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.

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.

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.

口腔鳞状细胞癌中TRPV2的表达及其临床病理意义

目的探究口腔鳞状细胞癌(OSCC)中TRPV2的表达及其临床病理意义。方法免疫组化及Western blot检测TRPV2在OSCC中的表达水平,Pearsonχ^(2)检验分析TRPV2表达与OSCC患者临床病理的关系,Log-rank检验分析TRPV2对生存期的影响。生物信息学方法探索TRPV2的潜在生物学功能,及免疫组化验证TRPV2与巨噬细胞浸润水平的关系。结果免疫组化及Western blot证实TRPV2在OSCC中高表达(P<0.05),且TRPV2高表达与OSCC患者T分期、肿瘤组织学分级、预后不佳具有相关性(P<0.05)。生物信息学方法发现TRPV2参与PI3K/AKT等通路及调控免疫细胞浸润水平,免疫组化验证TRPV2高表达与巨噬细胞浸润水平呈正相关(R=0.562,P<0.001)。结论TRPV2在OSCC中高表达且与预后不佳相关,可通过PI3K/AKT等通路及调控巨噬细胞免疫浸润水平参与OSCC的发生及发展。

Effect of exogenous hydrogen sulfide in the nucleus tractus solitarius on gastric motility in rats

BACKGROUND Hydrogen sulfide(H2S)is a recently discovered gaseous neurotransmitter in the nervous and gastrointestinal systems.It exerts its effects through multiple signaling pathways,impacting various physiological activities.The nucleus tractus solitarius(NTS),a vital nucleus involved in visceral sensation,was investigated in this study to understand the role of H2S in regulating gastric function in rats.AIM To examine whether H2S affects the nuclear factor kappa-B(NF-κB)and transient receptor potential vanilloid 1 pathways and the neurokinin 1(NK1)receptor in the NTS.METHODS Immunohistochemical and fluorescent double-labeling techniques were employed to identify cystathionine beta-synthase(CBS)and c-Fos co-expressed positive neurons in the NTS during rat stress.Gastric motility curves were recorded by inserting a pressure-sensing balloon into the pylorus through the stomach fundus.Changes in gastric motility were observed before and after injecting different doses of NaHS(4 nmol and 8 nmol),physiological saline,Capsazepine(4 nmol)+NaHS(4 nmol),pyrrolidine dithiocarbamate(PDTC,4 nmol)+NaHS(4 nmol),and L703606(4 nmol)+NaHS(4 nmol).RESULTS We identified a significant increase in the co-expression of c-Fos and CBS positive neurons in the NTS after 1 h and 3 h of restraint water-immersion stress compared to the expressions observed in the control group.Intra-NTS injection of NaHS at different doses significantly inhibited gastric motility in rats(P<0.01).However,injection of saline,first injection NF-κB inhibitor PDTC or transient receptor potential vanilloid 1(TRPV1)antagonist Capsazepine or NK1 receptor blockers L703606 and then injection NaHS did not produce significant changes(P>0.05).CONCLUSION NTS contains neurons co-expressing CBS and c-Fos,and the injection of NaHS into the NTS can suppress gastric motility in rats.This effect may be mediated by activating TRPV1 and NK1 receptors via the NF-κB channel.

Effect of TRPV1 Channel on Proliferation and Apoptosis of Airway Smooth Muscle Cells of Rats

