Transient receptor potential vanilloid 4-dependent calcium influx and ATP release in mouse and rat gastric epithelia

AIM: To explore the expression of transient receptor potential vanilloid 4(TRPV4) and its physiological meaning in mouse and rat gastric epithelia. METHODS: RT-PCR and immunochemistry were used to detect TRPV4 m RNA and protein expression in mouse stomach and a rat normal gastric epithelial cell line(RGE1-01), while Ca2+-imaging and electrophysiology were used to evaluate TRPV4 channel activity. ATP release was measured by a luciferin-luciferase assay. Gastric emptying was also compared between WT and TRPV4 knockout mice. RESULTS: TRPV4 m RNA and protein were detected in mouse tissues and RGE1-01 cells. A TRPV4-specific agonist(GSK1016790A) increased intracellular Ca2+ concentrations and/or evoked TRPV4-like current activities in WT mouse gastric epithelial cells andRGE1-01 cells, but not TRPV4 KO cells. GSK1016790 A or mechanical stimuli induced ATP release from RGE1-01 cells while TRPV4 knockout mice displayed delayed gastric emptying in vivo. CONCLUSION: TRPV4 is expressed in mouse and rat gastric epithelium and contributes to ATP release and gastric emptying.

Increased gene and protein expressions of the transient receptor potential vanilloid receptor 4 following sustained pure mechanical pressure on rat dorsal root ganglion neurons

Dorsal root ganglion （DRG） neurons from newborn Wistar rats cultured in vitro were pressurized with 20, 40, 80 or 120 mm Hg compressive Ioadings （1 mm Hg = 0.133 kPa） for 12, 24, 48 or 72 hours, respectively. The 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide test showed that pressures less than 80 mm Hg had no obvious impact on the activity of DRG neurons. The protein expression levels of transient receptor potential vanilloid receptor 4 （TRPV4）, transient receptor potential vanilloid receptor 1, transient receptor potential channel of melastatin type 8, and transient receptor potential subtype ankyrin 1 were assessed by western blot analysis. The mRNA expression of TRPV4 was assessed by real-time PCR. The results showed that sustained mechanical compression up-regulated TRPV4 mRNA and protein expression in the rat DRG neurons, in a time-dependent fashion. Similar changes were not found in the protein expression of transient receptor potential vanilloid receptor 1, transient receptor potential channel of melastatin type 8, and transient receptor potential subtype ankyrin 1. Images of cells using a laser scanning confocal microscope showed that the sustained mechanical pressure increased the number of responsive DRG neurons and was synergistic on the enhanced Ca^2＋ responses to the TRPV4 phorbol ester agonist 4a-phorbo112, 13-didecanoate and hypotonic solutions. These findings demonstrate that sustained mechanical compressive loading in vitro increases the expression of TRPV4 mRNA and protein in DRG neurons and sensitizes TRPV4 Ca^2＋ signals. Mechanical compression does not impact other ion channels in the transient receptor potential family.

Distribution of transient receptor potential vanilloid-1 channels in gastrointestinal tract of patients with morbid obesity

