Objective: To examine the effects of Tribulus terrestris L.(T. terrestris) extract on the modulation of calcium channels to evaluate its use in topical agents for treatment of atopic dermatitis.Methods: The 70% methan...Objective: To examine the effects of Tribulus terrestris L.(T. terrestris) extract on the modulation of calcium channels to evaluate its use in topical agents for treatment of atopic dermatitis.Methods: The 70% methanol extract of T. terrestris was prepared. Human HEK293 T cells with over-expressed calcium release-activated calcium channel protein 1(Orai1),transient receptor potential vanilloid 1, or transient receptor potential vanilloid 3(TRPV3)were treated with T. terrestris extract. Modulation of ion channels was measured using a conventional whole-cell patch-clamp technique.Results: T. terrestris extract(100 mg/m L) significantly inhibited Orai1 activity in Orai1-stromal interaction molecule 1 co-overexpressed HEK293 T cells. In addition, T. terrestris extract significantly increased the TRPV3 activity compared with 2-Aminoethyl diphenylborinate(100 mmol/L), which induces the full activation of TRPV3.Conclusions: Our results suggest that T. terrestris extract may have a therapeutic potential for recovery of abnormal skin barrier pathologies in atopic dermatitis through modulating the activities of calcium ion channels, Orai1 and TRPV3. This is the first study to report the modulatory effect of a medicinal plant on the function of ion channels in skin barrier.展开更多
To develop a cell-based high-throughput calcium fluorescent assay for screening of TRPV3 channel modulators, we employed the FlexStation3 microplate reader and optimized conditions of the high-throughput screening （...To develop a cell-based high-throughput calcium fluorescent assay for screening of TRPV3 channel modulators, we employed the FlexStation3 microplate reader and optimized conditions of the high-throughput screening （HTS） adaptable calcium fluorescent assay, such as cell density, incubation time and concentration of Cal-520 calcium fluorescent dye. The optimized FlexStation3 assay included the cell density of 40 000 cells per well, 5.0% of Cal-520 calcium fluorescent dye and 1.5 h of incubation time. Using the FlexStation3 assay in a 96-well format, we screened and identified a natural neferine from Nymphaeaceae plant that inhibited TRPV3 channel. To further confirm the inhibitory effect of neferine on TRPV3, we utilized the whole-cell patch clamp recordings and determined the dose-dependent inhibition of TRPV3 current by neferine with an ICs0 at 24.5~4.1 p.M （n = 6） without inhibition of TRPV1 or TRPV4 channels. Taken together, our data showed that this validated HTS adaptable calcium fluorescent assay in FlexStation3 format could be used for screening and identification of modulators for either TRPV3 channel or other calcium permeable TRP channels. The identified natural neferine could be used as a tool for pharmacological investigations of TRPV3 channel function.展开更多
Mitochondria play an important role in pressure overload-induced cardiac hypertrophy.The present study aimed to investigate the role of mitochondrial transient receptor potential vanilloid 3(TRPV3)in myocardial hypert...Mitochondria play an important role in pressure overload-induced cardiac hypertrophy.The present study aimed to investigate the role of mitochondrial transient receptor potential vanilloid 3(TRPV3)in myocardial hypertrophy.A 0.7 mm diameter U-shaped silver clip was used to clamp the abdominal aorta of Sprague Dawley(SD)rats and establish an animal model of abdominal aortic constriction(AAC).Rat H9C2 myocardial cells were treated with angiotensin II(Ang II)to establish a hypertrophic myocardial cell model,and TRPV3 expression was knocked down using TRPV3 small interfering RNA(siRNA).JC-1 probe was used to detect mitochondrial membrane potential(MMP).DHE probe was used to detect ROS generation.Enzyme activities of mitochondrial respiratory chain complex I and III and ATP production were detected by assay kits.Immunofluorescence staining was used to detect TRPV3 expression in H9C2 cells.Western blot was used to detect the protein expression levels ofβ-myosin heavy chain(β-MHC),mitochondrial TRPV3 and mitochondrial NOX4.The results showed that,in the rat AAC model heart tissue and H9C2 cells treated with Ang II,the protein expression levels ofβ-MHC,mitochondrial TRPV3 and mitochondrial NOX4 were up-regulated,MMP was decreased,ROS generation was increased,mitochondrial respiratory chain complex I and III enzyme activities were decreased,and ATP production was reduced.After knocking down mitochondrial TRPV3 in H9C2 cells,the protein expression levels ofβ-MHC and mitochondrial NOX4 were down-regulated,MMP was increased,ROS generation was decreased,mitochondrial respiratory chain complex I and III enzyme activities were increased,and ATP production was increased.These results suggest that mitochondrial TRPV3 in cardiomyocytes exacerbates mitochondrial dysfunction by up-regulating NOX4,thereby participating in the process of pressure overload-induced myocardial hypertrophy.展开更多
