Identification of transient receptor potential channel genes and functional characterization of TRPA1 in Spodoptera frugiperda

Spodoptera frugiperda is a highly destructive pest that has become a global problem due to its robust reproductive and migratory capabilities.Transient receptor potential(TRP)channels,which constitute a vast ion channel family,play pivotal roles in sensing the external environment and maintaining internal homeostasis in insects.TRP channels have been widely investigated for their critical roles in regulating various insect behaviors in recent years.In this study,we identified 15 TRP gene loci encoding 26 transcripts in the genome of S.frugiperda and analyzed their expression profiles at different developmental stages.The results revealed that S.frugiperda possesses four TRPC genes,six TRPA genes,one TRPM gene,two TRPV genes,one TRPN gene,and one TRPML gene,while a canonical TRPP is absent.Moreover,the SfruTRPA1 was functionally characterized using the Xenopus oocyte expression system.The results showed that SfruTRPA1 is activated by temperature increases from 20 to 45℃,and there is no significant desensitization after repeated stimuli within the same temperature range.Additionally,SfruTRPA1 is activated by certain natural chemicals,including allyl isothiocyanate(AITC)and cinnamaldehyde(CA).These findings provide valuable insights to the TRP genes in S.frugiperda.

Transient receptor potential channels as predictive marker and potential indicator of chemoresistance in colon cancer

Transient receptor potential(TRP)channels are strongly associated with colon cancer development and progression.This study leveraged a multivariate Cox regression model on publicly available datasets to construct a TRP channels-associated gene signature,with further validation of signature in real world samples from our hospital treated patient samples.Kaplan-Meier(K-M)survival analysis and receiver operating characteristic(ROC)curves were employed to evaluate this gene signature’s predictive accuracy and robustness in both training and testing cohorts,respectively.Additionally,the study utilized the CIBERSORT algorithm and single-sample gene set enrichment analysis to explore the signature’s immune infiltration landscape and underlying functional implications.The support vector machine algorithm was applied to evaluate the signature’s potential in predicting chemotherapy outcomes.The findings unveiled a novel three TRP channels-related gene signature(MCOLN1,TRPM5,and TRPV4)in colon adenocarcinoma(COAD).The ROC and K-M survival curves in the training dataset(AUC=0.761;p=1.58e-05)and testing dataset(AUC=0.699;p=0.004)showed the signature’s robust predictive capability for the overall survival of COAD patients.Analysis of the immune infiltration landscape associated with the signature revealed higher immune infiltration,especially an increased presence of M2 macrophages,in high-risk group patients compared to their low-risk counterparts.High-risk score patients also exhibited potential responsiveness to immune checkpoint inhibitor therapy,evident through increased CD86 and PD-1 expression profiles.Moreover,the TRPM5 gene within the signature was highly expressed in the chemoresistance group(p=0.00095)and associated with poor prognosis(p=0.036)in COAD patients,highlighting its role as a hub gene of chemoresistance.Ultimately,this signature emerged as an independent prognosis factor for COAD patients(p=6.48e-06)and expression of model gene are validated by public data and real-world patients.Overall,this bioinformatics study provides valuable insights into the prognostic implications and potential chemotherapy resistance mechanisms associated with TRPs-related genes in colon cancer.

New perspectives in prognostication of hepatocellular carcinoma:The role and clinical implications of transient receptor potential family genes

The study titled“Transient receptor potential-related risk model predicts prognosis of hepatocellular carcinoma patients”is a significant contribution to hepatocellular carcinoma(HCC)research,highlighting the role of transient receptor potential(TRP)family genes in the disease’s progression and prognosis.Utilizing data from The Cancer Genome Atlas database,it establishes a new risk assessment model,emphasizing the interaction of TRP genes with tumor proliferation pathways,key metabolic reactions like retinol metabolism,and the tumor immune microenvironment.Notably,the overexpression of the TRPC1 gene in HCC correlates with poorer patient survival outcomes,suggesting its potential as a prognostic biomarker and a target for personalized therapy,particularly in strategies combining immunotherapy and anti-TRP agents.

