This study investigated the effect of Lianmei Qiwu Decoction(LMQWD) on the improvement of cardiac autonomic nerve remodeling in the diabetic rat model induced by the high-fat diet and explored the underlying mechanism of LMQWD through the AMP-activated protein kinase(AMPK)/tropomyosin receptor kinase A(TrkA)/transient receptor potential melastatin 7(TRPM7) signaling pathway. The diabetic rats were randomly divided into a model group, an LMQWD group, an AMPK agonist group, an unloaded TRPM7 adenovirus group(TRPM7-N), an overexpressed TRPM7 adenovirus group(TRPM7), an LMQWD + unloaded TRPM7 adenovirus group(LMQWD+TRPM7-N), an LMQWD + overexpressed TRPM7 adenovirus group(LMQWD+TRPM7), and a TRPM7 channel inhibitor group(TRPM7 inhibitor). After four weeks of treatment, programmed electrical stimulation(PES) was employed to detect the arrhythmia susceptibility of rats. The myocardial cell structure and myocardial tissue fibrosis of myocardial and ganglion samples in diabetic rats were observed by hematoxylin-eosin(HE) staining and Masson staining. The immunohistochemistry, immunofluorescence, real-time quantitative polymerase chain reaction(RT-PCR), and Western blot were adopted to detect the distribution and expression of TRPM7, tyrosine hydroxylase(TH), choline acetyltransferase(ChAT), growth associated protein-43(GAP-43), nerve growth factor(NGF), p-AMPK/AMPK, and other genes and related neural markers. The results showed that LMQWD could significantly reduce the arrhythmia susceptibility and the degree of fibrosis in myocardial tissues, decrease the levels of TH, ChAT, and GAP-43 in the myocardium and ganglion, increase NGF, inhibit the expression of TRPM7, and up-regulate p-AMPK/AMPK and p-TrkA/TrkA levels. This study indicated that LMQWD could attenuate cardiac autonomic nerve remodeling in the diabetic state, and its mechanism was associated with the activation of AMPK, further phosphorylation of TrkA, and inhibition of TRPM7 expression.
Rats ; Animals ; AMP-Activated Protein Kinases/metabolism* ; Nerve Growth Factor/metabolism* ; Diabetes Mellitus, Experimental/drug therapy* ; TRPM Cation Channels/metabolism* ; GAP-43 Protein/metabolism* ; Signal Transduction ; Diabetic Neuropathies/genetics* ; Fibrosis

Transient receptor potential M2 (TRPM2) ion channel is a non-selective cationic channel that can permeate calcium ions, and plays an important role in neuroinflammation, ischemic reperfusion brain injury, neurodegenerative disease, neuropathic pain, epilepsy and other neurological diseases. In ischemic reperfusion brain injury, TRPM2 mediates neuronal death by modulating the different subunits of glutamate N-methyl-D-aspartic acid receptor in response to calcium/zinc signal. In Alzheimer's disease, TRPM2 is activated by reactive oxygen species generated by β-amyloid peptide to form a malignant positive feedback loop that induces neuronal death and is involved in the pathological process of glial cells by promoting inflammatory response and oxidative stress. In epilepsy, the TRPM2-knockout alleviates epilepsy induced neuronal degeneration by inhibiting autophagy and apoptosis related proteins. The roles of TRPM2 channel in the pathogenesis of various central nervous system diseases and its potential drug development and clinical application prospects are summarized in this review.
Amyloid beta-Peptides/metabolism* ; Humans ; Neurodegenerative Diseases ; Neuroglia ; TRPM Cation Channels/genetics* ; Transient Receptor Potential Channels