BACKGROUND Transient receptor potential vanilloid-1(TRPV1),a nonselective cation channel,is activated by capsaicin,a pungent ingredient of hot pepper.Previous studies have suggested a link between obesity and capsaicin-associated pathways,and activation of TRPV1 may provide an alternative approach for obesity treatment.However,data on the TRPV1 distribution in human gastric mucosa are limited,and the degree of TRPV1 distribution in the gastric and duodenal mucosal cells of obese people in comparison with normal-weight individuals is unknown.AIM To clarify gastric and duodenal mucosal expression of TRPV1 in humans and compare TRPV1 expression in obese and healthy individuals.METHODS Forty-six patients with a body mass index(BMI)of>40 kg/m^(2) and 20 patients with a BMI between 18-25 kg/m^(2) were included.Simultaneous biopsies from the fundus,antrum,and duodenum tissues were obtained from subjects between the ages of 18 and 65 who underwent esophagogastroduodenoscopy.Age,sex,history of alcohol and cigarette consumption,and past medical history regarding chronic diseases and medications were accessed from patient charts and were analyzed accordingly.Evaluation with anti-TRPV1 antibody was performed separately according to cell types in the fundus,antrum,and duodenum tissues using an immunoreactivity score.Data were analyzed using SPSS 17.0.RESULTS TRPV1 expression was higher in the stomach than in the duodenum and was predominantly found in parietal and chief cells of the fundus and mucous and foveolar cells of the antrum.Unlike foveolar cells in the antrum,TRPV1 was relatively low in foveolar cells in the fundus(4.92±0.49 vs 0.48±0.16,P<0.01,Mann-Whitney U test).Additionally,the mucous cells in the duodenum also had low levels of TRPV1 compared to mucous cells in the antrum(1.33±0.31 vs 2.95±0.46,P<0.01,Mann-Whitney U test).TRPV1 expression levels of different cell types in the fundus,antrum,and duodenum tissues of the morbidly obese group were similar to those of the control group.Staining with TRPV1 in fundus chief cells and antrum and duodenum mucous cells was higher in patients aged≥45 years than in patients<45 years(3.03±0.42,4.37±0.76,2.28±0.55 vs 1.9±0.46,1.58±0.44,0.37±0.18,P=0.03,P<0.01,P<0.01,respectively,Mann-Whitney U test).The mean staining levels of TRPV1 in duodenal mucous cells in patients with diabetes and hypertension were higher than those in patients without diabetes and hypertension(diabetes:2.11±0.67 vs 1.02±0.34,P=0.04;hypertension:2.42±0.75 vs 1.02±0.33,P<0.01 Mann-Whitney U test).CONCLUSION The expression of TRPV1 is unchanged in the gastroduodenal mucosa of morbidly obese patients demonstrating that drugs targeting TRPV1 may be effective in these patients.

Blockade of transient receptor potential cation channel subfamily V member 1 promotes regeneration after sciatic nerve injury

The transient receptor potential cation channel subfamily V member 1（TRPV1） provides the sensation of pain（nociception）. However, it remains unknown whether TRPV1 is activated after peripheral nerve injury, or whether activation of TRPV1 affects neural regeneration. In the present study, we established rat models of unilateral sciatic nerve crush injury, with or without pretreatment with AMG517（300 mg/kg）, a TRPV1 antagonist, injected subcutaneously into the ipsilateral paw 60 minutes before injury. At 1 and 2 weeks after injury, we performed immunofluorescence staining of the sciatic nerve at the center of injury, at 0.3 cm proximal and distal to the injury site, and in the dorsal root ganglia. Our results showed that Wallerian degeneration occurred distal to the injury site, and neurite outgrowth and Schwann cell regeneration occurred proximal to the injury. The number of regenerating myelinated and unmyelinated nerve clusters was greater in the AMG517-pretreated rats than in the vehicle-treated group, most notably 2 weeks after injury. TRPV1 expression in the injured sciatic nerve and ipsilateral dorsal root ganglia was markedly greater than on the contralateral side. Pretreatment with AMG517 blocked this effect. These data indicate that TRPV1 is activated or overexpressed after sciatic nerve crush injury, and that blockade of TRPV1 may accelerate regeneration of the injured sciatic nerve.

Serotonin receptor 2B induces visceral hyperalgesia in rat model and patients with diarrhea-predominant irritable bowel syndrome