Roles of transient receptor potential channel 6 in glucose-induced cardiomyocyte injury

BACKGROUND Diabetic cardiomyopathy(DCM) is a serious complication of end-stage diabetes that presents symptoms such as cardiac hypertrophy and heart failure. The transient receptor potential channel 6(TRPC6) protein is a very important selective calcium channel that is closely related to the development of various cardiomyopathies.AIM To explore whether TRPC6 affects cardiomyocyte apoptosis and proliferation inhibition in DCM.METHODS We compared cardiac function and myocardial pathological changes in wild-type mice and mice injected with streptozotocin(STZ), in addition to comparing the expression of TRPC6 and P-calmodulin-dependent protein kinase Ⅱ(P-CaMKⅡ) in them. At the same time, we treated H9C2 cardiomyocytes with high glucose and then evaluated the effects of addition of SAR, a TRPC6 inhibitor, and KN-93, a CaMKⅡ inhibitor, to such H9C2 cells in a high-glucose environment.RESULTS We found that STZ-treated mice had DCM, decreased cardiac function, necrotic cardiomyocytes, and limited proliferation. Western blot and immunofluorescence were used to detect the expression levels of various appropriate proteins in the myocardial tissue of mice and H9C2 cells. Compared to those in the control group, the expression levels of the apoptosis-related proteins cleaved caspase 3 and Bax were significantly higher in the experimental group, while the expression of the proliferation-related proteins proliferating cell nuclear antigen(PCNA) and CyclinD1 was significantly lower. In vivo and in vitro, the expression of TRPC6 and P-CaMKⅡ increased in a high-glucose environment. However, addition of inhibitors to H9C2 cells in a high-glucose environment resulted in alleviation of both apoptosis and proliferation inhibition.CONCLUSION The inhibition of apoptosis and proliferation of cardiomyocytes in a high-glucose environment may be closely related to activation of the TRPC6/P-CaMKⅡ pathway.

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.

Melastatin-related transient receptor potential 2 channel in Aβ_(42)-induced neuroinflammation: implications to Alzheimer's disease mechanism and development of therapeutics

Alzheimer’s disease （AD） is an age-related eurodegenerative disease that represents the most common cause of dementia among the elderly people. With the increasingly aging population, AD has presented an overwhelming healthcare challenge to modern society; the World Alzheimer Report 2015 has estimated that 46.8 million people worldwide lived with dementia in 2015 and this number will rise to 74.7 million in 2030 and that the total cost of dementia was 818 billion in US$ in 2015 and will reach two trillion in 2030. Post-mortem studies have identified two histopathological hallmarks in the brains of AD patients; extracellular senile plaque with elevated deposition of amyloid β （Aβ） peptides, and intracellular neurofibrillary tangle composed of hyper-phosphorylated microtubule-associated protein tau.Etiologically, progressive neuronal loss within the cerebral cortex and hippocampus regions of the brain leads to irreversible decline in, and eventually complete loss of, memory and other cognitive functions that afflict AD patients. The widely-accepted amyloid cascade hypothesis for AD pathogenesis holds that accumulation and aggregation of neurotoxic Aβ peptides, due to imbalance of their generation and clearance as a result of changes in genetic makeup, aging and/or exposure to environmental risk factors, is a major and early trigger of AD. This hypothesis has continuously gained support by preclinical and clinical studies （Selkoe and Hardy, 2016）. However, the intensive and costly drug discovery efforts over the past decades based on such a hypothesis have proved extremely frustrating in developing effective therapeutics to treat or slow down the progress of AD, highlighting the need for more research to improve our understanding towards the cellular and molecular mechanisms by which Aβ peptides bring about neurotoxicity and cognitive dysfunction.