OBJECTIVES: To investigate the role of transient receptor potential cation channel subfamily M member 2 (TRPM2) in hepatic ischemia-reperfusion injury of mouse (HIRI) and the possible mechanisms. METHODS: Sixty adult male C57BL/6 mice were randomly divided into 4 groups: a sham group (S group), a HIRI model group (M group), a TRPM2 adenovirus interference vector group (T group), and a TRPM2 adenovirus control vector group (C group) (=15 in each group). The liver tissues of mice before perfusion were obtained. The efficiency of adenovirus infection was detected by fluorescence microscopy, and the silencing efficiency of adenovirus against TRPM2 was detected by real-time PCR.The abdominal aorta blood and liver tissues were collected from mice at 2, 4 and 8 h after reperfusion. The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum of mice were detected. Hepatic pathological changes were examined by hematoxylin-eosin (HE) staining. The protein expression of TRPM2 and Rac family small GTPase 1 (RAC1) in liver tissues was detected by Western blotting. Changes of malondialdehyde (MDA), superoxide dismutase (SOD) and myeloperoxidase (MPO) activities in liver tissues were detected by enzyme-linked immunosorbent assay. RESULTS: A strong signal of green fluorescence was observed in the liver tissues of mice in the T and C groups compared to the S or M group. Compared with the S, M or C group, the expression of TRPM2 mRNA in liver tissue in the T group was significantly down-regulated (all <0.05). The morphology of hepatocytes was normal in the S group under light microscope.Hepatic sinus dilatation, congestion, hepatocyte degeneration, central necrosis of lobule, and massive inflammatory granulocyte infiltration were observed in the M and C group, respectively. The degree of hepatocyte damage in the T group was significantly reduced compared with that in the M and C group, respectively. Compared with the S group, the serum ALT and AST activities in the M, T and C groups were significantly increased at 2, 4 and 8 h after reperfusion (all <0.05). Compared with the M or C group, the serum ALT and AST activities in the T group were significantly lower in serum of mice at 2, 4, and 8 h after reperfusion (all <0.05). Compared with the M or C group, the serum SOD activity in the T group was significantly increased at 2, 4, and 8 h after reperfusion (all <0.05), while the serum MDA and MPO activities were significantly decreased (all <0.05). The protein expression of TRPM2 and RAC1 in liver tissues in the T group were significantly lower than those in the M and C groups at 2, 4 and 8 h after reperfusion (all <0.05). CONCLUSIONS Pretreatment with TRPM2 adenovirus interference vector can effectively silence TRPM2 gene expression in liver tissues of mice and attenuate HIRI, which may be related to inhibiting oxidative stress and reducing the expression of RAC1 protein.
Alanine Transaminase ; Animals ; Aspartate Aminotransferases ; Liver ; Male ; Mice ; Mice, Inbred C57BL ; Neuropeptides ; Rats, Sprague-Dawley ; Reperfusion Injury ; TRPM Cation Channels ; genetics ; Transient Receptor Potential Channels ; rac1 GTP-Binding Protein

OBJECTIVE: To develop methods of extraction and purification of Cterminal NUDT9 homology domain of human transient receptor potential melastatin 2 (TRPM2) channel. METHODS: After sonication and centrifuge of strain Rosetta (DE3) which was induced by isopropylthio-β-D-galactoside, GST-NUDT9-H was collected after the binding of supernatant with GST beads and eluted with reduced glutathione. Then the elution buffer containing fusion protein was purified by size exclusion chromatography after concentration and centrifuge. Finally, with the cleavage of thrombin and binding with the GST beads, NUDT9-H with high purity in supernatant was collected. RESULTS: The GST-NUDT9-H fusion protein was stabilized with lysis buffer containing 0.5% n-dodecyl -β-d-maltoside (DDM), and wash buffer containing 0.025% DDM in size-exclusion chromatography system, and finally the NUDT9-H with high purity was obtained after cleaved by thrombin (1 U/2 mg fusion protein) for 24 h. CONCLUSIONS Due to the poor stability of NUDT9-H, it is necessary to add DDM in extraction and purification buffer to stabilize the conformation of NUDT9-H, so as to increase its yields and purity.
Escherichia coli ; genetics ; Glucosides ; chemistry ; Humans ; Protein Domains ; Protein Stability ; Pyrophosphatases ; chemistry ; genetics ; isolation & purification ; Recombinant Fusion Proteins ; chemistry ; isolation & purification ; TRPM Cation Channels ; chemistry ; isolation & purification ; Thrombin ; metabolism

OBJECTIVE: To explore the clinical features and mutations of the TRPM6 gene in an infant featuring hypomagnesemia and secondary hypocalcemia. METHODS: Clinical data of the patient was collected. Genomic DNA was extracted from peripheral blood samples from the patient and her parents. Targeted exome sequencing was carried out to screen the potential mutations. Suspected mutations were verified by Sanger sequencing. RESULTS: A novel homozygous c.5538delA (p.Q1846Qfs*2) mutation in the TRPM6 gene was identified in the proband, for which both of her parents were heterozygous carriers. CONCLUSION The homozygous frameshift mutation of TRPM6 gene (c.5538delA) probably underlies the disease in the proband. The finding has expanded the mutation spectrum of TRPM6 gene.
DNA Mutational Analysis ; Female ; Frameshift Mutation ; Humans ; Hypocalcemia ; genetics ; Infant ; Magnesium Deficiency ; congenital ; genetics ; TRPM Cation Channels ; genetics