BACKGROUND Serotonin receptor 2B(5-HT2B receptor)plays a critical role in many chronic pain conditions.The possible involvement of the 5-HT2B receptor in the altered gut sensation of irritable bowel syndrome with diarrhea(IBS-D)was investigated in the present study.AIM To investigate the possible involvement of 5-HT2B receptor in the altered gut sensation in rat model and patients with IBS-D.METHODS Rectosigmoid biopsies were collected from 18 patients with IBS-D and 10 patients with irritable bowel syndrome with constipation who fulfilled the Rome IV criteria and 15 healthy controls.The expression level of the 5-HT2B receptor in colon tissue was measured using an enzyme-linked immunosorbent assay and correlated with abdominal pain scores.The IBS-D rat model was induced by intracolonic instillation of acetic acid and wrap restraint.Alterations in visceral sensitivity and 5-HT2B receptor and transient receptor potential vanilloid type 1(TRPV1)expression were examined following 5-HT2B receptor antagonist adminis-tration.Changes in visceral sensitivity after administration of the TRPV1 antago-INTRODUCTION Irritable bowel syndrome(IBS)is a chronic functional bowel disorder characterized by recurrent abdominal pain with altered bowel habits that affects approximately 15%of the population worldwide[1].IBS significantly impacts the quality of life of patients.Although the pathogenesis of IBS is not completely understood,the role of abnormal visceral sensitivity in IBS has recently emerged[2,3].5-Hydroxytryptamine(5-HT)is known to play a key role in the physiological states of the gastrointestinal tract.Plasma 5-HT levels in IBS with diarrhea(IBS-D)patients were greater than those in healthy controls[4],suggesting a possible role of 5-HT in the pathogenesis of IBS-D.The serotonin receptor 2(5-HT2 receptor)family comprises three subtypes:5-HT2A,5-HT2B,and 5-HT2c.All 5-HT2 receptors exhibit 46%-50%overall sequence identity,and all of these receptors preferentially bind to Gq/11 to increase inositol phosphates and intracellular calcium mobilization[5].5-HT2B receptors are widely expressed throughout the gut,and experimental evidence suggests that the primary function of 5-HT2B receptors is to mediate contractile responses to 5-HT through its action on smooth muscle[6].The 5-HT2B receptor is localized to both neurons of the myenteric nerve plexus and smooth muscle in the human colon.The 5-HT2B receptor mediates 5-HT-evoked contraction of longitudinal smooth muscle[6].These findings suggest that the 5-HT2B receptor could play an important role in modulating colonic motility,which could affect sensory signaling in the gut.Other laboratories have shown that the 5-HT2B receptor participates in the development of mechanical and formalin-induced hyperalgesia[7,8].A 5-HT2B receptor antagonist reduced 2,4,6-trinitrobenzene sulfonic acid(TNBS)and stress-induced visceral hyperalgesia in rats[9,10].However,the role of the 5-HT2B receptor in IBS-D patients and in acetic acid-and wrap restraint-induced IBS-D rat models was not investigated.

Transient receptor potential vanilloid 1 involved in the analgesic effects of total flavonoids extracted from Longxuejie(Resina Dracaenae Cochinchinensis)

OBJECTIVE: To evaluate the analgesic effects of total flavonoids of Longxuejie(Resina Dracaenae Cochinchinensis)(TFDB) and explore the possible analgesic mechanism associated with transient receptor potential vanilloid 1(TRPV1).METHODS: Whole-cell patch clamp technique was used to observe the effects of TFDB on capsaicin-induced TRPV1 currents. Rat experiments in vivo were used to observe the analgesic effects of TFDB. Western blot and immunofluorescence experiments were used to test the change of TRPV1 expression in DRG neurons induced by TFDB.RESULTS: Results showed that TFDB inhibited capsaicin-induced TRPV1 receptor currents in acutely isolated dorsal root ganglion(DRG) neurons of rats and the half inhibitory concentration was(16.7 ± 1.6) mg/L.TFDB(2-20 mg/kg) showed analgesic activity in the phase Ⅱ of formalin test and(0.02-2 mg per paw)reduced capsaicin-induced licking times of rats. TFDB(20 mg/kg) was fully efficacious on complete Freund's adjuvant(CFA)-induced inflammatory thermal hyperalgesia and capsaicin could weaken the analgesic effects. The level of TRPV1 expressions of DRG neurons was also decreased in TFDB-treated CFA-inflammatory pain rats.CONCLUSION: All these results indicated that the analgesic effect of TFDB may contribute to their modulations on both function and expression of TRPV1 channels in DRG neurons.

Role of transient receptor potential vanilloid subetype 1 in the increase of thermal pain threshold by moxibustion

OBTECTIVE:To explore the role of transient receptor potential vaniiloid subetype 1(TRPV1) in the increase of the thermal pain threshold by moxibustion.METHODS:Forty Kunming mice(20 ± 2) g were randomized into control group,capsaicin group,capsazepine group,moxibustion group and moxibustion + capsazepine(MC) group with 8 mice in each,and 16 C57BL/6 wild-type mice(18 ± 2) g were randomized into wild-type(WT) control group and WT moxibustion group with 8 mice in each,and 14 TRPV1 knockout mice(18 ± 2) g were randomized into knockout(KO) control group and KO moxibustion group with 7 in each.Each mouse in the capsaicin group was subcutaneously injected with the amount of 0.1 mL/10 g into L5 and L6 spinal cords;each mouse in the capsazepine group was intraperitoneally injected with the amount of0.1 mL/10 g.Similarly,each mouse in the moxibustion group was given a suspended moxibustion with specially-made moxa-stick for 20 min on L5 and L6 spinal cords.Each mouse in MC group was intraperitoneally injected with the amount of 0.1 mL/10 g first,then after 15 min was given a suspended moxibustion for 20 min on L5 and L6 spinal cords.Each mouse in WT moxibustion group and KO moxibustion group was given a suspended moxibustion with specially-made moxa-stick for 20 min on L5 and L6 spinal cords.The control group,WT control group and KO control group were of no treatment in any way.After all treatments were completed,the digital-display measurement instrument for thermal pain was used to measure the threshold of thermal pain in each group respectively.RESULTS:Compared with the control group,the thresholds of thermal pain in the moxibustion group and MC group were significantly increased(P <0.01);no significant changes in the thresholds in the capsaicin group and the capsazepine group(P > 0.05);compared with moxibustion group,he threshold of thermal in MC group was obviously decreased(P < 0.01).Compared with WT control group,the threshold of thermal pain in WT moxibustion group was significantly increased(P <0.01);compared with KO control group,no changes in the threshold in KO moxibustion group(P > 0.05).CONCLUSION:TRPV1 participated in the process of increasing the threshold of thermal pain by stimulating L5 and L6 of mice spinal cord with burning mosa-stick.