Distribution profiles of transient receptor potential melastatin-related and vanilloid-related channels in prostatic tissue in rat

Aim： To investigate the expression and distribution of the members of the transient receptor potential （TRP） channel members of TRP melastatin （TRPM） and TRP vanilloid （TRPV） subfamilies in rat prostatic tissue. Methods： Prostate tissue was obtained from male Sprague-Dawley rats. Reverse transcription polymerase chain reaction （RT-PCR） and quantitative real-time polymerase chain reaction （PCR） were used to check the expression of all TRPM and TRPV channel members with specific primers. Immunohistochemistry staining for TRPM8 and TRPV1 were also performed in rat tissues. Results： TRPM2, TRPM3, TRPM4, TRPM6, TRPM7, TRPMS, TRPV2 and TRPV4 mRNA were detected in all rat prostatic tissues. Very weak signals for TRPM1, TRPVI and TRPV3 were also detected. The mRNA of TRPM5, TRPV5 and TRPV6 were not detected in all RT-PCR experiments. Quantitative real-time RT-PCR showed that TRPM2, TRPM3, TRPM4, TRPMS, TRPV2 and TRPV4 were the most abundantly expressed TRPM and TRPV subtypes, respectively. Fluorescence immunohistochemistry indicated that TRPM8 and TRPV 1 are highly expressed in both epithelial and smooth muscle cells. Conclusion： Our results demonstrate that mRNA or protein for TRPM1, TRPM2, TRPM3, TRPM4, TRPM6, TRPM7, TRPMS, TRPV1, TRPV2, TRPV3 and TRPV4 exist in rat prostatic tissue. The data presented here assists in elucidating the physiological function of TRPM and TRPV channels.

Transient receptor potential channel A1 involved in calcitonin gene-related peptide release in neurons

Transient receptor potential channel A1 is one of the important transducers of noxious stimuli in the primary afferents, which may contribute to generation of neurogenic inflammation and hyperalgesia. The present study was designed to investigate if activation of transient receptor potential channel A1 may induce calcitonin gene-related peptide release from the primary afferent neurons. We found that application of allyl isothiocyanate, a transient receptor potential channel A1 activator, caused calcitonin gene-related peptide release from the cultured rat dorsal root ganglion neurons. Knock- down of transient receptor potential channel A1 with an antisense oligodeoxynucleotide prevented calcitonin gene-related peptide release by allyl isothiocyanate application in cultured dorsal root ganglion neurons. Thus, we concluded that transient receptor potential channel A1 activation caused calcitonin gene-related peptide release in sensory neurons.

Transient Receptor Potential Ion Channels in the Etiology and Pathomechanism of Chronic Fatigue Syndrome/Myalgic Encephalomyelitis

Chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) is a disabling condition of unknown cause having multi-system manifestations. Our group has investigated the potential role of transient receptor potential (TRP) ion channels in the etiology and pathomechanism of this illness. Store-operated calcium entry (SOCE) signaling is the primary intracellular calcium signaling mechanism in non-excitable cells and is associated with TRP ion channels. While the sub-family (Canonical) TRPC has been traditionally associated with this important cellular mechanism, a member of the TRPM sub-family group (Melastatin), TRPM3, has also been recently identified as participating in SOCE in white matter of the central nervous system. We have identified single nucleotide polymorphisms (SNPs) in TRP genes in natural killer (NK) cells and peripheral blood mononuclear cells (PBMCs) in CFS/ME patients. We also describe biochemical pathway changes and calcium signaling perturbations in blood cells from patients. The ubiquitous distribution of TRP ion channels and specific locations of sub-family group members such as TRPM3 suggest a contribution to systemic pathology in CFS/ME.

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.

2-aminoethoxydiphenyl borate or lanthanum potentiates transient receptor potential-like channels in rat CA1 hippocampal neurons

Expression of transient receptor potential （TRP） channels is widespread with transcripts distributed throughout the brain. All TRP channel subunits are activated following phospholipase C activation and form cation-selective ion channels. Previous studies examining the existence of TRP channels in hippocampal CA1 pyramidal neurons were based on cultured neurons. Therefore, their relevance for living tissue remains unclear. In the present study, patch-clamp recordings were conducted from CA1 pyramidal neurons in hippocampal slices from 7-day-old rats. Whole-cell currents were obtained from CA1 hippocampal neurons with potentiation effects of 2-aminoethoxydiphenyl borate and lanthanum, revealing that recorded experimental currents were characteristic TRP-like channel currents. Identification of rat hippocampal mRNA transcripts of TRPC4, TRPC5, TRPV1, TRPV2, and TRPV3 channels further verified the expression of characteristic TRP-like channels on rat CA1 hippocampal neurons.