Genetic gain-of-function mutations of warm temperature-sensitive transient receptor potential vanilloid 3(TRPV3)channel cause Olmsted syndrome characterized by severe itching and keratoderma,indicating that pharmacolo...Genetic gain-of-function mutations of warm temperature-sensitive transient receptor potential vanilloid 3(TRPV3)channel cause Olmsted syndrome characterized by severe itching and keratoderma,indicating that pharmacological inhibition of TRPV3 may hold promise for therapy of chronic pruritus and skin diseases.However,currently available TRPV3 tool inhibitors are either nonselective or less potent,thus impeding the validation of TRPV3 as therapeutic target.Using whole-cell patch-clamp and single-channel recordings,we report the identification of two natural dicaffeoylquinic acid isomers isochlorogenic acid A(IAA)and isochlorogenic acid B(IAB)that selectively inhibit TRPV3 currents with IC50 values of 2.7±1.3 and 0.9±0.3μmol/L,respectively,and reduce the channel open probability to 3.7±1.2%and 3.2±1.1%from 26.9±5.5%,respectively.In vivo evaluation confirms that both IAA and IAB significantly reverse the ear swelling of dermatitis and chronic pruritus.Furthermore,the isomer IAB is able to rescue the keratinocyte death induced by TRPV3 agonist carvacrol.Molecular docking combined with site-directed mutations reveals two residues T636 and F666 critical for the binding of the two isomers.Taken together,our identification of isochlorogenic acids A and B that act as specific TRPV3 channel inhibitors and gating modifiers not only provides an essential pharmacological tool for further investigation of the channel pharmacology and pathology,but also holds developmental potential for treatment of dermatitis and chronic pruritus.展开更多
Objective： To observe the effects of different moxibustion times on proteins of transient receptor potential vanilloid 3 （TRPV3） ion channel protein and synovial cell apoptosis in rats with rheumatoid arthritis （R...Objective： To observe the effects of different moxibustion times on proteins of transient receptor potential vanilloid 3 （TRPV3） ion channel protein and synovial cell apoptosis in rats with rheumatoid arthritis （RA）, to provide a new basis for the anti-inflammatory mechanism of moxibustion. Methods： A total of 50 Sprague-Dawley （SD） rats were divided into a normal group, a model group, moxibustion group Ⅰ, moxibustion group Ⅱ and moxibustion group III by complete randomization, with 10 rats in each group. Rats in the normal group were bred routinely, and rats in the model group were also bred routinely after successful modeling. After successful modeling, rats in moxibustion group I, Ⅱ and Ⅲ accepted consecutive moxibustion at Zusanli （ST 36） and Shenshu （BL 23） for 15 d, once a day, respectively 5 min, 20 min and 30 min for each session. The degree of paw edema was observed and recorded. Immunohistochemical assay was used to detect the protein expression of TRPV3 ion channel in dorsal root ganglia and spinal cord dorsal horn. Terminal-deoxynucleoitidyl transferase-mediated nick end labeling （TUNEL） was used to detect apoptotic synovial cell number. Results： At the end of treatment, paw circumference of rats in moxibustion groupⅡ and Ill were significantly reduced as compared with that in the model group （P〈0.05）. TRPV3 ion channel protein expression of dorsal root ganglia and spinal cord dorsal horn was higher in the model group than that in the normal group （P〈0.05）; the TRPV3 ion channel protein expressions of dorsal root ganglia and spinal cord dorsal horn in moxibustion group [[ and []I were higher than that in moxibustion group Ⅰ （P〈0.05）; apoptotic synovial cell number in the model group was larger than that in the normal group （P〈0.05）, and apoptotic synovial cell numbers in moxibustion group Ⅱ and Ⅲ were significantly higher than that in the model group （P〈0.05）. Conclusion： Moxibustion of appropriate time could induce TRPV3 expression, and promote svnovial cell apoptosis.展开更多
基金Supported by the Convergence of Conventional Medicine and Traditional Koran Medicine R&D Program funded by the Ministry of Health&Welfare through the Korean Health Industry Development Institute(Grant No.HI15C0256)