Airway remodeling is an important pathological feature of asthma and the basis of severe asthma. Proliferation of airway smooth muscle cells （ASMCs） is a major contributor to airway remod- eling. As an important Ca2＋ channel, transient receptor potential vanilloid 1 （TRPV1） plays the key role in the cell pathological and physiological processes. This study investigated the expression and activity of TRPV1 channel, and further clarified the effect of TRPV1 channel on the ASMCs proliferation and apoptosis in order to provide the scientific basis to treat asthmatic airway remodeling in clinical practice Immunofluorescence staining and reverse transcription polymerase chain reaction （RT-PCR） were used to detect the expression of TRPVI in rat ASMCs. Intracellular Ca2＋ was detected using the single cell confocal fluorescence microscopy measurement loaded with Fluo-4/AM. The cell cycles were observed by flow cytometry. MTT assay and Hoechst 33258 staining were used to detect the proliferation and apoptosis of ASMCs in rats respectively. The data showed that： （1） TRPV1 channel was present in rat ASMCs. （2） TRPV1 channel agonist, capsaicin, increased the Ca2~ influx in a concentration-dependent manner （EC50=284.3＋58 nmol/L）. TRPV1 channel antagonist, capsazepine, inhibited Ca2＋ influx in rat ASMCs. （3） Capsaicin significantly increased the percentage of S＋G2M ASMCs and the absorbance of MTT assay. Capsazepine had the opposite effect. （4） Capsaicin significantly inhibited the apoptosis, whereas capsazepine had the opposite effect. These results suggest that TRPV1 is present and mediates Ca2＋ influx in rat ASMCs. TRPV1 activity stimulates proliferation of ASMCs in rats.

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.

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.

Regulatory effects of anandamide on intracellular Ca^(2+) concentration increase in trigeminal ganglion neurons

Activation of cannabinoid receptor type 1 on presynaptic neurons is postulated to suppress neu- ~ ~ ~ 2＋ ~ ~ 2＋ rotransmlsslon by decreasing Ca reflux through high voltage-gated Ca channels. However, recent studies suggest that cannabinoids which activate cannabinoid receptor type 1 can increase neurotransmitter release by enhancing Ca2＋ influx in vitro. The aim of the present study was to investigate the modulation of intracellular Ca2＋ concentration by the cannabinoid receptor type 1 agonist anandamide, and its underlying mechanisms. Using whole cell voltage-damp and calcium imaging in cultured trigeminal ganglion neurons, we found that anandamide directly caused Ca2＋ influx in a dose-dependent manner, which then triggered an increase of intracellular Ca2＋ concentration. The cyclic adenosine and guanosine monophosphate-dependent protein kinase systems, but not the protein kinase C system, were involved in the increased intracellular Ca2＋concentration by anandamide. This result showed that anandamide increased intracellu- lar Ca2＋ concentration and inhibited high voltage-gated Ca2＋ channels through different signal transduction pathways.

Ion Channel Dysregulation Following Intracerebral Hemorrhage

Injury to the brain after intracerebral hemorrhage(ICH)results from numerous complex cellular mechanisms.At present,effective therapy for ICH is limited and a better understanding of the mechanisms of brain injury is necessary to improve prognosis.There is increasing evidence that ion channel dysregulation occurs at multiple stages in primary and secondary brain injury following ICH.Ion channels such as TWIK-related K+channel 1,sulfonylurea 1 transient receptor potential melastatin 4 and glutamate-gated channels affect ion homeostasis in ICH.They in turn participate in the formation of brain edema,disruption of the blood-brain barrier,and the generation of neurotoxicity.In this review,we summarize the interaction between ions and ion channels,the effects of ion channel dysregulation,and we discuss some therapeutics based on ion-channel modulation following ICH.

ASIC2 Synergizes with TRPV1 in the Mechano-Electrical Transduction of Arterial Baroreceptors