Baicalein and Salvia officinalis Extract Upregulate Transglutaminase 1 mRNA Expression via the Activation of Transient Receptor Potential Channel V4

Background: It is important to maintain skin homeostasis for cosmetic and medical reasons. Many ceramide-related ingredients and cosmetics have been developed to improve the skin barrier function and skin hydration. Similar to extracellular lipids, the cornified envelope, which is a structure formed beneath the plasma membrane, contributes to the skin barrier function as a scaffold for extracellular lipids. Therefore, in this study, we focused on transglutaminase 1 (TGM1) which is the key enzyme for formation of the cornified envelope Objective: The objectives of this study were to identify compounds that could upregulate the expression of TGM1 and evaluate their underlying action mechanisms. Methods: Expression of the transient receptor potential channel vanilloid subfamily member 4 (TRPV4) at the mRNA and protein levels was estimated by PCR and western blotting. Effects of baicalein and Salvia officinalis (SO) extract on TGM1 mRNA expression were measured by PCR. The involvement of TRPV4 in TGM1 mRNA expression was evaluated by the inhibition and silencing of TRPV4. Results: TRPV4 was expressed in both basal cell-like HaCaT cells and suprabasal cell-like HaCaT cells. Baicalein and SO extract upregulated TGM1 mRNA expression in basal cell-like HaCaT cells. However, inhibition and silencing of TRPV4 abrogated the effects of baicalein and SO extract. Conclusion: Baicalein and SO extract upregulated the expression of TGM1 mRNA via the activation of TRPV4, suggesting that it may improve the skin barrier function by enhancing cornified envelope formation.

The transient receptor potential melastatin 2:a new therapeutical target for Parkinson's disease?

The transient receptor potential melastatin 2 is a calcium-permeable cation channel member of the TRP family. Also known as an oxidative stress-activated channel, the transient receptor potential melastatin 2 gating mechanism is dependent on reactive oxygen species. In pathological conditions, transient receptor potential melastatin 2 is overactivated, leading to a Ca~(2+) influx that alters cell homeostasis and promotes cell death. The role of transient receptor potential melastatin 2 in neurodegenerative diseases, including Alzheimer's disease and ischemia, has already been described and reviewed. However, data on transient receptor potential melastatin 2 involvement in Parkinson's disease pathology has emerged only in recent years and the issue lacks review studies that focus specifically on this topic. The present review aims to elucidate the role of the transient receptor potential melastatin 2 channel in Parkinson's disease by reviewing, summarizing, and discussing the in vitro, in vivo, and human studies published until August 2022. Here we describe fourteen studies that evaluated the transient receptor potential melastatin 2 channel in Parkinson's disease. The Parkinson's disease model used, transient receptor potential melastatin 2 antagonist and genetic approaches, and the main outcomes reported were discussed. The studies described transient receptor potential melastatin 2 activation and enhanced expression in different Parkinson's disease models. They also evidenced protective and restorative effects when using transient receptor potential melastatin 2 antagonists, knockout, or silencing. This review provides a literature overview and suggests where there is a need for more research. As a perspective point, this review shows evidence that supports transient receptor potential melastatin 2 as a pharmacological target for Parkinson's disease in the future.

Transient Receptor Potential Melastatin 3 and Intracellular Calcium in Natural Killer Cells in Multiple Sclerosis

