to its effects of increasing E2 synthesis, enhancing aromatase activity, and improving GJIC between Leydig cells.</p>
ABSTRACT
Background Doxycycline is a broad spectrum antibiotic,and it is frequently used in the treatment of ocular surface diseases.Objective The purpose of the present study was to investigate the effect of doxycycline on the inhibition of cell proliferation in bovine corneal myofibroblasts in vitro and assess its contribution to ocular surface repairing mechanism.Methods Six fresh bovine corneas were collected.The corneal stromal layer was isolated by two-step method of 1.0 g/L and 2.0 g/L collegenase-1.Isolated cells were plated at mantaryay culture flask in 10％ FBS of RPMI-1640.Vimentin and alpha-smooth muscle actin (α-SMA) organization were evaluated by immunocytochemistry,and the cells with influoresccence staining for vimentin and α-SMA were identified as the corneal myofibroblasts.Doxycycline at the concentrations of 10,20,40,60,80 mg/L was added to the medium,respectively,in different concentrations of doxycycline groups.Dexamethasone (120 mg/L)was used in the same way in the positive control group,and no drug was used in the negative control group.Cell proliferation was evaluated by MTT and the cell cycle was analyzed by BD FACScan flow cytometer assay 24 hours and 48 hours after addition of any drug.Results The cells grew well and showed the positive response for vimentin and α-SMA.MTT assay showed that the A570values of bovine corneal myofibroblasts were gradually declined with the increase of the concentration of doxycycline and lapse of active time,showing statistically significant difference (Fconcentration =1233.778,P＜0.001 ; Ftime =227.564,P ＜ 0.001).And the difference between the two factors was also statistically significant (Ftime*concentration =51.656,P＜0.001).Flow cytometry cell cycle analysis showed that 24 hours after 10,20,40,60,80 mg/L doxycycline treated,the perentage of of corneal myofibroblast cell in G0-G1 phase was 82.85％,84.36％,85.18％,87.12 ％ and 89.31％,showing significant increase in comparison with 63.89％ of the negative control group (all P＜0.05),and that of 40 mg/L doxycycline group was near the positive control group.Forty-eight hours after 10,20,40,60,80 mg/L doxycycline treated,the perentage of of corneal myofibroblast cell in G0-G1 phase was 82.78％,86.15％,88.23％,89.57％,93.00％,with significant increase in comparison with 70.17％ of the negative control group (all P ＜ 0.01),and that of 40 mg/L doxycycline group was near the positive control group.Conclusions The growth of the bovine corneal myofibroblasts is inhibited by doxycycline in time-and dosedependent manner in the range from 10 mg/L to 80 mg/L,and 40 mg/L of doxycycline has an obviously inhibitory action as 120 mg/L dexamethasone.
ABSTRACT
<p><b>OBJECTIVE</b>To observe the effects of TGF-beta on the expression of connexin43 (Cx43) and Cx43-mediated gap junctional intercellular communication (GJIC) in rat Leydig cells, and investigate the association of its effects on Leydig cells with its ability of changing GJIC.</p><p><b>METHODS</b>Primarily cultured purified Leydig cells were divided into a blank control group, a positive control group (treated with the GJIC inhibitor Carbenoxolone), and four TGF-beta1 groups (treated with TGF-beta1 at the concentration of 1, 2, 5 and 10 ng/ml, respectively, for 20 hours). The localization and expression of Cx43 were detected by immunofluorescence and Western blot, and the changes in GJIC analyzed by FRAP assay.</p><p><b>RESULTS</b>Cx43 was expressed as scattered bright spots in the cytoplasm and membrane of Leydig cells. TGF-beta1 significantly elevated the expression of Cx43 in the cytoplasm, but caused no evident change in the membrane. Western blot showed an evident increase in the phosphorylation of Cx43 with the increased concentration of TGF-beta1 as compared with that of the blank control group (P < 0.05). After 20 hours of treatment with TGF-beta1 at 5 ng/ml, the fluorescence intensity of Leydig cells was markedly reduced (P < 0.01), with a mean fluorescence recovery rate of merely (43.58 +/- 1.87)%.</p><p><b>CONCLUSION</b>TGF-beta1 could significantly down-regulate GJIC between adjacent Leydig cells, and this inhibitory effect may be achieved by promoting the expression of Cx43 in the cytoplasm and elevating the phosphorylation of Cx43.</p>
Subject(s)
Animals , Male , Rats , Cell Communication , Cells, Cultured , Connexin 43 , Metabolism , Gap Junctions , Metabolism , Leydig Cells , Metabolism , Phosphorylation , Transforming Growth Factor beta1 , PharmacologyABSTRACT
<p><b>OBJECTIVE</b>To study the effects of Wenyang Shengjing Decoction (WSD) containing serum on the estradiol (E2) secretion, the synthesized cytochrome P450 aromatase (P450arom) activities, as well as the expression of its encode gene CYP19 in Leydig cells of male sterile rats of adenine induced Shen-yang deficiency (SYD).</p><p><b>METHODS</b>Experimental rats were randomly divided into 4 groups, i.e., the normal control group, the high, middle, and low dose WSD groups, 5 in each group. The normal saline, low, middle, and high dose WSD were respectively given to rats of all groups for 10 successive days. Blood was drawn from rats' heart 2 h after the last gastrogavage. The serum was separated after centrifuge. Leydig cells isolated and purified from SYD rats were primary cultured in vitro and divided into 5 groups in random, i. e., the blank control group, the model group, the high, middle, and low dose WSD groups (1.2, 1.0, and 0.8 g/mL, respectively). The content of E2 released in the culture supernate was determined by radioimmunoassay. The P450arom activity was detected by tritium release assay. Meanwhile, the mRNA and protein expressions of CYP19 were analyzed using fluorescent quantitative PCR and Western blot respectively.</p><p><b>RESULTS</b>Compared with the blank control group, the E2 secretion of the supernate of Leydig cells obviously decreased in the model groups, accompanied with the inhibition of P450arom activities, significant decreased protein and mRNA expressions of CYP19 (P < 0.01, P < 0.05). Compared with the model group, after intervened by WSD containing serum, the E2 secretion in the Leydig cells could be significantly increased, the P450arom activities up-regulated, the CYP19 expressions up-regulated at the protein and mRNA levels partially in a dose-dependent manner (P < 0.01, P < 0.05).</p><p><b>CONCLUSIONS</b>WSD containing serum could effectively elevate the E2 secretion in Leydig cells, which might be partially achieved through up-regulating P450arom activities and enhancing the gene expression of CyP19. This might be one of its mechanisms of action for treating male infertility of SYD.</p>