去盆腔神经支配对大鼠远端结肠黏膜瞬时受体电位通道V1表达的影响

Effects of pelvic nerves denervation on the expression of transit receptor potential vanilloid 1 in distalcolonic mucosa of rats

目的：探讨去盆腔神经支配（PND）对大鼠远端结肠黏膜瞬时受体电位通道V1（TRPV1）的影响。 方法：采用实验研究方法。将108只成年雄性大鼠按数字表法随机分成对照组、假手术组及PND组3组：（1）对照组36只，大鼠不做特殊处理，常规饲养；（2）假手术组36只，大鼠开腹后旷置15 min，不施行手术，关闭腹腔；（3）PND组36只，大鼠开腹后剥离出盆腔神经，显微剪剪断，缝合切口，关闭腹腔。采用免疫组织化学染色、Western blot检测各组大鼠术后第1、3、7天远端结肠黏膜中TRPV1蛋白表达，反转录实时定量PCR（RTqPCR）检测各组大鼠术后第1、3、7天远端结肠黏膜中TRPV1 mRNA水平。正态分布的计量资料以±s表示。重复测量数据采用重复测量方差分析。同时相点组间比较采用单因素方差分析，两两比较采用独立样本t检验。 结果：（1）免疫组织化学染色检测TRPV1表达情况：对照组大鼠远端结肠黏膜TRPV1平均光密度值术后第1、3、7天分别为0.180±0.016、0.179±0.015、0.183±0.026，3者比较，差异无统计学意义（F=0.088，P〉0.05）。假手术组大鼠远端结肠黏膜TRPV1平均光密度值术后第1、3、 7天分别为0.132±0.017、0.160±0.023、0.173±0.020，3者比较，差异有统计学意义（F=8.699，P〈0.05）。PND组大鼠远端结肠黏膜TRPV1平均光密度值术后第1、3、7天分别为0.057±0.009、0.122±0.016、0.180±0.016，3者比较，差异有统计学意义（F=113.315，P〈0.05）。对照组、假手术组及PND组大鼠远端结肠黏膜TRPV1平均光密度值术后第1、3天比较，差异均有统计学意义（F=108.960，15.218，P〈0.05）。术后第 1天对照组分别与假手术组、PND组比较，差异均有统计学意义（t=5.025，15.979，P〈0.05）；假手术组与PND组比较，差异有统计学意义（t=9.590，P〈0.05）。术后第3天对照组与假手术组比较，差异无统计学意义（t=1.670，P〉0.05）；对照组与PND组比较，差异有统计学意义（t=6.543，P〈0.05）；假手术组与PND组比较，差异有统计学意义（t=3.361，P〈0.05）。3组大鼠远端结肠黏膜TRPV1平均光密度值术后第7天比较，差异无统计学意义（F=0.518，P〉0.05）。（2）Western blot检测TRPV1表达情况：对照组大鼠远端结肠黏膜TRPV1蛋白相对表达量术后第1、3、7天分别为1.02±0.13、1.00±0.15、1.00±0.10，3者比较，差异无统计学意义（F=0.084，P〉0.05）。假手术组大鼠远端结肠黏膜TRPV1蛋白相对表达量术后第1、3、7天分别为0.51±0.13、0.93±0.14、1.01±0.16，3者比较，差异有统计学意义（F=20.930，P〈0.05）。PND组大鼠远端结肠黏膜TRPV1蛋白相对表达量术后第1、3、7天分别为0.30±0.10、0.70±0.10、1.07±0.16，3者比较，差异有统计学意义（F=61.441，P〈0.05）。对照组、假手术组及PND组大鼠远端结肠黏膜TRPV1蛋白相对表达量术后第1、3天比较，差异均有统计学意义（F=58.014，8.841，P〈0.05）。术后第1天对照组分别与假手术组、PND组比较，差异均有统计学意义（t=6.677，11.145，P〈0.05）；假手术组与PND组比较，差异有统计学意义（t=3.287，P〈0.05）。术后第3天对照组与假手术组比较，差异无统计学意义（t=0.798， P〉0.05）；对照组与PND组比较，差异有统计学意义（t=4.127，P〈0.05）；假手术组与PND组比较，差异有统计学意义（t=3.398，P〈0.05）。3组大鼠远端结肠黏膜TRPV1蛋白相对表达量术后第7天比较，差异无统计学意义（F=0.428，P〉0.05）。（3）RTqPCR检测TRPV1表达情况：对照组大鼠远端结肠黏膜TRPV1 mRNA水平术后第1、3、7天分别为1.00±0.15、1.10±0.21、1.09±0.18，3者比较，差异无统计学意义（F=0.489，P〉0.05）。假手术组大鼠远端结肠黏膜TRPV1 mRNA水平术后第1、3、7天分别为0.58 ±0.12、0.99±0.19、1.13±0.23，3者比较，差异有统计学意义（F=13.964，P〈0.05）。PND组大鼠远端结肠黏膜TRPV1 mRNA水平术后第1、3、7天分别为0.31±0.10、0.67±0.12、1.09±0.19，3者比较，差异有统计学意义（F=44.642，P〈0.05）。对照组、假手术组及PND组大鼠远端结肠黏膜TRPV1 mRNA水平术后第1、3天比较，差异均有统计学意义（F=44.653，9.700，P〈0.05）。术后第1天对照组分别与假手术组、PND组比较，差异 均有统计学意义（t=5.233，9.264，P〈0.05）；假手术组与PND组比较，差异有统计学意义（t=4.127，P〈0.05）。术后第3天对照组与假手术组比较，差异无统计学意义（t=0.995，P〉0.05）；对照组与PND组比较，差异有统计学意义（t=4.411，P〈0.05）；假手术组与PND组比较，差异有统计学意义（t=3.505，P〈0.05）。3组大鼠远端结肠黏膜TRPV1 mRNA水平术后第7天比较，差异无统计学意义（F=0.099，P〉0.05）。 结论：PND后，大鼠远端结肠黏膜TRPV1表达显著下调，但随时间推移逐步回升，这与盆腔神经损伤后结肠传输功能先减慢后逐步恢复的趋势一致。