Overexpression of TRPV1 activates autophagy in human lens epithelial cells under hyperosmotic stress through Ca^(2+)-dependent AMPK/mTOR pathway

●AIM:To explore whether autophagy functions as a cellular adaptation mechanism in lens epithelial cells(LECs)under hyperosmotic stress.●METHODS:LECs were treated with hyperosmotic stress at the concentration of 270,300,400,500,or 600 mOsm for 6,12,18,24h in vitro.Polymerase chain reaction(PCR)was employed for the mRNA expression of autophagyrelated genes,while Western blotting detected the targeted protein expression.The transfection of stub-RFP-sens-GFPLC3 autophagy-related double fluorescence lentivirus was conducted to detect the level of autophagy flux.Scanning electron microscopy was used to detect the existence of autolysosome.Short interfering RNA of autophagy-related gene(ATG)7,transient receptor potential vanilloid(TRPV)1 overexpression plasmid,related agonists and inhibitors were employed to their influence on autophagy related pathway.Flow cytometry was employed to test the apoptosis and intracellular Ca^(2+)level.Mitochondrial membrane potential was measured by JC-1 staining.The cell counting kit-8 assay was used to calculate the cellular viability.The wound healing assay was used to evaluate the wound closure rate.GraphPad 6.0 software was utilized to evaluate the data.●RESULTS:The hyperosmotic stress activated autophagy in a pressure-and time-dependent manner in LECs.Beclin 1 protein expression and conversion of LC3B II to LC3B I increased,whereas sequestosome-1(SQSTM1)protein expression decreased.Transient Ca^(2+)influx was stimulated caused by hyperosmotic stress,levels of mammalian target of rapamycin(mTOR)phosphorylation decreased,and the level of AMP-activated protein kinase(AMPK)phosphorylation increased in the early stage.Based on this evidence,autophagy activation through the Ca^(2+)-dependent AMPK/mTOR pathway might represent an adaptation process in LECs under hyperosmotic stress.Hyperosmotic stress decreased cellular viability and accelerated apoptosis in LECs and cellular migration decreased.Inhibition of autophagy by ATG7 knockdown had similar results.TRPV1 overexpression increased autophagy and might be crucial in the occurrence of autophagy promoted by hyperosmotic stress.●CONCLUSION:A combination of hyperosmotic stress and autophagy inhibition may be a promising approach to decrease the number of LECs in the capsular bag and pave the way for improving prevention of posterior capsular opacification and capsular fibrosis.

The relationship between the expression of transient receptor potential vanilloid 1 and the airway remodeling in elderly patients with chronic obstructive pulmonary disease

Objective To investigate the relationship between the expression of trannsient receptor potential vanilloid(TRPV1)and the severity of airway remodeling in elderly patients with chronic obstructive pulmonary disease(COPD).Methods According to airflow obstruction severity,totally 100 cases of elderly patients with