文摘Objective: To examine the effects of Tribulus terrestris L.(T. terrestris) extract on the modulation of calcium channels to evaluate its use in topical agents for treatment of atopic dermatitis.Methods: The 70% methanol extract of T. terrestris was prepared. Human HEK293 T cells with over-expressed calcium release-activated calcium channel protein 1(Orai1),transient receptor potential vanilloid 1, or transient receptor potential vanilloid 3(TRPV3)were treated with T. terrestris extract. Modulation of ion channels was measured using a conventional whole-cell patch-clamp technique.Results: T. terrestris extract(100 mg/m L) significantly inhibited Orai1 activity in Orai1-stromal interaction molecule 1 co-overexpressed HEK293 T cells. In addition, T. terrestris extract significantly increased the TRPV3 activity compared with 2-Aminoethyl diphenylborinate(100 mmol/L), which induces the full activation of TRPV3.Conclusions: Our results suggest that T. terrestris extract may have a therapeutic potential for recovery of abnormal skin barrier pathologies in atopic dermatitis through modulating the activities of calcium ion channels, Orai1 and TRPV3. This is the first study to report the modulatory effect of a medicinal plant on the function of ion channels in skin barrier.
基金The National Natural Sciences Foundation of China(Grant No.81573410)the Ministry of Science and Technology of China(Grant No.2014ZX09507003-006-004)the Natural Sciences Foundation of Shandong Province(Grant No.ZR2015QL008)
文摘To develop a cell-based high-throughput calcium fluorescent assay for screening of TRPV3 channel modulators, we employed the FlexStation3 microplate reader and optimized conditions of the high-throughput screening （HTS） adaptable calcium fluorescent assay, such as cell density, incubation time and concentration of Cal-520 calcium fluorescent dye. The optimized FlexStation3 assay included the cell density of 40 000 cells per well, 5.0% of Cal-520 calcium fluorescent dye and 1.5 h of incubation time. Using the FlexStation3 assay in a 96-well format, we screened and identified a natural neferine from Nymphaeaceae plant that inhibited TRPV3 channel. To further confirm the inhibitory effect of neferine on TRPV3, we utilized the whole-cell patch clamp recordings and determined the dose-dependent inhibition of TRPV3 current by neferine with an ICs0 at 24.5~4.1 p.M （n = 6） without inhibition of TRPV1 or TRPV4 channels. Taken together, our data showed that this validated HTS adaptable calcium fluorescent assay in FlexStation3 format could be used for screening and identification of modulators for either TRPV3 channel or other calcium permeable TRP channels. The identified natural neferine could be used as a tool for pharmacological investigations of TRPV3 channel function.
基金supported by the National Natural Science Foundation of China (No. 30872716)the Natural Science Foundation of Hubei Province,China (No. 2015CFB288)Open Foundation of Hubei Province Key Laboratory of Tumor Microenvironment and immunotherapy (No. 2023KZL06)。
文摘Mitochondria play an important role in pressure overload-induced cardiac hypertrophy.The present study aimed to investigate the role of mitochondrial transient receptor potential vanilloid 3(TRPV3)in myocardial hypertrophy.A 0.7 mm diameter U-shaped silver clip was used to clamp the abdominal aorta of Sprague Dawley(SD)rats and establish an animal model of abdominal aortic constriction(AAC).Rat H9C2 myocardial cells were treated with angiotensin II(Ang II)to establish a hypertrophic myocardial cell model,and TRPV3 expression was knocked down using TRPV3 small interfering RNA(siRNA).JC-1 probe was used to detect mitochondrial membrane potential(MMP).DHE probe was used to detect ROS generation.Enzyme activities of mitochondrial respiratory chain complex I and III and ATP production were detected by assay kits.Immunofluorescence staining was used to detect TRPV3 expression in H9C2 cells.Western blot was used to detect the protein expression levels ofβ-myosin heavy chain(β-MHC),mitochondrial TRPV3 and mitochondrial NOX4.The results showed that,in the rat AAC model heart tissue and H9C2 cells treated with Ang II,the protein expression levels ofβ-MHC,mitochondrial TRPV3 and mitochondrial NOX4 were up-regulated,MMP was decreased,ROS generation was increased,mitochondrial respiratory chain complex I and III enzyme activities were decreased,and ATP production was reduced.After knocking down mitochondrial TRPV3 in H9C2 cells,the protein expression levels ofβ-MHC and mitochondrial NOX4 were down-regulated,MMP was increased,ROS generation was decreased,mitochondrial respiratory chain complex I and III enzyme activities were increased,and ATP production was increased.These results suggest that mitochondrial TRPV3 in cardiomyocytes exacerbates mitochondrial dysfunction by up-regulating NOX4,thereby participating in the process of pressure overload-induced myocardial hypertrophy.
