CGRP protects bladder smooth muscle cells stimulated by high glucose through inhibiting p38 MAPK pathway in vitro

Abstract

This study aimed to explore the effect of calcitonin gene-related peptide (CGRP) on bladder smooth muscle cells (BSMCs) under high glucose (HG) treatment in vitro. BSMCs from Sprague-Dawley rat bladders were cultured and passaged in vitro. The third-generation cells were cultured and divided into control group, HG group, HG + CGRP group, HG + CGRP + asiatic acid (AA, p-p38 activator) group, CGRP group, AA group, HG + CGRP + CGRP-8-37 (CGRP receptor antagonist) group and HG + LY2228820 (p38 MAPK inhibitor) group. The cell viability, apoptosis, malondialdehyde (MDA) and superoxide dismutase (SOD) levels of BSMCs were observed by the relevant detection kits. The expressions of α-SM-actin, p38 and p-p38 were detected by qRT-PCR or Western blot analysis. Compared with the control group, the cell viability, SOD and α-SM-actin levels of BSMCs were decreased and apoptotic cells, MDA and p-p38 levels were increased after HG treatment, while these changes could be partly reversed when BSMCs were treated with HG and CGRP or LY2228820 together. Moreover, AA or CGRP-8-37 could suppress the effect of CGRP on BSMCs under HG condition. Our data indicate that CGRP protects BSMCs from oxidative stress induced by HG in vitro, and inhibit the α-SM-actin expression decrease through inhibiting the intracellular p38 MAPK signaling pathway.

Figure 1

(a) The viability of BSMCs was detected by MTT. Compared with the control, CGRP and AA groups, the OD values of the other groups decreased but the OD values of the HG + CGRP and HG + LY2228820 groups were higher than that in the HG, HG + CGRP + AA and HG + CGRP + CGRP-8-37 groups. (b) TUNEL assay results showed that there were almost no apoptotic cells in the control, CGRP and AA groups. More apoptotic cells were observed in the HGgroup, after CGRP or LY2228820 treatment, the number of apoptotic cells was decreased. AA or CGRP-8-37 reversed the effect of CGRP on the cells. (c) Statistical histogram of the percentage of apoptotic cells in each group. *VS. control group, p < 0.05; ^# VS. HG group, p < 0.05; ^@ VS. HG + CGRP group, p < 0.05; ^ VS. HG + CGRP + AA group, p < 0.05; $ VS. CGRP group, p < 0.05; & VS. AA group, p < 0.05; ! VS. HG + CGRP + CGRP-8-37 group, p < 0.05. Bar = 100 μm. n = 6. The bar charts were created by GraghPad Prism 7.0 software (Version 7.00, URL: www.graphpad.com).

Figure 2

The oxidative stress level of BSMCs was measured by SOD and MDA kits. (a) Compared with the control, CGRP and AA groups, the SOD levels in the other groups decreased, but the SOD levels in the HG + CGRP and HG + LY2228820 groups were higher than that in the HG group, HG + CGRP + AA and HG + CGRP + CGRP-8-37 groups. (b) Compared with the control, CGRP and AA groups, the MDA level in the other groups increased but the MDA levels in the HG + CGRP and HG + LY2228820group were lower than that in the HG group, HG + CGRP + AA and HG + CGRP + CGRP-8-37 groups. * VS. control group, p < 0.05; ^# VS. HG group, p < 0.05; ^@ VS. HG + CGRP group, p < 0.05; ^ VS. HG + CGRP + AA group, p < 0.05; $ VS. CGRP group, p < 0.05; & VS. AA group, p < 0.05; ! VS. HG + CGRP + CGRP-8-37 group, p < 0.05. n = 6. The bar charts were created by GraghPad Prism 7.0 software (Version 7.00, URL: www.graphpad.com).

Figure 3

The expression of α-SM-actin in BSMCs was measured by immunofluorescence, qRT-PCR and Western blot. (a) The result of immunofluorescence showed the visible α-SM-actin fibers in BSMCs were found in the control, CGRP, AA, HG + CGRP and HG + LY2228820 groups while they were unclear in the HG, HG + CGRP + AA and HG + CGRP + CGRP-8-37 groups. (b,c) Compared with the control, CGRP and AA groups, the mRNA and protein expressions of α-SM-actin in the HG group, the HG + CGRP + AA and HG + CGRP + CGRP-8-37 groups were significantly decreased. Though the expression of α-SM-actin mRNA and protein in the HG + CGRP and HG + LY2228820 groups also deceased, it was higher than that in the HG, HG + CGRP + AA and HG + CGRP + CGRP-8-37 groups. * VS. control group, p < 0.05; ^# VS. HG group, p < 0.05; ^@ VS. HG + CGRP group, p < 0.05; ^ VS. HG + CGRP + AA group, p < 0.05; $ VS. CGRP group, p < 0.05; & VS. AA group, p < 0.05; ! VS. HG + CGRP + CGRP-8-37 group, p < 0.05. Bar = 25 μm. n = 6. The bar charts were created by GraghPad Prism 7.0 software (Version 7.00, URL: www.graphpad.com). The Western blot bands were created by Odyssey 3.0 image analysis system software (Version 1.2.15, URL: www.licor.com/bio/supportsupport).

Figure 4

The expressions of p38 MAPK in BSMCs were detected by qRT-PCR and Western blot. (a) The expression of p38 mRNA in the groups was no significant change. (b) The expression of p38 protein in the groups was no significant change. Compared with the control and CGRP groups, the expression of p-p38 protein in AA group increased. The expression of p-p38 protein in the HG group was notably increased, the degree of the change was reduced when BSMCs were treated with CGRP or LY2228820 at the same time, and AA or CGRP-8-37 could suppress the effect of CGRP on the expression of p-p38 protein in BSMCs under HG. . * VS. control group, p < 0.05; ^# VS. HG group, p < 0.05; ^@ VS. HG + CGRP group, p < 0.05; ^ VS. HG + CGRP + AA group, p < 0.05; $ VS. CGRP group, p < 0.05; & VS. AA group, p < 0.05; ! VS. HG + CGRP + CGRP-8-37 group, p < 0.05. n = 6. The bar charts were created by GraghPad Prism 7.0 software (Version 7.00, URL: www.graphpad.com). The Western blot bands were created by Odyssey 3.0 image analysis system software (Version 1.2.15, URL: www.licor.com/bio/supportsupport).