瞬时受体电位阳离子通道8型及瞬时受体电位香草酸亚型1对急性结肠炎小鼠肠道炎症及感觉传导的影响

Effects of transient receptor potential cation channel subfamily V member 1 and transient receptor potential cation channel subfamily M member 8 on intestinal inflammation and sensory conduction inmicewithacutecolitis

目的探讨瞬时受体电位香草酸亚型1(TRPV1)及瞬时受体电位阳离子通道8型(TRPM8)蛋白在急性结肠炎小鼠肠道中的表达、相互作用及对炎症和内脏感觉的影响。方法本实验采用30只雄性C57BL/6小鼠采用随机数字表法分为3组, 分别为对照组、模型组、干预组, 每组10只。模型组与干预组小鼠使用质量浓度为30 g/L的DSS共7 d构建结肠炎模型, 同时干预组每天予以0.3%WS-12溶液灌肠, 连续7 d试剂处理。每天同一时间观察记录小鼠生命活力、毛发及体重变化、粪便性状, 测量粪便隐血情况, 进行疾病活动指数(DAI)评分, 并根据病理切片计算组织病理评分;腹壁反射撤退试验检测各组肠道敏感性;酶联免疫吸附试验(ELISA)检测结肠组织炎性因子:白细胞介素(IL)-1β、IL-6、肿瘤坏死因子-α(TNF-α)、缓激肽(BK)活性表达水平;蛋白质印迹法(Western blot)检测结肠组织紧密连接蛋白(ZO-1、Occludin), TRPV1、TRPM8、降钙素基因相关肽α亚单位(CGRP-Gαq)蛋白表达水平。实时定量反转录聚合酶链反应(RT-qPCR)法检测结肠组织炎性因子IL-1β、IL-6 mRNA及内脏敏感蛋白TRPV1、TRPM8及Gαq蛋白mRNA表达水平;免疫组织化学法检测结肠组织炎性细胞CD4+T淋巴细胞水平。两组间比较采用t检验。结果 (1)WS-12可以改变肠道TRPV1、TRPM8及Gαq蛋白表达量:模型组小鼠肠道TRPV1蛋白表达水平高于对照组(2.58±0.25比1.00±0, t=12.130, P<0.01)、模型组Gαq蛋白表达水平明显高于对照组(2.33±0.51比1.00±0, t=6.984, P<0.01)。模型组TRPM8蛋白表达低于对照组(0.70±0.10比1.00±0, t=8.001, P<0.01), 而经WS-12干预后, 干预组TRPV1蛋白表达水平低于模型组(1.49±0.21比2.58±0.25, t=11.580, P<0.01)、干预组Gαq蛋白蛋白表达水平低于模型组(1.34±0.14比2.33±0.51, t=5.021, P<0.01), 而干预组TRPM8蛋白表达高于模型组(1.60±0.32比0.70±0.10, t=6.914, P<0.01)。(2)WS-12可减轻肠道炎症程度:模型组小鼠结肠长度较对照组变短[(4.01±0.72) cm比(7.47±0.55) cm, t=11.960, P<0.01]、模型组DAI评分高于对照组[(3.70±0.48)分比(0.20±0.42)分, t=17.26, P<0.01], 模型组苏木精-伊红(HE)组织病理评分高于对照组[(7.10±0.74)分比(0.20±0.42)分, t=25.680, P<0.01], 组织间CD4^(+)T淋巴细胞表达水平升高, 浸润明显。而经WS-12干预后, 干预组结肠长度变长[(5.95±0.85) cm比(4.01±0.72) cm, t=6.734, P<0.01], 干预组DAI评分低于模型组[(2.10±0.74)分比(3.70±0.48)分, t=5.737, P<0.01]及干预组HE组织病理评分低于模型组[(4.80±0.79)分比(7.10±0.74)分, t=6.734, P<0.01], CD4^(+)T淋巴细胞表达量降低(P均<0.01)。模型组小鼠肠道IL-1β、IL-6、TNF-α、BK表达水平明显高于对照组[IL-1β:(107.70±7.86) ng/ml比(23.59±2.26) ng/ml, t=30.190;IL-6:(62.49±6.61) pg/ml比(5.26±1.13) pg/ml, t=27.190;TNF-α:(184.40±11.19) pg/ml比(33.45±6.37) pg/ml, t=37.060;BK:(65.69±7.53) pg/ml比(12.84±4.12) pg/ml, t=19.470, P均<0.01], WS-12干预组上述指标低于模型组[IL-1β:(42.84±10.45) ng/ml比(107.70±7.86) ng/ml, t=14.230;IL-6:(14.18±5.91) pg/ml比(62.49±6.61) pg/ml, t=17.190;TNF-α:(92.71±3.39) pg/ml比(184.40±11.19) pg/ml, t=8.569;BK:(16.46±3.96) pg/ml比(65.69±7.53) pg/ml, t=18.31, P均<0.01]。(3)WS-12可改善肠道上皮通透性:模型组小鼠肠道ZO-1、Occludin蛋白表达水平明显低于对照组(0.58±0.18比1.00±0, t=10.180;0.64±0.19比1.00±0, t=6.466, P<0.01)。而经WS-12干预后, 干预组ZO-1、Occludin蛋白表达水平明显高于模型组(0.88±0.11比0.58±0.18, t=6.674;0.82±0.12比0.64±0.19, t=3.314, P均<0.01)。(4)WS-12干预后可改善肠道敏感性:模型组小鼠在不同压力(20、40、60、80 mmHg)直肠扩张下AWR评分均明显高于对照组[(0.8±0.4)分比(0.1±0.3)分, t=4.200;(1.8±0.4)分比(0.5±0.5)分, t=6.091;(2.7±0.5)分比(1.7±0.4)分, t=4.629;(3.8±0.4)分比(2.8±0.6)分, t=4.160, P均<0.01], 予WS-12干预后, 小鼠肠道敏感性明显降低, 其AWR评分虽然高于对照组, 但在不同压力下均较模型组有不同程度的降低[(0.4±0.3)分比(0.8±0.4)分, t=1.897;(1.1±0.3)分比(1.8±0.4)分, t=4.200;(1.9±0.3)分比(2.7±0.5)分, t=4.382;(2.9±0.3)分比(3.8±0.4)分, t=5.400, P均<0.01]。结论 TRPV1和TRPM8之间可能存在平衡机制维持肠道正常功能。WS-12可以通过激活TRPM8通道对小鼠急性结肠炎起一定的治疗作用。

