Dynamic Expression of Hyperpolarization-activated Cyclic Nucleotide-gated Cation Channel 4 Involved in Microwave Induced Pacemaker Cell Injuries

To investigate the mechanisms of microwave induced pacemaker cell injuries, Wistar rats and the primary pacemaker cells of newborn Wistar rats were exposed to microwave at average power density of 50 mW/cm2. Slower spontaneous beating rate, intercellular Ca2＋ aggregation and cell membrane perforation were detected immediately after the exposure. Moreover, hyperpolarizationactivated cyclic nucleotide-gated cation channel 4 （HCN4） was down-regulated immediately after the exposure and up-regulated at 12 h after the exposure. In the sinoatrial node （SAN） of the rats,

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.

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.

TRPA1 channel mediates organophosphate-induced delayed neuropathy

OBJECTIVE We want to investigate the mechanism of organophosphate-induced delayed neuropathy(OPIDN) and find appropriate therapeutic medicine.OPIDN,often leads to paresthesias,ataxia and paralysis,occurs in the late-stage of acute poisoning or after repeated exposures to organophosphate(OP) insecticides or nerve agents,and may contribute to the Gulf War Syndrome.METHODS FDSS Ca2^(+)-influx assays,single-cell calcium imaging and patch-clamp electrophysiology were the major testing techniques.Transfected HEK293 cells and dorsal root ganglion(DRG) neurons were used to evaluate the effects of compounds.Wild type and trpa1 knockout mice and adult hyline brown hens were used to evaluate the neuropathological damages caused by the OPs.Transmission electron microscopy imaging was used to observe the nerve injuries ultrastructurally.High-throughput screen for TRPA1 inhibitors was accomplished by Ion Works Barracuda(IWB) automated electrophysiology assay.RESULTS TRPA1(Transient receptor potential cation channel,member A1) channel mediates OPIDN.A variety of OPs,exemplified by malathion,activates TRPA1 but not other neuronal TRP channels.Malathion increases the intracellular calcium levels and upregulates the excitability of mouse DRG neurons in vitro.Mice with repeated exposures to malathion also develop local tissue nerve injuries and pain-related behaviors,which resembles the early symptoms of OPIDN.Both the neuropathological changes and the nocifensive behaviors can be attenuated by treatment of TRPA1 antagonist HC030031 or abolished by knockout of Trpa1 gene.In the classic hens OPIDN model,malathion causes nerve injuries and ataxia to a similar level as the positive inducer tri-ortho-cresyl phosphate(TOCP),which also activates TRPA1 channel.Treatment with HC030031 reduces the damages caused by malathion or TOCP.Duloxetine and Ketotifen,two commercially available drugs exhibiting TRPA1 inhibitory activity,show neuroprotective effects against OPIDN and might be used in emergency situations.CONCLUSION TRPA1 is the major mediator of OPIDN and targeting TRPA1 is an effective way for the treatment of OPIDN.

MLKL forms cation channels

The mixed lineage kinase domain-like(MLKL)protein is a key factor in tumor necrosis factor-induced necroptosis.Recent studies on necroptosis execution revealed a commitment role of MLKL in membrane disruption.However,our knowledge of how MLKL functions on membrane remains very limited.Here we demonstrate that MLKL forms cation channels that are permeable preferential y to Mg2+rather than Ca2+in the presence of Na+and K+.Moreover,the N-terminal domain containing six helices(H1-H6)is sufficient to form channels.Using the substituted cysteine accessibility method,we further determine that helix H1,H2,H3,H5 and H6 are transmembrane segments,while H4 is located in the cytoplasm.Finally,MLKL-induced membrane depolarization and cell death exhibit a positive correlation to its channel activity.The Mg2+-preferred permeability and five transmembrane segment topology distinguish MLKL from previously identified Mg2+-permeable channels and thus establish MLKL as a novel class of cation channels.

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.

