A Study Of The Effect Of H₂S And NO On Different Human Pregnant Myometrium Contractile Activity

In this experiment, we hope to take a study of regulation of H2S and NO on human pregnant myometrium contractile activity by determination of isometric tension of human myometrial tissue and then observe the expression of ATP-sensitive potassium channel in human myometrium of pregnancy in different states, to realize the possible mechanism of its regulation to uterine contraction. Thus we can have further understanding on the mechanism of start-up and premature, provide a theoretical basis for the prevention and treatment of preterm labor and other abnormal uterine smooth muscle contraction-related diseases.PartⅠA study of the effect of H2S on human pregnant myometrium contractile activityObjective The aim is to study the effect of H2S on human pregnant myometrium contractile activity and the expression of ATP-sensitive potassium channel in different human pregnant myometrium.Methods 1. For determinate the isometric tension of human myometrial tissue, we divided four groups to do experiments as follow: group A is term non-labor (TNL), group B is term labor (TL), group C is term non-labor (TNL), myometrium contractile induced by oxytocin(OT), group D is term labor (TL), myometrium contractile induced by oxytocin (OT). 1.1 Adding directly in the progressive manner in accordance with the concentration in the bath of the establishment of uterine muscle contraction rule using sodium hydrogen sulphide (NaHS)[10-8 to 10-3mmol/l], a donor of hydrogen sulphide (H2S), observing and recording the state of myometrium contractile.1.2 Adding Glibenclamide[10-2mol/L], the blocking agent of ATP-sensitive potassium channel directly into the myometrial tissue bath which had already set up regular myometrium contractile. After 5 min, adding NaHS with 10-3mol/L , and observing and recording the state of myometrium contractile. 2. After extracting uterine smooth muscle RNA, RT–PCR are used todetect the presence of mRNA for three subunits of KATP channels (Kir6.1, Kir6.2 and SUR2B) in the two tissue types. Quantitative analysis of these subunits was achieved with real-time PCR using Lightcycler? technology. 3. Using immunohistochemical method to detect the distribution of the three types of KATP subunits protein in the TL and TNL groups.Results (1) Among the concentration of 10-8～10-3mol/L, the depression effect of hydrogen sulphide (H2S) with 10-7mol/L on myometrial tissue contractile are signigficant in the group TL and TL(OT). This inhibition effects of H2S on human pregnant uterin strips showed in the area under the response curve(AUC) and contraction frequency, and a dose-dependent manner, whereas no significant effect on contraction tension. (2) AUC inhibition rate and contraction frequency between the TL group and the TL (OT) group are no statistically significant difference(P>0.05).(3)The medium effective concentration(EC50) of group TL and group TL(OT) are 42.62±11.03μmol/l, 4.75±1.32μmol/l, have significant difference(P=0.029). (4) Both in TNL group and the TNL (OT) group, AUC inhibition rate and contraction frequency between the pre-H2S and post-H2S are no statistically significant difference(P>0.05). H2S had no significant relaxation effect in term non-labor uterine smooth muscle.(5) Using KATP channel blocker glibenclamide with 5min, then adding sodium hydrosulfide, uterine smooth muscle of the area under the curve, contraction frequency and intensity compared to the dosing ago is no significant difference (P>0.05). Inhibition of H2S on contraction is disappeared. (6) Results of real-time PCR show that both of the group TNL and TL have the expression of the three subunits of KATP channels. In TNL, the level of SUR2B transcript is higher than the level of Kir6.2 and Kir6.1, respectively(P=0.016, 0.072). There are no differences among the three subunits in TL(P>0.05). There are higher levels of SUR2B and Kir6.2 transcripts in TNL myometrium compared with those measured in TL(P=0.045,0.047).The level of Kir6.1 between TNL and TL has no statistical significance(P>0.05). (7) The results of immunohistochemistry reveal that both of the group TNL and TL have the expression of Kir6.1,Kir6.2 and SUR2B proteins.Summary H2S has a depress effect on human term labor myometrium contractile. Their EC50 have differences between contractile OT and without OT induce contractile groups,it shows the term labor uterin strips with OT may be more sensitive than self-contraction myometrium. After using sodium hydrogen sulphide combine wih Glibenclamide, the depress effect of H2S disappeared, it indicates that KATP channels exist in human pregnancy myometrium, and H2S make smooth muscle cell relaxation may through exciting KATP channels.Regulation of Kir6.2 and SUR2B transcripts may facilitate myometrial function during pregnancy.PartⅡA study of the effect of NO on different human pregnant myometrium contractile activityObjective The aim is to study the effect of NO on different human pregnant myometrium contractile activity. Methods For determinate the isometric tension of human myometrial tissue, we divided four groups to do experiments as follow: group A is term non-labor (TNL), group B is term labor (TL), group C is term non-labor (TNL), myometrium contractile induced by oxytocin(OT), group D is term labor (TL), myometrium contractile induced by oxytocin (OT). Adding directly in the progressive manner in accordance with the concentration in the bath of the establishment of uterine muscle contraction rule using or sodium nitroprusside(SNP)[10-8 to 10-5mol/L], a donor of nitric oxide, observing and recording the state of myometrium contractile.Results (1) Among the concentration of 10-8～10-5mol/L, NO can inhibit the uterine muscle contraction in group TL and TNL with dose-dependent manner. (2) In TNL group, NO has the depression effect on the AUC with the concentration of 10-6 mol/L, whereas the depression effect on the AUC is disappeared in the concentration of 10-5 mol/L(P=0.007). (3) In the TL group, when sodium nitroprusside with the concentration of 10-7mol/L, it presents AUC inhibition; when the concentration of 10-5mol/L, the inhibitory effect is most evident (P = 0.001). (4) In the TNL (OT) and TL (OT) group, it is not observed that sodium nitroprusside has the obvious inhibitory effect in uterine smooth muscle contraction induced by oxytocin. (5) The medium effective concentration(EC50) of group TNL and group TL are 1.165±0.651μmol/l,0.325±0.102μmol/l, it has statistical significance(P=0.033). It indicates that the uterine smooth muscle of TL may be more sensitivity to NO.Summary Nitric oxide can inhibit the uterine muscle contraction in group TL and TNL with dose-dependent manner. The inhibition effect of NO on TL group is high than TNL group, it shows that uterine smooth muscle after labor is more sensitivity to NO.Conclusion1. H2S has a depress effect on human term labor myometrium contractile. It shows the term labor uterin strips with OT is more sensitive than self-contraction myometrium. After using sodium hydrogen sulphide combine wih Glibenclamide, the depress effect of H2S disappeared, it indicates that KATP channels exist in human pregnancy myometrium, and H2S make smooth muscle cell relaxation may through exciting KATP channels.2. The regulation of Kir6.2 and SUR2B may facilitate myometrial function during pregnancy.3. Nitric oxide can inhibit the uterine muscle contraction in group TL and TNL with dose-dependent manner. Compared with H2S, they had the similar inhibitory effect on TL uterine smooth muscle, but different on TNL uterine smooth muscle.4. The inhibition effects of NO on TL group are high than TNL group, and the uterine smooth muscle after labor is more sensitivity to NO.