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.

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.

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.

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.

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.

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.

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.

TRPC1与BK-α的表达对大鼠糖尿病肾病的影响

目的 探究瞬时受体电位C1(transient receptor potential channel 1,TRPC1)蛋白和大电导钙离子激活钾通道α亚单位(large conductance Ca^(2+)-activated K^(+)channel α subunit,BK-α)蛋白对大鼠糖尿病肾病(diabetic kidney disease,DKD)的影响。方法 将SD大鼠随机分为对照组(n=15)和模型组(n=15)。利用高脂饲料和链脲佐菌素(streptozocin,STZ)构建DKD模型。采用血糖分析仪检测大鼠血糖变化;采用全自动生化分析仪检测大鼠肾功能水平;HE染色检测肾组织的病理变化以确定造模成功。实时荧光定量PCR(RT-qPCR)和蛋白免疫印迹分别检测肾组织TRPC1和BK-α的mRNA和蛋白表达水平;免疫组化检测TRPC1和BK-α的分布和表达情况。结果 模型组大鼠空腹血糖(fasting plasma glucose,FPG)、尿白蛋白排泄率(urinary albumin excretion rates,UAER)、血尿素氮(blood urea nitrogen,BUN)和肌酐(creatinine,Cr)均显著高于对照组(P <0.01);模型组大鼠肾小管内壁细胞出现膨胀现象,部分细胞脱离;可见肾小管发生病变或死亡;此外,在许多肾小管及肾间质区域发现有中性白细胞及其残骸;以上HE染色结果提示,DKD模型复制成功。TRPC1和BK-α在肾小球部位最为丰富,且模型组大鼠肾组织中TRPC1和BK-α的mRNA和蛋白水平都显著高于对照组(P <0.05)。结论 大鼠糖尿病肾病影响TRPC1和BK-α在肾组织中的分布和表达。

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。

新生大鼠缺氧缺血性脑损伤模型中TRPC3通路介导的神经自噬研究

目的:探讨新生大鼠缺氧缺血性脑损伤模型中TRPC3通路介导的神经自噬作用。方法:7日龄Sprague-Dawley(SD)雄性幼鼠随机分为4组,每组20只,分别为Sham组、HIBD组、HIBD+Vector组和HIBD+TRPC3组。HIBD+Vector组和HIBD+TRPC3组在诱导HIBD模型前24 h,分别将Vector或TRPC3注射到左侧脑室中。评估各组幼鼠脑梗死面积、神经系统评分、脑水肿体积、神经元凋亡和线粒体自噬。体外将小鼠海马神经元细胞系(HT22)分为以下6组:Control组、OGD/R组、OGD/R+TRPC3组、OGD/R+NC组、OGD/R+si-Pink1组和OGD/R+TRPC3+si-Pink1组。除Control组外,其他组使用氧气-葡萄糖剥夺/再灌注(Oxygen-glucose deprivation/reperfusion,OGD/R)方法建立体外脑缺血再灌注模型。通过线粒体膜电位的检测线粒体损伤情况。结果:与Sham组相比,HIBD组和HIBD+Vector组显示出较大的梗死面积、脑含水量、神经学评分和神经元凋亡(P<0.05)。HIBD+TRPC3组的梗死面积、脑含水量、神经学评分和神经元凋亡较HIBD+Vector组明显降低(P<0.05)。与Sham组相比,HIBD组和HIBD+Vector组线粒体自噬相关蛋白PTEN诱导假定激酶1(PTEN induced putative kinase 1,Pink1)、E3泛素连接酶(Parkin RBR E3 ubiquitin protein ligase,Parkin)和微管相关蛋白轻链3(light chain 3,LC3)Ⅱ的表达水平增加(P<0.05)。与HIBD+Vector组相比,HIBD+TRPC3组Pink1、Parkin和Lc3Ⅱ的表达水平进一步增加(P<0.05)。体外实验显示,TRPC3上调可以强烈增加Pink1、Parkin的表达和Lc3Ⅱ/Lc3Ⅰ比值,以及减少选择性自噬接头蛋白(sequestosome-1,p62)的表达。Pink1沉默可以部分阻止TRPC3对线粒体自噬的影响。TRPC3上调增加了健康线粒体的数量,减少了受损线粒体的数量,提高了HT-22细胞的生存率。然而,Pink1沉默阻止了TRPC3对线粒体功能和增殖能力的恢复。结论:TRPC3通过激活Pink1/Parkin通路介导的线粒体自噬,在HIBD早期阶段中发挥了重要的保护作用。