MLKL forms cation channels

OBJECTIVE To investigate how MLKL functions on the membrane and explore its electrophysiological characters and structure.METHODS The full-length human MLKL were expressed in SF21 cells and purified using glutathione-sepharose affinity chromatography.The currents of purified MLKL proteins were recorded in avoltage-clamp mode using a Warner BC-535 bilayer clamp amplifier.The currents were digitized using p CLAMP 10.2 software.HEK293 cells were cultured and transfected with MLKL plasmid.Cell viability was examined using the Cell Titer-Glo Luminescent Cell Viability Assay kit.RESULT MLKL forms cation channels that are permeable preferentially to Mg2+rather than Ca2+in the presence of Na+and K+.Moreover,each MLKL monomer contains five transmembrane helices:H1,H2,H3,H5 and H6 of the N-terminal domain which is sufficient to form channels.Finally,MLKL-induced membrane depolarization and cell death exhibit a positive correlation to its channel activity.

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.

The expression and significance of CATSPER1 in human testis and ejaculated spermatozoa

Aim： To investigate the distribution of cation channel of sperm 1 （CATSPER1） protein and the presence of CATSPER1 mRNA in human testis and ejaculated spermatozoa. The influence of anti-human CATSPER1 antibody upon human sperm motility was used to evaluate the function of human CATSPER1 and to estimate its possible use as a target for immunocontraception. Methods： Human ejaculated sperm from normozoospermic donors （n = 12） and liquid nitrogen frozen human testis were used for the study of mRNA and protein expression of CATSPER1 by reverse transcription polymerase chain reaction （RT-PCR） and immunohistochemistry, respectively. Spermatozoa from normozoospermic donors （n = 12） were individually processed using a swim-up procedure and were then incubated with CATSPER1 antibody at final concentrations of 20, 4 and 0.8 μg/mL. After 1, 2 and 6 h incubation, progressive motility and fast progressive motility were measured by means of computer-assisted semen analysis. Results： CATSPER1 transcript was detected in both human testis and each human ejaculated semen sample. CATSPER1 protein expressed in the membrane of spermatid and was localized in the principal piece of the sperm tail. The application of CATSPER1 antibody at all concentrations significantly inhibited both progressive motility and fast progressive motility after 1, 2 and 6 h incubation, and significant dose-dependent changes were observed. Conclusion： CATSPER1 is meiotically and post-meiotically expressed in human testis tissue. CATSPER1 mRNA in human ejaculated spermatozoa could be a more feasible target for study and infertility screening than testis biopsy. In addition, our results suggest that human CATSPER1 could be a possible target for immunocontraception.

Membrane-Potential Alteration During K+ Uptake of Different Salt-Tolerant Wheat Varieties

K＋ is the most abundant cation in plant cells and plays an important role in many ways.K＋ uptake of plant has respect to its salt resistant capacity.There are two categories of channel transportation for plants to uptake K＋,one is through K＋ channels and the other is through nonselective cation channels（NSCCs）.The transmembrane localization of K＋ may change membrane potential（MP）.In this paper,three wheat varieties with different salt tolerance were selected and the MP was measured by microelectrode during K＋ uptake.The results showed that the effects of K＋ uptake on MP through K＋ channels or NSCCs were distinct.K＋ influx through K＋ channels led to MP hyperpolarization,while K＋ influx through NSCCs resulted in depolarization.Diverse MP alteration of wheat varieties with different salt tolerance was mainly due to NSCCs-mediated K＋ uptake.Compared with the salt-tolerant wheat,the MP hyperpolarization during K＋ uptake of saltsensitive wheat was much more evident,probably because of the cation outflux through NSCCs during this process.