Background: Natural killer (NK) cell phenotypes have reported to be implicated in the pathomechanism of Multiple Sclerosis (MS). Several investigators have observed reduced peripheral numbers, reduced cytotoxic activity, and altered CD56Dim and CD56Bright NK cell phenotypes. This current project, for the first time, investigates the NK cell cytotoxicity, calcium mobilisation and transient receptor potential melastatin 3 (TRPM3) surface expression. Methods: NK cell cytotoxic activity and calcium signaling were examined in CD56Dim and CD56Bright NK cells before and after stimulation using Ionomycin, Pregnenolone sulphate, 2-Aminoethoxydiphenyl borate and Thapsigargin. Purified NK cells were labelled with antibodies to determine TRPM3, CD69 and CD107a surface expression using flow cytometry. Results: Twenty-two MS patients and 22 healthy controls were recruited for this project. Twelve of the 22 previously received Alemtuzumab (Lemtrada&reg;) and the remaining ten reported nil medication. We report TRPM3 was significantly increased in untreated MS patients compared with healthy controls and treated MS patients (p-value 0.034). There was a significant decrease in CD69 surface expression on CD56Dim NK cell phenotype for untreated MS patients (p-value 0.031) and treated MS patients (p-value 0.036). We report altered calcium mobilisation in CD56Bright NK cells and to a lesser extent CD56Dim NK cells between healthy controls, treated and untreated MS patients. Conclusion: This investigation suggests variations in TRPM3 expression and calcium mobilisation of NK cells may be implicated in the pathogenesis of MS. Further investigation is required to determine the mechanism by which alemtuzumab alters calcium signaling in NK cells.

瞬时感受电位通道6在同型半胱氨酸诱导的小鼠肾脏足细胞自噬中的作用

目的探讨在同型半胱氨酸(Hcy)作用下瞬时感受电位通道6(TRPC6)对小鼠肾脏足细胞自噬的调控作用。方法将小鼠肾脏足细胞分为对照组与Hcy组(40、60、80、100μmol/L的Hcy刺激48 h),采用qRT-PCR检测TRPC6mRNA水平,筛选出Hcy的最佳浓度后,后续均以此浓度进行实验。采用Western blotting检测自噬相关蛋白LC3Ⅱ、p62,以及足细胞结构蛋白Nephrin、Podocin的表达水平;采用qRT-PCR、Western blotting及免疫荧光检测两组TRPC6 mRNA及蛋白的表达水平。转染过表达或干扰TRPC6的细胞分别设置为:(1)对照组(不做处理)、TRPC6过表达阴性对照组、TRPC6过表达组;(2)对照组(不做处理)、TRPC6干扰阴性对照组和TRPC6干扰组(si-1、si-2、si-3),并采用qRT-PCR检测TRPC6表达水平。再将过表达或干扰TRPC6后的细胞分别设置为:(1)对照组(不做处理)、Hcy组(加入80μmol/L Hcy)、TRPC6过表达对照+Hcy组、TRPC6过表达+Hcy组;(2)对照组(不做处理)、Hcy组、TRPC6干扰对照+Hcy组、TRPC6干扰+Hcy组,并采用Western blotting检测p62、LC3Ⅱ、TRPC6蛋白的表达水平。结果qRT-PCR检测结果显示,与对照组比较,TRPC6 mRNA表达水平随Hcy浓度增加而升高,其中80μmol/L Hcy干预后的表达水平最高,因此将80μmol/L作为Hcy的最佳干预浓度。此时,自噬相关蛋白LC3Ⅱ的表达水平升高、p62表达水平降低(P<0.05)。Western blotting检测结果显示,与对照组比较,Hcy组中足细胞相关蛋白Nephrin、Podocin的表达水平明显降低(P<0.05)。qRT-PCR检测结果显示,与对照组比较,Hcy组中TRPC6 mRNA表达水平明显升高(P<0.05);与TRPC6过表达阴性对照组比较,TRPC6过表达组的TRPC6 mRNA及蛋白表达水平均明显升高(P<0.05);与TRPC6干扰阴性对照组比较,TRPC6干扰组的TRPC6 mRNA及蛋白表达水平均明显降低(P<0.05)。Western blotting检测结果显示,与TRPC6过表达对照组相比,TRPC6过表达+Hcy组中自噬相关蛋白LC3Ⅱ表达水平明显升高,p62表达水平明显降低(P<0.05);与TRPC6干扰对照+Hcy组相比,TRPC6干扰+Hcy组中自噬相关蛋白LC3Ⅱ表达水平明显降低,p62表达水平明显升高(P<0.05)。结论Hcy可诱导小鼠肾脏足细胞自噬,抑制TRPC6的表达可明显减轻足细胞的自噬性损伤。