OBJECTIVE: To explore the molecular pathogenesis for a pedigree affected with hypocalcemia secondary to hypomagnesemia. METHODS: Sanger sequencing was used to detect potential variant of the TRPM6 gene in the patient and their parents. RESULTS: The results showed that the patient has carried novel homozygous c.3311C>T (p.Pro1104Leu) variant of the TRMP6 gene, for which both of his parents were heterozygous carriers. Analysis of protein functions using software predicted high risk of pathogenicity. CONCLUSION The homozygous c.3311C>T (p.Pro1104Leu) variant of the TRPM6 gene probably underlies the disease in this patient.
Heterozygote ; Humans ; Hypocalcemia ; genetics ; Magnesium ; Magnesium Deficiency ; genetics ; Male ; Pedigree ; TRPM Cation Channels ; genetics

To investigate the association between single nucleotide polymorphism (SNP) in the 11th exon of transient receptor potential melastatin 2 (TRPM2) gene with the susceptibility and outcome of sepsis.A total of 119 septic patients and 112 normal subjects were enrolled from the First Affiliated Hospital, Zhejiang University School of Medicine. Among 119 septic patients, 62 died (fatal group) and 57 survived (survival group) within 28 days of disease onset. The genotypes of these individuals were detected using TaqMan allelic discrimination assays, and its correlations with susceptibility and outcome of sepsis were analyzed.There was no significant difference in genotype frequencies and allelic frequencies of TRPM2 SNP rs1556314 between septic patients and the controls (all>0.05). And no significant difference in genotype frequencies and allelic frequencies of TRPM2 SNP rs1556314 was observed between the survivors and fatal cases of septic patients (all>0.05).The TRPM2 SNP rs1556314 does not have significant association with sepsis, but this result need to be confirmed by large scale studies.
Exons ; genetics ; physiology ; Female ; Gene Frequency ; Genetic Predisposition to Disease ; genetics ; Genotype ; Humans ; Male ; Polymorphism, Single Nucleotide ; genetics ; physiology ; Sepsis ; genetics ; mortality ; TRPM Cation Channels ; genetics

OBJECTIVETo compare the intervention effects of four traditional Chinese medicines (TCMs) with typical cold or hot property on body temperature and temperature-sensitive transient receptor potential ion channel proteins (TRPs) of rats with yeast-induced fever.

METHODThe pyrexia model was induced by injecting yeast suspension subcutaneously. Totally 108 male SD rats were randomly divided into the normal group, the model group, the Rhei Radix et Rhizoma treated group, the Coptidis Rhizoma treated group, the Euodiae Fructus treated group, and the Alpiniae Officinarum Rhizoma treated group, with 18 rats in each group. At the 4 h, 8 h and 12 h after injection of yeast, the rats were sacrificed to collect their hypothalamus and dorsal root ganglion. The expressions of TRPV1 and TRPM8 were detected by immunohistochemistry and Western blot method.

RESULTCompared with the normal group, after injection of yeast, the temperature of rats in the model group notably increased, and reached the peak at 8 h (P < 0.01). The TRPV1 level in hypothalamus and dorsal root ganglia (DRG) of the model group significantly increased, whereas the TRPM8 level significantly reduced. Compared with the model group, the Rhei Radix et Rhizoma group and the Coptidis Rhizoma group showed significant decrease in the high body temperature of rats caused by yeast, down-regulation in the expression of TRPV1, and up-regulation in the expression of TRPM8 (P < 0.05 or P < 0.01). Euodiae Fructus and Alpiniae Officinarum Rhizoma had no significant effect on either temperature or TRPs of fever rats.

CONCLUSIONRhei Radix et Rhizoma and Coptidis Rhizoma, both are TCMs with cold property, can reduce the temperature of fever rats induced by yeast, which may be related to their effective regulation of TRPV1 and TRPM8 in hypothalamus and DRG, while Euodiae Fructus and Alpiniae Officinarum Rhizoma had no relevant effect.