基金supported by National Natural Science Foundation of China(81903734,81973299 and 81573410)the Ministry of Science and Technology of the People’s Republic of China(2018ZX09711001-004-006)。
文摘Genetic gain-of-function mutations of warm temperature-sensitive transient receptor potential vanilloid 3(TRPV3)channel cause Olmsted syndrome characterized by severe itching and keratoderma,indicating that pharmacological inhibition of TRPV3 may hold promise for therapy of chronic pruritus and skin diseases.However,currently available TRPV3 tool inhibitors are either nonselective or less potent,thus impeding the validation of TRPV3 as therapeutic target.Using whole-cell patch-clamp and single-channel recordings,we report the identification of two natural dicaffeoylquinic acid isomers isochlorogenic acid A(IAA)and isochlorogenic acid B(IAB)that selectively inhibit TRPV3 currents with IC50 values of 2.7±1.3 and 0.9±0.3μmol/L,respectively,and reduce the channel open probability to 3.7±1.2%and 3.2±1.1%from 26.9±5.5%,respectively.In vivo evaluation confirms that both IAA and IAB significantly reverse the ear swelling of dermatitis and chronic pruritus.Furthermore,the isomer IAB is able to rescue the keratinocyte death induced by TRPV3 agonist carvacrol.Molecular docking combined with site-directed mutations reveals two residues T636 and F666 critical for the binding of the two isomers.Taken together,our identification of isochlorogenic acids A and B that act as specific TRPV3 channel inhibitors and gating modifiers not only provides an essential pharmacological tool for further investigation of the channel pharmacology and pathology,but also holds developmental potential for treatment of dermatitis and chronic pruritus.
基金supported by 2014 Undergraduate Innovation and Entrepreneurship Training Program(No.2014041)National Basic Research Program of China 973 Program(No.2015CB554500)~~
文摘Objective： To observe the effects of different moxibustion times on proteins of transient receptor potential vanilloid 3 （TRPV3） ion channel protein and synovial cell apoptosis in rats with rheumatoid arthritis （RA）, to provide a new basis for the anti-inflammatory mechanism of moxibustion. Methods： A total of 50 Sprague-Dawley （SD） rats were divided into a normal group, a model group, moxibustion group Ⅰ, moxibustion group Ⅱ and moxibustion group III by complete randomization, with 10 rats in each group. Rats in the normal group were bred routinely, and rats in the model group were also bred routinely after successful modeling. After successful modeling, rats in moxibustion group I, Ⅱ and Ⅲ accepted consecutive moxibustion at Zusanli （ST 36） and Shenshu （BL 23） for 15 d, once a day, respectively 5 min, 20 min and 30 min for each session. The degree of paw edema was observed and recorded. Immunohistochemical assay was used to detect the protein expression of TRPV3 ion channel in dorsal root ganglia and spinal cord dorsal horn. Terminal-deoxynucleoitidyl transferase-mediated nick end labeling （TUNEL） was used to detect apoptotic synovial cell number. Results： At the end of treatment, paw circumference of rats in moxibustion groupⅡ and Ill were significantly reduced as compared with that in the model group （P〈0.05）. TRPV3 ion channel protein expression of dorsal root ganglia and spinal cord dorsal horn was higher in the model group than that in the normal group （P〈0.05）; the TRPV3 ion channel protein expressions of dorsal root ganglia and spinal cord dorsal horn in moxibustion group [[ and []I were higher than that in moxibustion group Ⅰ （P〈0.05）; apoptotic synovial cell number in the model group was larger than that in the normal group （P〈0.05）, and apoptotic synovial cell numbers in moxibustion group Ⅱ and Ⅲ were significantly higher than that in the model group （P〈0.05）. Conclusion： Moxibustion of appropriate time could induce TRPV3 expression, and promote svnovial cell apoptosis.