Effects of different flow velocities on behavior and TRPA1 expression in the sea cucumber Apostichopus japonicas

A water flow simulation device capable of adjusting flow velocity was designed in flow velocity range of 0–30 cm/s,with which an indoor experiment was conducted to simulate the movement and adhesive behaviors of different-sized Apostichopus japonicus under different flow velocities.Observation showed that,in slow flow(~5 cm/s),A.japonicus moved more distance than in still water,and hardly moved in the riptide(~30 cm/s);and the adhesive capacity of A.japonicus was related to the flow velocity and attachment time.A.japonicus were able to attach to the bottom after any attachment time in the slow flow,after 10 s in the medium flow(~15 cm/s),and after 60 s in the riptide(~30 cm/s).In addition,larger A.japonicus were stronger with adhesive ability than smaller ones.The transcriptome data showed that the expression of transient receptor potential cation channel subfamily A,member 1(TRPA1)in the tube feet was increased significantly in a flowing water,but those in the tentacles and tube feet were not significantly changed.Fluorescence in-situ hybridization results showed that TRPA1 was expressed around the watervascular of tentacles,tube feet,body wall,and spines.Therefore,tube feet were important for sea cucumbers to keep themselves stable in relatively swift flow with adhesion ability.

Polymorphisms of AZIN1 rs2679757 and TRPM5 rs886277 are Associated with Cirrhosis Risk in Chinese Patients with Chronic Hepatitis B

Objective Genome-wide association studies(GWAS)have linked many single nucleotide polymorphisms(SNPs)to the outcomes of a variety of liver diseases.The aim of the present study was to evaluate the association of several candidate SNPs with the risk and severity of cirrhosis due to chronic hepatitis B in a Chinese population.Methods A total of 714 Chinese participants with persistent HBV infection were studied.Patients were divided into cirrhotic(n=429)and non-cirrhotic(n=285)groups based on clinical and pathological evidence.The progression rate and severity of liver cirrhosis were evaluated with an arbitrary t-score system.Genotypes of six SNPs in five candidate genes were detected with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS).The genotypic distributions of the SNPs were compared between the age-matched cirrhotic and non-cirrhotic subjects.The association between the risk of SNPs and the severity and progression rate of cirrhosis was further analyzed.Results Rs2679757 polymorphism of the antizyme inhibitor 1(AZIN1)gene and Rs886277 in the transient receptor potential cation channel subfamily M,member 5 gene(TRPM5)were found to be associated with cirrhosis risk in CHB.They were also correlated with the overall severity and progression rate of cirrhosis.Genotype frequencies of other SNPs were not different between the cirrhosis and non-cirrhosis groups.Conclusions AZIN1 rs2679757 and TRPM5 rs886277 are associated with the risk and the progression rate of HBV-related liver fibrosis in Chinese patients.The emerging SNPs associated with cirrhosis prognosis warrant further clinical validation in other CHB cohorts or ethnic groups,and merit mechanistic studies to reveal their roles in fibrosis progression.

Neuronal Fc gamma receptor I as a novel mediator for IgG immune complex-induced peripheral sensitization

Chronic pain often accompanies immune-related diseases with an elevated level of IgG immune complex （IgG-IC） in the serum and/or the affected tissues though the underlying mechanisms are largely unknown. Fc gamma receptors （FcyRs）, known as the receptors for the Fc domain of immunoglobulin G （IgG）, are typically expressed on immune cells. A general consensus is that the activation of FcyRs by IgG-IC in such immune cells induces the release of proinflammatory cytokines from the immune cells, which may contribute to the IgG-IC-mediated peripheral sensitization. In addition to the immune cells, recent studies have revealed that FcyRI, but not FcyRII and FcyRIII, is also expressed in a subpopulation of primary sensory neurons. Moreover, IgG-IC directly excites the primary sensory neurons through neuronal FcyRI. These findings indicate that neuronal FcyRI provides a novel direct linkage between immunoglobulin and primary sensory neurons, which may be a novel target for the treatment of pain in the immune-related disorders. In this review, we summarize the expression pattern, functions, and the associated cellular signaling of FcyRs in the primary sensory neurons.