Objective：To explore the effects of pelvic nerves denervation （PND） on the expression of transit potential receptor vanilloid 1 （TRPV1） in distal colonic mucosa of rats. Methods：The experimental study was conducted. One hundred and eight adult male rats were randomly divided into the control group, sham operation group and PND group： （1） 36 rats in the control group remained untreated and were fed regularly; （2） 36 in the sham operation group received open exclusion for 15 minutes, and then sew up the incision; （3） 36 in the PND group received laparotomy with pelvic nerve transection before abdominal closure. The expression of TRPV1 protein in distal colonic mucosa was respectively detected by Western blot at postoperative day 1, 3 and 7. Reverse transcription quantitative realtime polymerase chain reaction （RTqPCR） was used to detect the mRNA level of TRPV1 in the distal colonic mucosa. Measurement data with normal distribution were represented as ±s. Repeated measurement data were analyzed by repeated measures ANOVA. Comparisons at the same time intervals among the 3 groups were analyzed using oneway ANOVA. Pairwise comparison was done by the independentsamples t test. Results：（1） The results of immunohistochemical staining： the average density of TRPV1 in distal colonic mucosa at postoperative day 1, 3 and 7 was respectively 0.180±0.016, 0.179±0.015 and 0.183±0.026 in the control group, with no statistically significant difference （F=0.088, P〉0.05）. The average density of TRPV1 in distal colonic mucosa at postoperative day 1, 3 and 7 was respectively 0.132±0.017, 0.160±0.023 and 0.173±0.020 in the sham operation group, with a statistically significant difference （F=8.699, P〈0.05）. The average density of TRPV1 in distal colonic mucosa at postoperative day 1, 3 and 7 was respectively 0.057±0.009, 0.122±0.016 and 0.180±0.016 in the PND group, with a statistically significant difference （F=113.315, P〈0.05）. There were statistically significant differences in the average density of TRPV1 at postoperative day 1 and 3 among the 3 groups （F=108.960, 15.218, P〈0.05）, while significant differences were respectively detected between the control group and the sham operation group or the PND group at postoperative day 1 （t=5.025, 15.979, P〈0.05）, and a significant difference was also detected between the sham operation group and the PND group （t=9.590, P〈0.05）. There was no statistically significant difference in the average density of TRPV1 between the control group and the sham operation group at postoperative day 3 （t=1.670, P〉0.05）, while significant differences were respectively detected between the control group and the PND group and between the sham operation group and the PND group （t=6.543, 3.361, P〈0.05）. There was no statistically significant difference in the average density of TRPV1 at postoperative day 7 among the 3 groups （F=0.518, P〉0.05）. （2） The results of Western blot： the relative expressions of TRPV1 in distal colonic mucosa at postoperative day 1, 3 and 7 were respectively 1.02±0.13, 1.00±0.15 and 1.00±0.10 in the control group, with no statistically significant difference （F=0.084, P〉0.05）. The relative expressions of TRPV1 in distal colonic mucosa at postoperative day 1, 3 and 7 were respectively 0.51±0.13, 0.93±0.14 and 1.01±0.16 in the sham operation group, with a statistically significant difference （F=20.930, P〈0.05）. The relative expressions of TRPV1 in distal colonic mucosa at postoperative day 