Reduced TRPV1, Kir6.1/SUR2B, and eNOS expression in severe preeclampsia
The localization of the TRPV1, Kir6.1/SUR2B, and eNOS channels was evaluated in placental arterioles by immunohistochemistry (Fig. 3), and the results showed that each channel was mainly distributed in the vascular endothelial layer. It was noted that the counterstained nuclei in the endothelial cell layer was significantly decreased, as was membrane staining of each channel in the SP group compared with the NP group. The optical densities of TRPV1, Kir6.1/SUR2B, eNOS in the SP group were 11660.0±1721.0, 2975.0±505.5, 10236.0±1355.0, 14663.0±2320.0, respectively, which were lower than those in the NP group (24917.0±2044.0, 4495.0±775.0, 14663.0±2320.0, 20988.0±2289.0, P<0.01) (Fig. 3). We performed quantitative PCR to examine the relative expression of the four channel genes. The relative expression of TRPV1, Kir6.1, SUR2B and eNOS in the SP group was 0.559±0.609, 0.419±0.281, 0.166±0.087 and 0.383±0.110, respectively, which was significantly lower than that in the NP group (1.098±0.341, 1.024±0.085, 1.243±0.335 and 1.219±0.247, P<0.01) (Fig. 4). Since gene expression does not fully reflect protein expression, we performed Western blotting to quantitatively examine whether this difference was also present at the protein level. The relative quantitative analysis results (Fig. 5) showed that the ratio of the gray value of each target protein to GAPDH in the SP group was 0.282±0.058, 0.058±0.005, 0.132±0.007, 0.059±0.023, respectively, which was lower than that in the NP group (0.688±0.145, 0.196±0.010, 0.514±0.018, 0.327±0.063, P<0.01), suggesting that the relative protein expression of TRPV1, KATP subtype Kir6.1/SUR2B and eNOS in the SP group was also significantly downregulated.
The roles ofeNOS and Kir6.1/SUR2B channels in capsaicin/capsazepine-induced relaxation in human umbilical vein endothelial cells
To explore the interaction of the four channels, we carried out further cytological experiments. The relative gene expression of TRPV1, Kir6.1, SUR2B and eNOS in the control group was 0.986±0.129, 1.439±0.358, 1.479±0.403 and 1.162±0.090, respectively; in the capsaicin group, it was 4.568±0.810, 4.014±0.781, 5.505±0.287 and 2.821±0.377, respectively; and in the capsazepine group, it was 0.077±0.010, 0.036±0.014, 0.046±0.010 and 0.083±0.005, respectively, and the differences were statistically significant (P < 0.01) (Fig. 6). The Western blot results showed that the relative protein expression of TRPV1, Kir6.1, SUR2B and eNOS in the control group was 0.266±0.026, 0.226±0.014, 0.166±0.013 and 0.277±0.025, respectively; expression in the capsaicin group was 0.763±0.044, 0.687±0.046, 0.493±0.035 and 0.498±0.022, respectively; and expression in the capsazepine group was 0.157±0.040, 0.057±0.005, 0.065±0.005 and 0.058±0.008, respectively (P<0.01), and these differences were statistically significant (P<0.01) (Fig. 7).