Effects of Shensong Yangxin capsule on pacemaker channels encoded by human HCN4 gene

Shensong Yangxin （SSYX） is one of the compound recipes of Chinese materia medica including 12ingredients such as Panax ginseng, dwarf lilyturf tuber,nardostachys root, etc. Small-scale randomized multi-centre clinical trials suggested that SSYX reduced the number of ventricular extrasystoles in patients with or without structural heart disease.1 Besides excellent antiarrhythmic efficacy,2 SSYX also improved bradycardia in some patients, which was evidenced by animal studies3 as well.

Wenyang Huazhuo Fang exerts transient receptor potential cation channel subfamily C member-dependent nephroprotection in a rat model of doxorubicin-induced nephropathy

OBJECTIVE:To determine the effect of Wenyang Huazhuo Fang(WHF),a Traditional Chinese Medicine decoction,on renal function in a rat model of doxorubicin-induced nephropathy,and to elucidate the underlying mechanism.METHODS:Sprague-Dawley rats were randomly divided into six groups:control,doxorubicin-nephropathy,and prednisone-treated(6.45 mg·kg^-1·d^-1)doxorubicin nephropathy groups,as well as high-(7.26 g·kg^-1·d^-1),medium-(2.42 g·kg-1·d-),and low-dose(0.81 g·kg^-1·d^-1)WHF-treated doxorubicin-nephropathy groups.The nephropathy rat model was established by two tail vein injections of doxorubicin,followed by prednisone or WHF treatment for 8 weeks.Body weights were monitored and urinary protein was measured every 2 weeks.After the end of the treatment period,the rats were euthanized.Serum biochemical indicators were determined and renal morphological alterations were assessed using histological staining.The expression of transient receptor potential cation channel subfamily C member 6(TRPC6),stromal interaction molecule 1(STIM1),and calcium release-activated calcium channel protein 1(Orai1)was detected using western blotting,and their mRNA levels were examined using quantitative real-time reverse transcription-polymerase chain reaction.RESULTS:WHF treatment was found to significantly ameliorate weight loss,proteinuria,hypoalbuminemia,and dyslipidemia in doxorubicin-nephropathy rats.The protein and mRNA levels of TRPC6,STIM1,and Orai1 were partially,but significantly suppressed by prednisone or WHF treatment.CONCLUSION:Treatment with WHF significantly ameliorates renal injury in a rat model of doxorubicin-induced nephropathy,which could be at least partially related to repression of the TRPC6 pathway.

Heteromerization of TRP channel subunits: extending functional diversity

Transient receptor potential(TRP)channels are widely found throughout the animal kingdom.By serving as cellular sensors for a wide spectrum of physical and chemical stimuli,they play crucial physiological roles ranging from sensory transduction to cell cycle modulation.TRP channels are tetrameric protein complexes.While most TRP subunits can form functional homomeric channels,heteromerization of TRP channel subunits of either the same subfamily or different subfamilies has been widely observed.Heteromeric TRP channels exhibit many novel properties compared to their homomeric counterparts,indicating that co-assembly of TRP channel subunits has an important contribution to the diversity of TRP channel functions.

Overexpression of connexin 45 in rat mesenchymal stem cells improves the function as cardiac biological pacemakers

