硫化氢对健康雄性大鼠胃运动的影响及其机制研究

发布时间：2018-05-19 03:14

本文选题：硫化氢 + 胃　；参考：《山东大学》2009年硕士论文

【摘要】： 目的: 一氧化氮(NO)和一氧化碳(CO)属于气体递质(gaseous transmitter),参与多种生理功能的调节。硫化氢(Hydrogen Sulfide,H_2S)是一种无色具有臭鸡蛋气味的有毒气体。有研究发现在人和大鼠的脑、血清、肾脏和肝脏中有相对高浓度的H_2S,这表明H_2S可能在这些组织中发挥生理作用,H_2S可能是除NO和CO外的第三种气体递质。机体可以通过胱硫醚β-合成酶(cystathionineβ-synthase,CBS)和胱硫醚γ-裂解酶(cystathionineγ-lyase,CSE)催化L-半胱氨酸(L-Cysteine)产生内源性的H_2S。而硫氢化钠(NaHS)作为H_2S的外源性供体已经广泛应用于H_2S的研究当中,因为在溶液中它能够分解为Na~+和HS~-,HS~-和H~+结合产生H_2S。有研究报道H_2S能够引起肠道平滑肌的舒张,但是机制尚不确定。RudolfSchicho等发现在豚鼠和人回肠的肠神经系统内存在CSE,H_2S可能会通过肠神经系统来调节胃肠道运动,但内源性H_2S是否调节胃的运动尚未见报道。本实验目的是研究H_2S对胃运动的影响及其可能机制。方法: 实验选用健康雄性Wistar大鼠,实验前禁食12小时,自由饮水。手术操作和胃内压记录 2%戊巴比妥钠(56mg/kg)麻醉大鼠,依次行气管插管、颈静脉插管、股动脉插管。将一塑料气囊从食管插入胃腔后固定。气囊内注入生理盐水使基础胃内压维持于25mmHg。大鼠自发胃收缩活动稳定1个小时后,正式开始实验。实验分2组:1)腹腔注射生理盐水作为对照组;2)腹腔注射L-Cysteine(12mg/kg)或者NaHS组(5.6mg/kg)。离体肌条制备和张力记录快速牺牲大鼠,取出胃放置于4℃Krebs液中。沿胃小弯剪开胃腔,冲洗胃腔,再沿胃纤维走向剪取胃体纵行肌肌条(4mm×10mm)。将肌条分别放置在含有5ml krebs液(37℃)的浴槽中孵育,并每隔15分钟更换一次浴槽中的krebs液。待肌条自发收缩稳定后开始实验。统计学处理在整体实验中,胃的运动强度用胃内压来表示,注射药物前3分钟的平均胃内压为基础值(baseline),注射药物后不同时间的平均胃内压为效应值;在离体肌条实验中,肌条运动强度的大小用张力来衡量,加药前3分钟的平均张力胃基础值(baseline),加药后不同时间的平均张力为效应值;效应值与基础值的比值为统计指标R。所有数据均采用均数±标准误表示,采用单因素方差分析和f检验进行统计学处理,以P0.05为显著性差异界值。结果: 1.腹腔注射NaHS(5.6 mg/kg)和L-Cysteine(12 mg/kg)后,胃内压都迅速出现降低,然后逐渐恢复正常。 2.低浓度的NaHS(10~(-4)M)不影响胃体纵行肌肌条的收缩活动。当NaHS的浓度升高至5×10~(-4)M—10~(-3)M时胃体纵行肌肌条收缩活动受到明显抑制,其中5×10~(-4)M时抑制作用最强,且在加入NaHS 10min时作用最强,然后逐渐恢复。 3. ATP依赖性的K~+通道(K_(ATP))阻断剂格列苯脲(glibenclamide)(10~(-7)M)能够明显逆转NaHS(10~(-3)M)对胃体纵行肌肌条收缩活动的抑制作用。但是,一氧化氮合酶非特异性阻断剂N-硝基-L-精氨酸(N-nitro-L-arginine methylester,L-NAME)(10~(-4)M)和电压依赖性的Na~+通道阻断剂河豚毒素(tetrodotoxin,TTX)(10~(-6)M)对NaHS(10~(-3)M)的这种作用没有影响。 4.低剂量的L-Cysteine(5×10~(-4)M)对胃体纵行肌肌条的收缩活动没有影响,当剂量增加至(10~(-3) M—5×10~(-3) M)时,胃体纵行肌肌条的收缩活动受到明显的剂量依赖性的抑制,该作用在加入L-Cysteine后1-2min时抑制作用最强,10min内恢复正常。 5.河豚毒素(tetrodotoxin,TTX)(10~(-6) M)和M受体阻断剂阿托品(atropine)(10~(-6) M)都能部分阻断L-Cysteine(10~(-3)M)对胃体纵行肌收缩活动的抑制作用。但是,N-硝基-L-精氨酸(N-nitro-L-arginine methyl ester,L-NAME)(10~(-4)M)和格列苯脲(glibenclamide)(10~(-7)M)对L-Cysteine(10~(-3)M)的这种作用没有影响。 6. CSE特异性抑制剂炔丙基甘氨酸(propargylglycine,PAG)(10~(-6)M-10~(-3)M)和CBS抑制剂氨基-羟乙酸(amino-oxyacetic acid,AOAA)(10~(-6)M-10~(-5)M)增强胃体纵行肌肌条收缩活动,且这种增强作用持续超过30min。 7. PAG(10~(-6)M)和AOAA(10~(-6)M)能够部分阻断L-Cysteine(10~(-3)M)对胃体纵行肌肌条收缩活动的抑制,但是不影响NaHS(10~(-3)M)的作用。结论: 1.外源性和内源性H_2S均抑制胃的运动。 2.外源性H_2S对胃运动的抑制主要是通过诱导胃平滑肌细胞膜上的K_(ATP)开放,而内源性的H_2S的作用主要是通过肠神经系统内的胆碱能神经实现的。 3.内源性的H_2S对胃运动起着紧张性抑制作用。