Objective To investigate the expression and interaction of transient receptor potential vanillate subtype 1(TRPV1)and transient receptor potential cationic channel 8(TRPM8)proteins in the intestinal tract of mice with acute colitis,and their effects on inflammation and visceral sensation.Methods A total of 30 male C57BL/6 mice were purchased from Beijing Huafukang Biotechnology,and divided into 3 groups by a random number table method:control group,model group and intervention group,with 10 mice in each group.Mice in model group and intervention group were treated with 30 g/L DSS for 7 days to construct colitis model.Meanwhile,the intervention group was treated with 0.3% WS-12 solution enema every day for 7 consecutive days.At the same time,the vital vitality,hair and body mass changes,fecal characteristics of mice were observed and recorded,the occult blood in feces was measured,and dis-ease activity index(DAl)score was recorded,and the histopathologic score was calculated according to the pathological sections every day.Abdominal wall reflex retreat test was used to detect intestinal sensitivity in each group.The expression levels of colon inflammatory factors including interleukin(IL)-1β,IL-6,tumor necrosis factor(TNF-α)and bradykinin(BK)were detected by enzyme linked immunosorbent assay(ELISA).The expression levels of ZO-1,Occludin,TRPV1,TRPM8,calcitonin gene-related peptideαsubunit(CGRP-Gαq)in colon tissue were detected by Western blotting.The mRNA expression levels of inflammatory factors IL-1β and IL-6 in colon tissues and visceral sensitive proteins TRPV1,TRPM8 and Gaq proteins were detected by real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR).The levels of CD4^(+)T lymphocytes in inflammatory cells were detected by immunohistochemis-try.Comparison between the two groups was performed by t test.Results(1)WS-12 could change the expression levels of intestinal TRPV1,TRPM8 and Gaq proteins:The expression level of intestinal TRPV1 protein in model group was significantly higher than that in control group(2.58±0.25 vs.1.00±0,t=12.130,P<0.01),and the expression level of Gαq protein in model group was significantly higher than that in control group(2.33±0.51 vs.1.00±0,t=6.984,P<0.01).The protein expression of TRPM8 in the model group was lower than that in the control group(0.70±0.10 vs.1.00±0,t=8.001,P<0.01),and the protein expression level of TRPV1 in the intervention group was lower than that in the mod-el group after WS-12 intervention(1.49±0.21 vs.2.58±0.25,t=11.580,P<0.01),the expression level of Gaq protein in the intervention group was lower than that in the model group(1.34±0.14 vs.2.33±0.51,t=5.021,P<0.01),and the expression of TRPM8 protein in the intervention group was higher than that in the model group(1.60±0.32 vs.0.70±0.10,t=6.914,P<0.01).