Background Extensive research toward creating a biological pacemaker by enhancement of inward depolarizing current has been performed. However, studies have mainly focused on inducing spontaneous activity and have not adequately addressed ways to improve pacemaker function. In this study we attempted to improve pacemaker function by altering connexin expression in rat mesenchymal stem cells （MSCs） to a phenotype similar to native sinus node pacemaker cells. Methods To generate a biological pacemaker, MSCs were transduced with a cardiac pacemaker gene- hyperpolarization-activated cyclic nucleotide-gated channel 4 （HCN4）, via transfection with a lentiviral vector. Funny current （If） in HCN4~ MSCs was recorded by voltage-clamp. Overexpression of connexin 45 （gene Gja7） in MSCs was achieved by transfection with the plasmid pDsRED2-N1-Gja7-RFP. Double-immunolabelling with anti-connexin 43 and anti-connexin 45 antibodies were used to identify the gap junction channels. The effects of the genetically modified MSCs on cardiomyocyte excitability were determined in MSCs cocultured with neonatal rat ventricular myocytes. Spontaneous action potentials of neonatal rat ventricular myocytes were recorded by current-clamp. Results High level time- and voltage-dependent inward hyperpolarization current that was sensitive to 4 mmol/L Cs＋ was detected in HCN4＋ MSCs, confirming that HCN4 acted as Ir channels in MSCs. Connexin 43 and connexin 45 were simultaneously detected in CX45＋ MSCs. Beating frequency was （82±8） beats per minute （n=-5） in myocytes cocultured with non-transfected control MSCs, versus （129±11） beats per minute （n=-5） in myocytes cocultured with HCN4＋ MSCs. Myocytes cocultured with MSCs cotransfected with HCN4 and connexin 45 had the highest beating frequency at （147±9） beats per minute （n=5）. Conclusion These findings demonstrate that overexpression of connexin 45 and subsequent formation of heteromeric connexin 45/connexin 43 gap junction channels in HCN4 expressing MSCs can improve their function as cardiac biological pacemakers in vitro.

Canonical transient receptor potential 4 and its small molecule modulators

Canonical transient receptor potential 4(TRPC4) forms non-selective cation channels that contribute to phospholipase C-dependent Ca2+ entry into cells following stimulation of G protein coupled receptors and receptor tyrosine kinases.Moreover,the channels are regulated by pertussis toxin-sensitive Gi/o proteins,lipids,and various other signaling mechanisms.TRPC4-containing channels participate in the regulation of a variety of physiological functions,including excitability of both gastrointestinal smooth muscles and brain neurons.This review is to present recent advances in the understanding of physiology and development of small molecular modulators of TRPC4 channels.

Yanghe decoction(阳和汤)attenuated pain hypersensitivity induced by michigan cancer foundation-7 injection in rats with bone metastases from breast cancer by inhibiting transient receptor potential ankyrin 1

OBJECTIVE:To study the effect and underlying mechanisms of Chinese medicine Yanghe decoction(阳和汤)on pain relief in a rat model of bone metastasis of breast cancer induced by michigan cancer foundation-7(MCF-7).METHODS:Bone pain was induced in the tibia of rats injected with MCF-7 cells.The Chinese herbal remedy was used to decoct Yanghe decoction for the treatment of bone pain rats.The behavior study was carried out to evaluate the paw mechanical withdraw threshold and thermal withdraw latency.Liquid chromatography-mass spectrometry,Western blotting,quantitative real-time polymerase chain reaction,enzyme-linked immunosorbent assay(ELISA),immunohistochemical(IHC)staining were performed for analysis.RESULTS:Yanghe decoction could improve the defensive behavior similar to the transient receptor potential ankyrin 1(TRPA1)inhibitor.In morphology study,Yanghe decoction could attenuate the cellular growth as well as inflammatory infiltration in the metastasis group.Furthermore,Yanghe decoction downregulated the TRPA1 expression on the dorsal root ganglion from the metastatic rats at both transcriptional and protein level.Yanghe decoction alleviated the inflammation in metastatic tissues by hematoxylin-eosin and IHC analysis,and Yanghe decoction also reduced the inflammatory cytokines production in the serum including tumor necrosis factor-αand interleukin-6,interleukin-1 beta by ELISA.As the cytochromec oxidase subunit II/prostaglandin E2(PGE2)is required for cancer development,Yanghe decoction reduced the expression of PGE2 in the tissue and serum.CONCLUSION:Taken together,Yanghe decoction protected the rats from breast cancer bone metastasis through TRPA1 signaling mediated neuropathic pain and additional immune modulation in tumor microenvironment.