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。

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。

低频脉冲磁场诱导TRPC1改善COVID-19患者康复期下肢的肌肉无力症状

背景:肌肉无力是新型冠状病毒(COVID-19)感染后的常见症状,影响康复期人体日常活动能力。在强度1.5 mT,频率3300 Hz的低频脉冲磁场刺激下可通过诱导和激活经典瞬时感受器电位通1(classical transient receptor potential channel 1,TRPC1),提升人体骨骼肌的最大自主收缩力与力量耐力,对肌肉组织产生一系列生理支持效应,该手段是否会改善新型冠状病毒肺炎患者康复期的肌无力症状尚无研究。目的:选用低频脉冲磁场对新型冠状病毒肺炎患者下肢肌群进行磁刺激,以观察该刺激对新型冠状病毒肺炎患者康复期下肢肌群肌无力改善的影响。方法:招募胶体金法抗原检测试剂(COVID-19)为阳性并伴有肌肉无力症状的新型冠状病毒(奥密克戎毒株)感染患者14例,将所有受试者随机分成2组,分别为接受磁场刺激的试验组和接受假治疗的对照组。试验总时长3周,试验组每隔48 h对腿部进行低频脉冲磁刺激,对照组与试验组干预流程一致但给予假刺激,两组患者均不被告知磁刺激仪器是否运行,两组患者共进行9次操作,随后观察两组患者下肢局部肌群最大自主收缩力、腿部爆发力与力量耐力的变化情况。结果与结论:①在采集的8个局部肌群中,试验组患者7个局部肌群在经过3周的低频脉冲磁场刺激,最大自主收缩力值均增长。对照组除3个肌群最大自主收缩力自行增长改善以外,其他肌群肌力无提升。②试验组的左腿前群与双腿后群提升率显著高于对照组。③两组的纵跳摸高高度与膝关节峰值角速度相比试验前测均提升,试验组摸高高度提升率高于对照组。④在疲劳状态下,试验组膝关节峰值角速度下降率显著下降,对照组膝关节峰值角速度下降率无显著性变化;试验组摸高高度下降率显著下降,而对照组摸高高度下降率无显著性变化。⑤上述数据证实,在强度1.5 mT,频率3300 Hz的低频脉冲磁场刺激方案下,新型冠状病毒肺炎患者在康复期经过3周的低频脉冲磁场刺激相比人体自愈过程可使更多的下肢局部肌群肌力获得提升,对基于腿部爆发力的全身协调发力能力及功能状态明显改善。因此,低频脉冲磁场刺激可作为一种改善新冠感染患者下肢肌肉无力症状的有效、非运动的康复手段。

TRP通道与炎症性肠病内脏高敏感相关研究进展

炎症性肠病(inflammatory bowel disease,IBD)是临床常见的慢性、复发性炎症性疾病,其主要特点是肠道弥漫性炎症和内脏敏感性改变,且病因及发病机制复杂。瞬时受体电位(transient receptor potential,TRP)通道于胃肠道分布广泛,作为多种刺激的检测器、效应器和信号转导器参与调控IBD肠道炎症和内脏高敏感(visceral hypersensitivity,VHS),并对其产生促进或抑制作用。本文就已知主要的TRP通道与IBD中VHS的相关研究进展作一综述。