Mechanosensitive ion channels(MSCs)are key molecules in the mechano-electrical transduction of arterial baroreceptors.Among them,acid-sensing ion channel 2(ASIC2)and transient receptor potential vanilloid subfamily member 1(TRPV1)have been studied extensively and documented to play important roles.In this study,experiments using aortic arch-aortic nerve preparations isolated from rats revealed that both ASIC2 and TRPV1 are functionally necessary,as blocking either abrogated nearly all pressure-dependent neural discharge.However,whether ASIC2 and TRPV1 work in coordination remained unclear.So we carried out cell-attached patch-clamp recordings in HEK293T cells co-expressing ASIC2 and TRPV1 and found that inhibition of ASIC2 completely blocked stretch-activated currents while inhibition of TRPV 1 only partially blocked these currents.Immunofluorescence staining of aortic arch-aortic adventitia from rats showed that ASIC2 and TRPV1 are co-localized in the aortic nerve endings,and co-immunoprecipitation assays confirmed that the two proteins form a compact complex in HEK293T cells and in baroreceptors.Moreover,protein modeling analysis,exogenous co-immunoprecipitation assays,and biotin pull-down assays indicated that ASIC2 and TRPV1 interact directly.In summary,our research suggests that ASIC2 and TRPV1 form a compact complex and function synergisti-cally in the mechano-electrical transduction of arterial baroreceptors.The model of synergism between MSCs may have important biological significance beyond ASIC2 and TRPV 1.

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

瞬时受体电位香草酸亚家族1介导针刺治疗病理性疼痛的作用

Acupuncture,a traditional oriental intervention for chronic pain,has been gaining worldwide popularity.Transient receptor potential vanilloid subfamily 1(TRPV1)is a key factor mediating pain production and sensitization.According to multiple studies,TRPV1 is involved the acupuncture-induced relief of pathological pain.Herein,we systematically screened the experimental reports on TRPV1 involvement in acupuncture analgesia,and reviewed the role of TRPV1 in acupuncture in inhibiting different pathological pain and unresolved problems,including inflammatory pain,neuropathic pain,visceral pain,fibromyalgia and cancer pain.At localized acupoints,TRPV1 was involved in the initiation of acupuncture signals.Acupuncture could inhibit the development of pathological pain as well as the transmission of pain signals by suppressing TRPV1 expression and opening activity from the peripheral dorsal root ganglia to the central spinal cord.Furthermore,acupuncture can not only inhibit the expression of TRPV1,but also promote the expression of TRPV1 in the brain to alleviate pain sensation and depression-like behavior.Moreover,the mechanism by which acupuncture regulates TRPV1 may involve neuro-immune crosstalk.In conclusion,the regulation of TRPV1 expression and function may be one of the primary mechanisms by which acupuncture relieves pathological pain,laying the groundwork for future basic research on acupuncture's pain-relieving effects.

Low Electrical Resistance Properties of Acupoints:Roles of NOergic Signaling Molecules and Neuropeptides in Skin Electrical Conductance

Early studies from several independent laboratories demonstrated that acupoints possess the characteristics of low electrical resistance.New devices are developing to increase the reliability of electrical skin impedance measurements for counteracting the factors including skin dryness,skin thickness,size of the sensing electrode,pressure applied on the electrode,interelectrode distance,room temperature,and humidity.Morphological studies have identified that blood vessels,hair follicles,and nervous components are enhanced in the meridians/acupoints,which represent areas of potentially high neuronal activity.Recent evidence shows that nitric oxide(NO)concentrations are enhanced in skin acupoints/meridians.L-arginine-derived NO synthesis modifies skin norepinephrine(NE)synthesis/release in acupoints/meridians,and NO-NE activations play an important role in mediating the skin conductance responses to electrical stimulation.NOergic signaling molecules interact with gap junction and transient receptor potential vanilloid type-1.Other studies reported that the high conductance at acupoints is a result of the release of the neuropeptides substance P and calcitonin gene-related peptide during neurogenic inflammation in the referred pain area.Pathological body conditions caused considerable changes in skin conductance or impedance at acupoints.Although systematic research with an improved equipment and research design to avoid the influencing factors are requested for a definite answer in this field,the results from anatomical and biochemical studies consistently show that acupoints exist higher levels of nervous components,and NOergic signaling molecules and neuropeptides involved in the skin low resistance at acupoints.The increased interest in the acupoints/meridians has led to an open-minded attitude towards understanding this system,which is fundamental important to establish the valid aspects of scientific basis of Chinese medicine mechanisms and therapies.