(2)WS-12 could reduce the degree of intestinal inflammation:The colon length of the model group was shorter than that of the control group[(4.01±0.72)cm vs.(7.47±0.55)cm,t=11.960,P<0.01],the DAI score of the model group was higher than that of the control group(3.70±0.48 vs.0.20±0.42,t=17.260,P<0.01).The histopathologic score of the model group was higher than that of the control group(7.10±0.74 vs.0.20±0.42,t=25.680,P<0.01),and the expression level of intertissue CD4^(+)T lymphocytes was increased,and the infiltration was obvious.After WS-12 intervention,colon length was longer[(5.95±0.85)cm vs.(4.01±0.72)cm,t=6.734,P<0.01],DAI score was lower(2.10±0.74 vs.3.70±0.48,t=5.737,P<0.01)and the histopathologic score of hematoxylin and eosin(HE)was lower(4.80±0.79 vs.7.10±0.74,t=6.734,P<0.01),and the expression of CD4^(+)T lymphocytes was lower in the intervention group than in the model group(all P<0.01).The expression levels of IL-1β,IL-6,TNF-αand BK in intestinal tract of model group were significantly higher than those of control group[IL-1β:(107.70±7.86)vs.(23.59±2.26)ng/ml,t=30.190;IL-6:(62.49±6.61)vs.(5.26±1.13)pg/ml,t=27.190;TNF-α:(184.40±11.19)vs.(33.45±6.37)pg/ml,t=37.060;BK:(65.69±7.53)vs.(12.84±4.12)pg/ml,t=19.470,P<0.01].The above indexes in WS-12 intervention group were lower than those in model group[IL-1β:(42.84±10.45)vs.(107.70±7.86)ng/ml,t=14.230;IL-6:(14.18±5.91)vs.(62.49±6.61)pg/ml,t=17.190;TNF-α:(92.71±3.39)vs.(184.40±11.19)pg/ml,t=8.569;BK:(16.46±3.96)vs.(65.69±7.53)pg/ml,t=18.310,P<0.01].(3)WS-12 could improve intestinal epithelial permeability:the expression level of ZO-1 and Occludin in the intestinal tract of model group was significantly lower than that of control group(0.58±0.18 vs.1.00±0,t=10.180;0.64±0.19 vs.1.00±0,t=6.466,P<0.01).After WS-12 intervention,the expression of ZO-1 and Occludin in the intervention group was significantly higher than that in the model group(0.88±0.11 vs.0.58±0.18,t=6.674;0.82±0.12 vs.0.64±0.19,t=3.314,P<0.01).(4)The intestinal sensitivity could be improved after the intervention of WS-12:AWR scores in the model group were significantly higher than those in the control group under different pressures(20,40,60,80 mmHg)rectal dilation(0.8±0.4 vs.0.1±0.3,t=4.200;1.8±0.4 vs.0.5±0.5,t=6.091;2.7±0.5 vs.1.7±0.4,t=4.629;3.8±0.4 vs.2.8±0.6,t=4.160,P<0.01),the intestinal sensitivity of mice was significantly decreased after WS-12 intervention,and AWR score was higher than that of the control group,but it was lower than that of the model group under different pressures(0.4±0.3 vs.0.8±0.4,t=1.897;1.1±0.3 vs.1.8±0.4,t=4.200;1.9±0.3 vs.2.7±0.5,t=4.382;2.9±0.3 vs.3.8±0.4;t=5.400,P<0.01).Conclusion There may be a balancing mechanism between TRPV1 and TRPM8 to maintain normal intestinal function.WS-12 can be used to treat acute colitis in mice by activating TRPM8 channel.