瞬时感受器电位通道vanilloid 4在机械牵张人脑微血管内皮细胞中对内皮型一氧化氮合酶的作用

目的:筛选人脑微血管内皮细胞(human brain microvascular endothelial cells,HBMEC)中瞬时感受器电位(transient receptor potential,TRP)通道亚型,检测该通道亚型在机械牵张刺激中对内皮型一氧化氮合酶(endothelial nitric oxide synthase,e NOS)蛋白表达的影响及探讨相关机制。方法:荧光定量RT-PCR(q RT-PCR)、Western印迹及细胞免疫荧光筛选HBMEC上TRP通道亚型。根据筛选的TRP通道亚型将细胞分组,应用多功能小型生物撞击机给予机械牵张刺激;流式细胞仪检测细胞牵张前后胞内Ca2+荧光强度变化;Western印迹检测e NOS蛋白表达情况。结果:q RT-PCR结果表明:TRPV4及TRPC1的m RNA相对表达量较多;细胞免疫荧光及Western印迹显示TRPV4通道蛋白表达;流式细胞仪检测结果显示,特异性TRPV4通道抑制组较单纯牵张组Ca2+相对浓度低(P<0.001),较未牵张组(P=0.072)、非特异性TRP通道抑制组(P=0.308)无明显差异。Western印迹检测结果显示:TRPV4通道抑制组和内皮型一氧化氮合酶抑制组e NOS蛋白表达水平较单纯牵张组明显降低(P<0.05),较未牵张组无明显差异(P>0.05)。结论:TRPV4在HBMEC上有表达,机械牵张将其激活后,可以引起e NOS蛋白表达增多,其机制可能与胞外Ca2+的内流有关。

Nerve growth factor in muscle afferent neurons of peripheral artery disease and autonomic function

In peripheral artery disease patients,the blood supply directed to the lower limbs is reduced.This results in severe limb ischemia and thereby enhances pain sensitivity in lower limbs.The painful perception is induced and exaggerate during walking,and is relieved by rest.This symptom is termed by intermittent claudication.The limb ischemia also amplifies autonomic responses during exercise.In the process of pain and autonomic responses originating exercising muscle,a number of receptors in afferent nerves sense ischemic changes and send signals to the central nervous system leading to autonomic responses.This review integrates recent study results in terms of perspectives including how nerve growth factor affects muscle sensory nerve receptors in peripheral artery disease and thereby alters responses of sympathetic nerve activity and blood pressure to active muscle.For the sensory nerve receptors,we emphasize the role played by transient receptor potential vanilloid type 1,purinergic P2X purinoceptor 3 and acid sensing ion channel subtype 3 in amplified sympathetic nerve activity responses in peripheral artery disease.

Capsaicin-sensitive afferentation represents an indifferent defensive pathway from eradication in patients with H.pylori gastritis

AIM:To study the role of capsaicin-sensitive afferent nerves in Helicobacter pylori (H.pylori) positive chronic gastritis before and after eradication.METHODS:Gastric biopsy samples were obtained from corpus and antrum mucosa of 20 healthy human subjects and 18 patients with H.pylori positive chronic gastritis (n=18) before and after eradication.Tradi-tional gastric mucosal histology (and Warthin-Starry silver impregnation) and special histochemical examina-tions were carried out.Immunohistochemistry for cap-saicin receptor (TRVP1),calcitonin gene-related peptide (CGRP) and substance P (SP) were carried out by the labeled polymer immunohistological method (Lab VisionCo.,USA) using polyclonal rabbit and rat monoclonal antibodies (Abcam Ltd.,UK).RESULTS:Eradication treatment was successful in 16 patients (89%).Seven patients (7/18,39%) re-mained with moderate complaints,meanwhile 11 pa-tients (11/28,61%) had no complaints.At histological evaluation,normal gastric mucosa was detected in 4 patients after eradication treatment (4/18,22%),and moderate chronic gastritis could be seen in 14 (14/18,78%) patients.Positive immuno-staining for capsaicin receptor was seen in 35% (7/20) of controls,89% (16/18,P < 0.001) in patients before and 72% (13/18,P < 0.03) after eradication.CGRP was positive in 40% (8/20) of controls,and in 100% (18/18,P < 0.001) of patients before and in 100% (18/18,P < 0.001) after eradication.The immune-staining of gastric mucosa for substance-P was positive in 25% (5/20) of healthy con-trols,and in 5.5% (3/18,P > 0.05) of patients before and in 0% of patients (0/18,P > 0.05) after H.pylori eradication.CONCLUSION:Distibution of TRVP1 and CGRP is altered during the development of H.pylori positive chronic gastritis.The immune-staining for TRVP1,CGRP and SP rwemained unchanged before and after H.py-lori eradication treatment.The capsaicin-sensitive affer-entation is an independent from the eradication treat-ment.The 6 wk time period might not be enough time for the restituion of chronic H.pylori positive chronic gastritis.The H.pylori infection might not represent the main pathological factor in the development of chronic