线粒体氧化应激及m6A表观遗传调控TRPC6钙通道在肾病综合征发病中的作用研究

目的 初步探讨N6-甲基腺嘌呤(m6A)表观遗传修饰瞬时受体电位阳离子通道6(TRPC6)通道失调在肾病综合征发病中的作用及潜在机理。方法 按照随机数字表法将小鼠足细胞分为4组:对照组、叔丁基对苯二酚(TBHQ)组、嘌呤霉素氨基核苷(PAN)处理组、TBHQ+PAN处理组。对照组为正常完全培养液,TBHQ组培养液中加入10 nmol/L TBHQ,PAN处理组培养液中加入PAN 50μg/mL,TBHQ+PAN处理组培养液中加入10 nmol/L TBHQ以及PAN 50μg/mL,刺激24 h收集细胞。应用膜片钳证实PAN损伤诱导TRPC6通道激活机理及1,4,5-肌醇三磷酸(IP3)受体拮抗剂TBHQ对电流的影响,检测对照组、PAN处理组、TBHQ组和TBHQ+PAN处理组细胞TRPC6通道电流变化。通过葡萄糖氧化酶(GO)建立足细胞氧化应激模型。另外按照随机数字表法将足细胞分为4组:空白对照组、GO组、姜黄素组、姜黄素+GO组。GO组给予GO 3.5 kU/L,姜黄素组给予Nrf2激动剂(姜黄素)40μmol/L,姜黄素+GO组给予姜黄素40μmol/L和GO 3.5 kU/L处理,给药处理8~12 h后收集细胞。检测各组Nrf2和特异性调控蛋白NAD(P)H:醌氧化还原酶1(NQO-1)、TRPC6及Transgelin蛋白和线粒体调控蛋白表达变化。通过SRAMP对TRPC6通道m6A位点进行精准预测,对PAN诱导足细胞损伤模型公共数据库GSE124622进行2次生物信息学分析。结果 对照组与TBHQ组电流比较,差异无统计学意义(P>0.05);与对照组比较,PAN处理组电流升高,而TBHQ+PAN组电流减小,差异均有统计学意义(P<0.05)。与空白对照组比较,GO组Nrf2、NQO-1、TRPC6及Transgelin蛋白表达均升高,差异均有统计学意义(P<0.01);与GO组比较,姜黄素+GO组Nrf2、NQO-1、TRPC6及Transgelin蛋白表达均降低,差异均有统计学意义(P<0.05)。与空白对照组比较,GO组线粒体调控蛋白Mfn2、Opa1蛋白表达均降低,Drp1蛋白表达升高,差异均有统计学意义(P<0.05);与GO组比较,姜黄素+GO组粒体调控蛋白Mfn2、Opa1蛋白表达升高,Drp1蛋白表达降低,差异均有统计学意义(P<0.05);空白对照组与姜黄素组TRPC6/Transgelin及线粒体调控蛋白表达比较,差异均无统计学意义(P>0.05)。TRPC6通道序列存在多个m6A修饰位点,均具有被甲基转移酶(METTL)3、METTL14、肾母细胞肿瘤1相关蛋白(WTAP)和去甲基化酶ALKB同源蛋白(ALKBH)、m6A去甲基化酶(FTO)调控的潜在可能。对12个m6A调节基因进行表达分析,发现m6A调节基因的表达在PAN诱导足细胞损伤中发生显著差异。结论 TRPC6介导钙离子内流可被氧化应激激活参与足细胞损伤,激活Nrf2可以减少钙过负荷所致线粒体损伤而保护足细胞。TRPC6序列中存在多个高m6A修饰靶点,肾病综合征发病机理可能通过m6A修饰足细胞TRPC6离子通道,m6A相关调控基因在肾病足细胞损伤中发生明显变化。