[Abstract]:Objective:
Nitric oxide (NO) and carbon monoxide (CO) belong to the gas transmitter (gaseous transmitter) and are involved in a variety of physiological functions. Hydrogen sulfide (Hydrogen Sulfide, H_2S) is a colorless toxic gas with odorless egg odor. Studies have found that there is a relatively high concentration of H_2S in the brain, blood, kidney and liver of the human and rat, which indicates that H_2S can be found H_2S can be the third gas transmitter except NO and CO, which can play a physiological role in these tissues.
The organism can catalyze L- cysteine (L-Cysteine) to produce endogenous H_2S. with cysteine beta synthetase (cystathionine beta -synthase, CBS) and cystthioether gamma lyase (cystathionine gamma -lyase, CSE) as a H_2S exogenous donor, which has been widely used in H_2S studies because it can be decomposed in the solution. The combination of Na~+, HS~-, HS~- and H~+ combined with H_2S. has reported that H_2S can cause intestinal smooth muscle relaxation, but the mechanism is not yet determined that.RudolfSchicho and so on are found in the gut nervous system in guinea pigs and human ileum in CSE, H_2S may regulate gastrointestinal motility through the intestinal nervous system, but whether endogenous H_2S regulates gastric motility No report has been reported. The purpose of this experiment is to study the effect of H_2S on gastric motility and its possible mechanism.
Method:
The healthy male Wistar rats were randomly fasted for 12 hours before the experiment.
Surgical operation and intragastric pressure recording
2% pentobarbital sodium (56mg/kg) rats were anesthetized with tracheal intubation, jugular intubation, and femoral artery intubation. A plastic balloon was inserted into the stomach cavity from the esophagus. The air bag was injected with saline to maintain the underlying gastric pressure in the spontaneous gastric contractile activity of 25mmHg. rats. The experiment was formally started. The experiment was divided into 2 groups: 1) intraperitoneal injection. Normal saline was used as control group; 2) intraperitoneal injection of L-Cysteine (12mg/kg) or NaHS group (5.6mg/kg).