TRPV4在眼病理生理功能中的研究进展

瞬时受体电位香草醛受体4(TRPV4)是一种非选择性阳离子通道,负责感知细胞肿胀、温度、机械牵张、剪切应力和渗透压的变化,通过调节跨膜钙信号进而影响基因表达、细胞形态和细胞骨架等构建。TRPV4在全身广泛表达。在眼内,TRPV4在角膜、晶状体、睫状体、小梁网和视网膜等组织均有功能性表达。本文就TRPV4在眼内各个组织中的表达及生理病理功能方面进行阐述。随着TRPV4在眼部病理生理功能中的深入研究,TRPV4在角膜损伤修复、青光眼及视网膜血管生成方面可能成为潜在的新兴药物靶点,但仍需进一步的深入研究。

基于NGF/PI3K/TRPV1信号通路探究参苓白术散对腹泻型肠易激综合征小鼠的干预机制

目的研究基于神经生长因子/磷脂酰肌醇3-激酶/瞬时受体电位香草酸受体1(NGF/PI3K/TRPV1)信号通路探究参苓白术散对腹泻型肠易激综合征小鼠的干预机制。方法选取40只SPF级SD雄性大鼠,其中空白组10只,模型组13只、研究1组7只、研究2组9只。对照组、模型组只进行生理盐水灌胃,不做其他任何处理。研究1组给予匹维溴铵片20mg/kg水溶液进行灌胃,研究2组给予参苓白术散4g/kg进行灌胃。评估Bristol评分情况;分析脾脏系数、胸腺系数情况;检测IgA、IgG、IgM、IL-1β、IL-6、TNF-α表达水平及NGF/PI3K/TRPV1蛋白表达情况。结果相比于空白组,模型组、研究1组、研究2组Bristol评分、脾脏系数、胸腺系数、IgA、IgG、IgM、IL-1β、IL-6、TNF-α水平及NGF、PI3K、TRPV1蛋白表达均上升(P<0.05);相比于模型组、研究1组,研究2组Bristol评分、脾脏系数、胸腺系数、IgA、IgG、IgM、IL-1β、IL-6、TNF-α水平及NGF、PI3K、TRPV1蛋白表达均下降(P<0.05)。结论参苓白术散抑制NGF/PI3K/TRPV1通路,改善大鼠胃肠功能及大便状态,减轻炎症反应,提高免疫功能稳态情况,干预效果显著。

参榆洗液对痔术后大鼠痛觉敏化及cAMP/PKA信号通路和TRPV1蛋白表达的影响

目的探讨参榆洗液对痔术后大鼠痛觉敏化及环磷酸腺苷/蛋白激酶A(cAMP/PKA)信号通路、香草酸瞬时受体亚型1(TRPV1)蛋白表达的影响。方法取40只雄性SD大鼠,随机选择其中8只作为空白组,其余大鼠采用冰醋酸建立痔术后创面模型。将造模成功大鼠随机分为模型组及参榆洗液低、中、高剂量组,每组8只。自成功造模后的第1天开始,模型组给予高压灭菌水局部熏洗,参榆洗液低、中、高剂量组分别予以参榆洗液0.4 g生药/mL、0.8 g生药/mL、1.6 g生药/mL局部熏洗,均2次/d,每次15 min,2次治疗间隔12 h,连续7 d。记录大鼠第1,4,7天换药刺激后3 min内首次舔舐肛周创面潜伏时间、扭体反应次数以及舔舐肛周创面总时间,观察造模后和干预3,5,7 d后创面情况并记录创面愈合率,HE染色观察干预7 d后创面组织病理学形态,Western blot法检测干预7 d后创面组织中cAMP、PKA蛋白和背根神经节中TRPV1、p-PKA蛋白表达情况。结果与空白组比较,模型组大鼠首次舔舐肛周创面潜伏时间明显缩短(P<0.05),扭体反应次数明显增加(P<0.05),舔舐肛周创面总时间明显延长(P<0.05);创面组织中有大量中性粒细胞与淋巴细胞浸润,组织水肿明显;创面组织中cAMP、PKA蛋白相对表达量及背根神经节中TRPV1、p-PKA蛋白相对表达量均明显升高(P均<0.05)。与模型组比较,参榆洗液中、高剂量组大鼠首次舔舐肛周创面潜伏时间明显延长(P均<0.05),扭体反应次数明显减少(P均<0.05),舔舐肛周创面总时间明显缩短(P均<0.05);参榆洗液各组干预5,7 d后创面愈合率更高(P均<0.05);参榆洗液各组大鼠创面组织中中性粒细胞、淋巴细胞浸润减少,成纤维细胞、新生血管增多;参榆洗液各组创面组织中cAMP、PKA蛋白相对表达量及背根神经节中TRPV1、p-PKA蛋白相对表达量均明显降低(P均<0.05),且呈剂量依赖性下降(P均<0.05)。结论参榆洗液可呈剂量依赖性改善痔术后大鼠痛觉敏化,促进创面愈合,机制可能与下调cAMP/PKA信号通路相关蛋白及TRPV1蛋白表达有关。