CaR调节TRPC6介导的自噬对软骨细胞SOD及NOX2的影响

目的观察钙敏感受体(CaR)调节瞬时感受器电位通道6(TRPC6)介导的自噬对软骨细胞超氧化物歧化酶(SOD)、NADPH氧化酶-2(NOX2)的影响。方法2023年7月将出生3~5 d SD大鼠关节软骨用胶原酶消化后进行传代培养,取第3代培养的细胞接种于96孔板,用白细胞介素-1β(10 ng/mL)诱导软骨细胞,分为对照组、CaR(10μg/L)组、U73122(5μg/L)组和CaR(10μg/L)+U73122(5μg/L)组,共培养24 h后应用酶联免疫吸附试验检测各组上清液SOD、NOX2水平,采用实时定量-聚合酶链反应检测各组SOD、NOX2、人自噬基因Beclin-1 mRNA表达,Western-blot检测SOD、NOX2、Beclin-1蛋白表达。结果原代关节软骨细胞在显微镜下呈三角形或不规则形或多角形,分布稀疏,培养24 h后可见贴壁生长;第3代软骨细胞显示其蓝染的细胞核,细胞间质及细胞质内偶见紫红色异染颗粒;细胞核染成蓝色,细胞质区呈棕黄色。对照组软骨细胞上清液SOD水平,SOD、Beclin-1 mRNA及其蛋白相对表达量,NOX2 mRNA相对表达量均明显低于CaR(10μg/L)组,NOX2水平、NOX2蛋白相对表达量均明显高于CaR(10μg/L)组,差异均有统计学意义(P<0.05);U73122(5μg/L)组和CaR(10μg/L)+U73122(5μg/L)组软骨细胞上清液SOD水平,SOD、Beclin-1 mRNA及蛋白相对表达量均明显低于对照组,NOX2水平、NOX2 mRNA及蛋白相对表达量均明显高于对照组,差异均有统计学意义(P<0.05)。结论CaR通过TRPC6介导的自噬降低软骨细胞氧化应激水平,可能是延缓骨关节炎发生、发展的重要机制。

桔梗素D通过下调成纤维细胞TRPC6表达改善小鼠肺纤维化

目的探究桔梗素D(PD)对肺纤维化的影响及其机制。方法博来霉素(BLM)气道内滴入构建C57BL/6J小鼠肺纤维化模型,再予PD(10 mg/kg)灌胃,28 d后处死小鼠。分组如下:对照组(control)、BLM(10 mg/kg)模型组、BLM(10 mg/kg)+PD(10 mg/kg)组。肺组织石蜡切片HE染色、免疫组化和天狼星红染色评估小鼠肺组织纤维化程度和瞬时受体电位离子通道亚族C成员6(TRPC6)的表达与分布。Westernblot检测肺组织α-平滑肌肌动蛋白(α-SMA)的表达水平。小鼠原代肺成纤维细胞体外培养,分别用PD、TRPC6抑制剂醋酸落叶松酯(LA)预处理后加入转化生长因子-β1(TGF-β1)刺激细胞,根据加入的TGF-β1和PD浓度不同分组如下:control组、TGF-β1(10 ng/mL)组、PD(2.5μmol/L)+TGF-β1组、PD(5μmol/L)+TGF-β1组和PD(10μmol/L)+TGF-β1组,LA处理分组如下:control组、TGF-β1(10 ng/mL)组和LA(10μmol/L)+TGF-β1组,测定细胞存活率、collagenⅠ、α-SMA和TRPC6的表达水平、活性氧(ROS)生成水平、线粒体膜电位、细胞增殖能力等指标。利用网络药理学方法结合文献分析PD作用于肺纤维化可能通过的机制。结果PD可明显减轻BLM诱导小鼠模型的肺部纤维化程度、减少α-SMA表达(P<0.05)。CCK-8结果显示PD、TGF-β1和LA的最适宜刺激浓度分别是10μmol/L、10 ng/mL和10μmol/L;5-乙炔基-2'-脱氧尿苷(EdU)染色结果显示PD能够有效抑制肺成纤维细胞增殖;2,7-二氯荧光素二乙酸酯(DCFH-DA)染色显示PD能有效减少TGF-β1诱导的细胞ROS生成(P<0.0001);线粒体膜电位检测(Jc-1染色)结果显示PD能有效缓解TGF-β1诱导的细胞线粒体膜电位下降(P<0.001);蛋白电泳结果显示PD能显著抑制TGF-β1诱导的α-SMA和collagenⅠ的表达(P<0.05)。网络药理学结合文献分析发现PD可能通过TRPC6作用于肺纤维化。免疫组织化学结果显示PD明显减轻了BLM诱导的肺组织TRPC6表达。蛋白电泳结果显示PD可明显抑制TGF-β1诱导的TRPC6表达(P<0.05);使用LA可明显抑制细胞TRPC6、α-SMA、collagenⅠ的表达(P<0.05)。结论PD可降低小鼠肺纤维化,其机制可能与下调TRPC6并减少ROS产生有关。