Preparation of isolated muscle strips and record of tension
The rats were sacrificed quickly and placed in the Krebs solution at 4 degrees centigrade. The gastric cavity was cut along the gastric small bend, the stomach cavity was washed, and then the muscle strips of the stomach were cut along the gastric fibers (4mm x 10mm). The muscle strips were incubated in the bath containing 5ml Krebs liquid (37 degrees C), and the Krebs solution in the bath was changed every 15 minutes. After the muscle strips were spontaneously contracted, the muscle strips were stable and stable. Start the experiment.
Statistical treatment
In the whole experiment, the exercise intensity of the stomach was expressed by intragastric pressure. The average intragastric pressure of 3 minutes before injection was the base value (baseline). The average intragastric pressure was the effect value at different time after injection. In the experiment of isolated muscle strips, the intensity of muscle strips was measured by tension, and the average tension of gastric base was 3 minutes before adding medicine (base Line) the average tension at different times after adding medicine is the effect value; the ratio of the effect value to the base value is the statistical index R..
All data were expressed by means of mean + standard error, and one-way ANOVA and F test were used for statistical analysis. P0.05 was a significant difference value.
Result:
1. intraperitoneal injection of NaHS (5.6 mg/kg) and L-Cysteine (12 mg/kg) resulted in a rapid decrease in intragastric pressure and gradually returned to normal.
2. the low concentration of NaHS (10~ (-4) M) did not affect the contractile activity of the longitudinal muscle strips of the stomach. When the concentration of NaHS increased to 5 * 10~ (-4) M 10~ (-3) M, the contractile activity of the longitudinal muscle strips of the stomach was obviously inhibited, and the inhibitory effect was strongest when 5 * 10~ (10~) was the strongest, and then gradually recovered.
3. ATP dependent K~+ channel (K_ (ATP)) blocker glibenclamide (glibenclamide) (10~ (-7) M) can obviously reverse the inhibition of NaHS (10~ (-3) M) on the contractile activity of the longitudinal muscle strips of the stomach. However, the nitric oxide synthase nonspecific blocking agent, nitroedible arginine, and voltage dependence The Na~+ channel blocker, tetrodotoxin (TTX) (10~ (-6) M), has no effect on NaHS (10~ (-3) M).
4. the low dose of L-Cysteine (5 x 10~ (-4) M) had no effect on the contractile activity of the longitudinal muscle strips of the gastric body. When the dose increased to (10~ (-3) M - 5 * 10~ (-3) M), the contraction activity of the muscle strips of the gastric body was obviously dose-dependent, and the inhibitory effect was strongest in L-Cysteine 1-2min.
5. tetrodotoxin (TTX) (10~ (-6) M) and M receptor blocking agent atropine (atropine) (10~ (-6) M) can partially block the inhibitory effect of L-Cysteine (10~) on the contractile activity of the longitudinal muscle of the stomach. The effect of ysteine (10~ (-3) M) is not affected.
The 6. CSE specific inhibitor propargylglycine (PAG) (10~ (-6) M-10~ (-3) M) and the CBS inhibitor amino hydroxyacetic acid (AMINO-OXYACETIC acid, AOAA) enhanced the contractile activity of the longitudinal muscle strips of the stomach body, and this enhancement continued to exceed that of the muscle.
7. PAG (10~ (-6) M) and AOAA (10~ (-6) M) can partially block the inhibitory activity of L-Cysteine (10~ (-3) M) on the contractile activity of the longitudinal muscle strips of the stomach body, but it does not affect the effect.
Conclusion:
1. both exogenous and endogenous H_2S inhibit the motion of the stomach.
2. the inhibitory effect of exogenous H_2S on gastric motility is mainly through the induction of K_ (ATP) on the membrane of the smooth muscle cells of the stomach, and the role of endogenous H_2S is realized mainly through the cholinergic nerve in the intestinal nervous system.
3. endogenous H_2S plays a tonic inhibitory role on gastric motility.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R333

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