含有TRPV1受体拮抗剂的修护霜对敏感性皮肤的改善作用

目的:评价含有TRPV1受体拮抗剂的修护霜对敏感性皮肤的改善作用及安全性。方法:采用半脸对照及使用前后自身对照研究,受试者在第一次随访根据随机号将修护霜涂抹于单侧面部,使用本品后进行即刻时间点TI敏感程度评估(30s,1 min、10 min)。随后研究期间全面部涂抹修护霜每天2次,共28天。分别在使用前(TO)、使用后48 h(T2)、使用后28d(T3)进行面部乳酸刺痛实验、生理指标检测及皮肤敏感状态主观评分。由皮肤科医生,眼科医生及耳鼻喉科医生分别在使用前和使用后10 min,48 h及28d对皮肤和眼部鼻部黏膜耐受性进行评估。结果:共收集39例受试者。受试者单侧面部涂抹修护霜10min,与未用修护霜侧相比,敏感评估量表各维度评分下降,具有统计学意义(P{<}0.001),受试者的皮肤刺激症状迅速改善。在使用修护霜30 s,1 min、10 min,48 h以及28 d后,各项敏感评估评分均较使用前下降,具有统计学意义(P{<}0.001)。使用修护霜后28天,乳酸刺痛实验评分和经表皮失水率,分别为1.46±1.52和14.91±3.90g/h·m^(2),较使用前(3.50±1.63,17.82±5.34 g/h.m^(2))降低。受试区角质层含水量(62.90±10.83)较使用前(53.91±16.07)水平增加(P{<}0.001),差异均具有统计学意义(P{<}0.01);红斑指数,pH值变化无统计学差异。研究期间未观察到与修护霜使用相关的皮肤、眼部和鼻部的任何刺激性反应及其他不良反应。结论:含有TRPV1受体拮抗剂的修护霜可快速缓解敏感性皮肤的多种不适症状并可以修复皮肤屏障,耐受性良好。

瞬时受体电位香草酸亚型1在急性呼吸窘迫综合征中的作用及研究进展

急性呼吸窘迫综合征(acute respiratory distress syndrome,ARDS)是由肺内和肺外病因引发的一种严重的、以顽固性低氧血症为显著特征的呼吸系统危重症,起病较急,发病率和死亡率较高。随着全球范围内的呼吸道病毒的流行和变异,ARDS的诊疗也变得更加复杂,因此临床上亟待探索有关ARDS发生发展的分子机制和有效的治疗方法。研究发现,ARDS的发病机制涉及炎症反应及氧化还原反应失衡、内皮细胞功能失调、肺泡毛细血管屏障的破坏、凝血功能异常等多个因素的相互作用。虽然基因组学、蛋白质组学等分子生物学技术的发展已为ARDS的发病机制提供了全新视角,但仍缺乏早期诊断ARDS的生物标志物和针对性治疗ARDS的有效药物。目前,越来越多的研究表明,瞬时受体电位香草酸亚型1(transient receptor potential vanilloid-1,TRPV1;即辣椒素受体)广泛分布于上呼吸道、气道平滑肌、肺泡和肺血管等部位,参与调解气道舒张和收缩、咳嗽反射、炎症和疼痛相关的炎症介质释放,以及呼吸系统对温度、化学物质和机械牵拉等刺激的感知并传递各种生物信号,在呼吸系统疾病中扮演着重要角色,且已成为肺炎、肺水肿、咳嗽、哮喘、急性肺损伤等呼吸系统疾病的研究热点。基于此,该文以脓毒症、创伤性脑损伤和呼吸道病毒引发的ARDS与TRPV1的相关性和分子机制为切入点进行综述,总结了调控TRPV1的表达对ARDS发病进程所发挥的积极作用,旨在为加强ARDS的早期诊断和有效干预措施提供参考。