Animals ; Antipyretics ; administration & dosage ; chemistry ; Body Temperature Regulation ; drug effects ; Drugs, Chinese Herbal ; administration & dosage ; chemistry ; Fever ; drug therapy ; immunology ; microbiology ; physiopathology ; Gene Expression Regulation ; drug effects ; Humans ; Male ; Rats ; Rats, Sprague-Dawley ; Saccharomyces cerevisiae ; immunology ; TRPM Cation Channels ; genetics ; immunology ; TRPV Cation Channels ; genetics ; immunology

OBJECTIVE: To construct phosphorylation sites domain (PSD) mutant of myristoylated alaninerich C kinase substrate (MARCKS) and explore the role of transient receptor potential melastatin 8 cation channels (TRPM8) and MARCKS in cold-induced synthesis and exocytosis of mucin (MUC) 5AC. METHODS: Human placental cDNA was used as a template to amplify the full coding region of MARCKS cDNA by PCR. Ser159, Ser 163, Ser 167, Ser 170 in the PSD were mutated to aspartic acids by an overlap PCR method. The resultant PSD mutant cDNA and the wild-type MARCKS cDNA were each subcloned into a mammalian expression vector pcDNA3.0. Recombinant constructs were confirmed by restriction enzyme digestion analysis and DNA sequencing. In intervention experiments, cells were pretreated with the TRPM8 channel antagonist BCTC and transfected with MARCKS-PSD mutant cDNA, and thereafter cold stimulation was applied. The levels of MUC5AC were measured by immunofluorescence and ELISA to clarify the roles of TRPM8 and PSD mutant on the synthesis and secretion of MUC5AC induced by cold, respectively. RESULTS: Restriction enzyme digestion analysis and DNA sequencing revealed that the pcDNA3.0- MARCKS and pcDNA3.0-MARCKS-PSD mutants were successfully constructed. The levels of intracellular and secreted MUC5AC of cold treated group were significantly higher than those of control group (P<0.05). BCTC attenuated the cold-induced synthesis and secretion of MUC5AC when compared with cold treated group (P<0.05). Transfection of 16HBE cells with the MARCKS-PSD mutant cDNA resulted in significant inhibition of mucin secretion in response to cold, and significantly higher level of intracellular MUC5AC than that of control group (P<0.01), whereas transfection with the vector DNA or the wild-type MARCKS cDNA had no effect on the mucin synthesis and secretion in response to cold (P>0.05). CONCLUSION TRPM8 and phosphorylation of MARCKS-PSD mediates the cold-induced exocytosis of MUC5AC by airway epithelial cells.
Base Sequence ; Cell Line ; Cold Temperature ; Epithelial Cells ; cytology ; metabolism ; Exocytosis ; physiology ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Membrane Proteins ; genetics ; metabolism ; Molecular Sequence Data ; Mucin 5AC ; metabolism ; Mutation ; Myristoylated Alanine-Rich C Kinase Substrate ; Phosphorylation ; TRPM Cation Channels ; metabolism ; Trachea ; cytology ; metabolism

OBJECTIVETo study the effects of the ingredients from Chinese herbs with the nature of cold or hot on the expression of TRPV1 and TRPM8.

METHODThe effects of ingredients from herbs on primary culture DRG neurons are observed in vitro. The expression quantity of gene is detected by the method of real time PCR. the 2 (-deltadeltaCT) method is applied to analyze the data.

RESULTIngredients from herbs with the nature of cold up-regulate the expression level of TRPV1 and down-regulate that of TRPM8, especially under the temperature condition of 39 degrees C; while ingredients from herbs with the nature of hot up-regulate the expression level of TRPM8 and down-regulated that of TRPV1, which is more significant under the temperature condition of 19 degrees C.

CONCLUSIONThe regulatory changes of TRPV1 and TRPM8 mRNA expression induced by the chemical ingredients might be related to the cold and hot natures of the herbs from which the ingredients are extracted. And this could be one of the therapeutic mechanisms for the treatment of Chinese herbal medicines to cold- and heat-related diseases.

Animals ; Drugs, Chinese Herbal ; administration & dosage ; analysis ; Gene Expression ; drug effects ; Male ; Rats ; Rats, Sprague-Dawley ; TRPM Cation Channels ; genetics ; metabolism ; TRPV Cation Channels ; genetics ; metabolism