Identification of TRPM7 channels in human intestinal interstitial cells of Cajal

AIM： To investigate the characteristics of slow electrical waves and the presence of transient receptor potential melastatin-type 7 （TRPM7） in the human gastrointestinal （GI） tract. METHODS： Conventional microelectrode techniques were used to record intracellular electrical responses from human GI smooth muscle tissue. Immunohistochemistry was used to identify TRPM7 channels in interstitial cells of Cajat （ICCs）. RESULTS： The human GI tract generated slow electrical waves and had ICCs which functioned as pacemak er cells. Flufenamic acid, a nonselective cation channel blocker, and 2-APB （2-aminoethoxydiphenyl borate） and La3＋, TRPM7 channel blockers, inhibited the slowwaves. Also, TRPM7 channels were expressed in ICCs in human tissue. CONCLUSION： These results suggest that the human GI tract generates slow waves and that TRPM7 channels expressed in the ICCs may be involved in the gen- eration of the slow waves.

麻杏石甘汤对咳嗽变异型哮喘大鼠IL-6/STAT3信号通路及TRPV1感受器的影响

【目的】探讨麻杏石甘汤对咳嗽变异型哮喘(CVA)大鼠的治疗作用及机制。【方法】将60只大鼠随机分为正常组,模型组,麻杏石甘汤低、高剂量组,麻杏石甘汤高剂量+信号转导和转录激活因子3(STAT3)激活剂Colivelin(Col)组,每组12只。除正常组外,其他各组大鼠采用腹腔注射卵清蛋白结合艾条熏蒸法构建CVA模型。对应治疗后,观察大鼠体征和咳嗽次数,肺功能仪检测气道阻力(RE),Diff-Quik染色计数嗜酸性粒细胞(EOS),苏木素-伊红(HE)染色法观察肺、支气管组织病理学特征,酶联免疫吸附分析(ELISA)检测肺组织单核细胞趋化蛋白1(MCP-1)、肿瘤坏死因子α(TNF-α)含量,Western Blot法检测肺组织白细胞介素6(IL-6)、STAT3、瞬时受体电位香草酸亚型1(TRPV1)蛋白表达水平。【结果】与正常组比较,模型组大鼠出现明显哮喘症状,肺组织可见炎性细胞浸润严重,支气管上皮细胞坏死、纤毛粘连、黏液多,RE,EOS数目,MCP-1、TNF-α含量,以及IL-6、STAT3、TRPV1蛋白表达水平均升高(P<0.05);与模型组比较,麻杏石甘汤低、高剂量组大鼠哮喘症状明显改善,肺及支气管损伤减轻,RE,EOS数目,MCP-1、TNF-α含量以及IL-6、STAT3、TRPV1蛋白表达水平呈剂量依赖性降低(P<0.05);与麻杏石甘汤高剂量组比较,麻杏石甘汤高剂量+Col组大鼠哮喘加重,肺及支气管损伤加重,RE,EOS数目,MCP-1、TNF-α含量以及IL-6、STAT3、TRPV1蛋白表达水平升高(P<0.05)。【结论】麻杏石甘汤可有效改善CVA大鼠症状,其机制与抑制IL-6/STAT3信号通路及TRPV1高表达有关。