靶向瞬时受体电位香草酸亚型1离子通道的Nplus-RhTx多肽抑制剂理性设计及功能验证

目的:获得靶向瞬时受体电位香草酸亚型1(TRPV1)通道的多肽抑制剂。方法:基于已有的靶向TRPV1通道的多肽激动剂红头蜈蚣毒素(RhTx)进行理性设计,在其氨基末端增加带正电荷的氨基酸,并在细胞上使用膜片钳电生理验证设计的多肽对TRPV1通道的抑制作用。结果:理性设计了8条Nplus-RhTx多肽,在膜片钳电生理记录中发现其中4条可以抑制TRPV1的辣椒素激活,4条Nplus-RhTx多肽的半数有效抑制浓度分别为(188.3±4.7)、(193.6±18.0)、(282.8±11.9)和(299.5±6.4)µmol/L。结论:通过对RhTx多肽的氨基端进行理性设计改变其性质,可获得靶向TRPV1的多肽抑制剂。

不同湿度条件下甲醛暴露致大鼠认知功能损伤及毒理学机制研究

探讨不同湿度条件下甲醛暴露对大鼠认知功能的影响及毒理学机制。取SPF级6~8周龄雌性SD大鼠48只,随机分为6组:对照组(Control组)、甲醛组(FA组)、低湿度组(60%Hum组)、中湿度组(75%Hum组)、高湿度组(90%Hum组)和高湿度联合甲醛组(90%Hum+FA组)。不同湿度组分别置于(60%/75%/90%)环境仓中暴露,FA组采用职业气态方式暴露(3 mg·m^(-3),8 h·d^(-1),5 d·周^(-1)),连续21 d后观察Morris水迷宫和旷场实验结果,苏木精-伊红染色法(HE)、尼氏染色观察各组大鼠海马神经元的形态学变化,免疫荧光法和免疫印迹实验(Western Blot)检测瞬时受体电位香草素受体4(TRPV4)、闭合蛋白(Occludin)和紧密连接蛋白5(Claudin-5)的蛋白表达水平。与Control组相比,不同湿度组和FA组大鼠的认知功能下降,海马神经元数量减少,且随湿度升高而加剧(P<0.05);与FA组、90%Hum组分别比较,90%Hum+FA组认知功能和海马神经元损伤加剧,且TRPV4表达升高,Occludin和Claudin-5表达降低,差异具有统计学意义(P<0.05)。高湿度和甲醛联合暴露可能通过激活TRPV4离子通道,下调Occludin和Claudin-5表达,加剧大鼠海马神经元的损伤,使认知功能进一步下降。

β-细辛醚通过抑制TRPV4的表达缓解谷氨酸诱导的Ca^(2+)超载

谷氨酸处理会导致Ca^(2+)超载,瞬时受体电位香草素受体4(transient receptor potential vanilloid 4,TRPV4)在其中的作用及可能机制尚不清楚。β-细辛醚能快速透过血脑屏障,对兴奋性毒性具有较强的神经保护作用。文章以高分化的PC12细胞为研究对象,探究β-细辛醚(15、30、60μmol/L)预处理4 h,40 mmol/L谷氨酸处理实时记录对PC12细胞Ca^(2+)浓度的影响,采用钙成像技术检测Ca^(2+)浓度的变化;采用实时荧光定量聚合酶链式反应(polymerase chain reaction,PCR)、Western Blot及免疫荧光技术检测TRPV4的mRNA和蛋白的表达;采用Lipofectiamine 2000脂质体实验转染TRPV4-siRNA和pEX-3-TRPV4,观察沉默和过表达TRPV4对谷氨酸引起Ca^(2+)超载的影响。结果表明:与正常对照组相比,谷氨酸处理5 min可诱导Ca^(2+)超载,显著提高TRPV4的mRNA和蛋白的表达;与模型组相比,β-细辛醚能够剂量依赖性地降低谷氨酸诱导的Ca^(2+)超载和TRPV4的表达;沉默TRPV4抑制细胞Ca^(2+)超载;过表达TRPV4则部分逆转β-细辛醚抑制谷氨酸诱导的Ca^(2+)超载。该研究证明,谷氨酸处理PC12细胞5 min通过上调TRPV4的表达诱导Ca^(2+)超载,β-细辛醚作为TRPV4的拮抗剂,是一种潜在的抑制兴奋性毒性的药物。