1, 3 and 7 were respectively 0.30±0.10, 0.70±0.10 and 1.07±0.16 in the PND group, with a statistically significant difference （F=61.441, P〈0.05）. There were statistically significant differences in the relative expressions of TRPV1 at postoperative day 1 and 3 among the 3 group （F=58.014, 8.841, P〈0.05）, while significant differences were respectively detected between the control group and the sham operation group or the PND group at postoperative day 1（t=6.677,11.145, P〈0.05）, and significant difference was also detected between the sham operation group and the PND group （t=3.287, P〈0.05）. There was no statistically significant difference in the relative expressions of TRPV1 between the control group and the sham operation group at postoperative day 3 （t=0.798, P〉0.05）, while significant differences were respectively detected between the control group and the PND group and between the sham operation group and the PND group （t=4.127, 3.398, P〈0.05）. There was no statistically significant difference in the relative expressions of TRPV1 at postoperative day 7 among the 3 group （F=0.428, P〉0.05）. （3） The results of RTqPCR： the mRNA levels of TRPV1 in distal colonic mucosa at postoperative day 1, 3 and 7 were respectively 1.00±0.15, 1.10±0.21 and 1.09±0.18 in the control group, with no statistically significant difference （F=0.489, P〉0.05）. The mRNA levels of TRPV1 in distal colonic mucosa at postoperative day 1, 3 and 7 were respectively 0.58±0.12, 0.99±0.19 and 1.13±0.23 in the sham operation group, with a statistically significant difference （F=13.964, P〈0.05）. The mRNA levels of TRPV1 in distal colonic mucosa at postoperative day 1, 3 and 7 were respectively 0.31±0.10, 0.67±0.12 and 1.09±0.19 in the PND group, with a statistically significant difference （F=44.642, P〈0.05）. There were statistically significant differences in the mRNA levels of TRPV1 at postoperative day 1 and 3 among the 3 group （F=44.653, 9.700, P〈0.05）, while significant differences were respectively detected between the control group and the sham operation group or the PND group at postoperative day 1 （t=5.233, 9.264, P〈0.05）, and significant difference was also detected between the sham operation group and the PND group （t=4.127, P〈0.05）. There was no significant difference in the mRNA levels of TRPV1 between the control group and the sham operation group at postoperative day 3 （t=0.995, P〉0.05）, while significant differences were respectively detected between the control group and the PND group and between the sham operation group and the PND group （t=4.411, 3.505, P〈0.05）. There was no statistically significant difference in the mRNA levels of TRPV1 at postoperative day 7 among the 3 group （F=0.099, P〉0.05）. Conclusion：The expression of TRPV1 in distal colonic mucosa of rats is significantly downregulated after pelvic nerves denervation, however, it is gradually recovered with passage of time, which is consistent with the trend of gradual recovery of colonic transit function after pelvic nerve injury.