Chemogenetics Modulation of Electroacupuncture Analgesia in Mice Spared Nerve Injury-Induced Neuropathic Pain through TRPV1 Signaling Pathway.

Neuropathic pain, which is initiated by a malfunction of the somatosensory cortex system, elicits inflammation and simultaneously activates glial cells that initiate neuroinflammation. Electroacupuncture (EA) has been shown to have therapeutic effects for neuropathic pain, although with uncertain mechanisms. We suggest that EA can reliably cure neuropathic disease through anti-inflammation and transient receptor potential V1 (TRPV1) signaling pathways from the peripheral to the central nervous system. To explore this, we used EA to treat the mice spared nerve injury (SNI) model and explore the underlying molecular mechanisms through novel chemogenetics techniques. Both mechanical and thermal pain were found in SNI mice at four weeks (mechanical: 3.23 ± 0.29 g; thermal: 4.9 ± 0.14 s). Mechanical hyperalgesia was partially attenuated by 2 Hz EA (mechanical: 4.05 ± 0.19 g), and thermal hyperalgesia was fully reduced (thermal: 6.22 ± 0.26 s) but not with sham EA (mechanical: 3.13 ± 0.23 g; thermal: 4.58 ± 0.37 s), suggesting EA's specificity. In addition, animals with Trpv1 deletion showed partial mechanical hyperalgesia and no significant induction of thermal hyperalgesia in neuropathic pain mice (mechanical: 4.43 ± 0.26 g; thermal: 6.24 ± 0.09 s). Moreover, we found increased levels of inflammatory factors such as interleukin-1 beta (IL1-ß), IL-3, IL-6, IL-12, IL-17, tumor necrosis factor alpha, and interferon gamma after SNI modeling, which decreased in the EA and Trpv1-/- groups rather than the sham group. Western blot and immunofluorescence analysis showed similar tendencies in the dorsal root ganglion, spinal cord dorsal horn, somatosensory cortex (SSC), and anterior cingulate cortex (ACC). In addition, a novel chemogenetics method was used to precisely inhibit SSC to ACC activity, which showed an analgesic effect through the TRPV1 pathway. In summary, our findings indicate a novel mechanism underlying neuropathic pain as a beneficial target for neuropathic pain.

Electroacupuncture ameliorates neuroinflammation by inhibiting TRPV4 channel in ischemic stroke.

AIMS: We investigated the potential mechanisms underlying the therapeutic efficacy of electroacupuncture (EA) at the Shuigou (GV26) and Baihui (GV20) acupoints in the treatment of ischemic stroke. METHODS: We assessed the therapeutic effects of EA on MCAO mice through behavioral studies and TTC staining. Various techniques, such as RT-PCR, immunofluorescence, and Western blots, were employed to evaluate the activation and polarization of microglia/macrophages, and changes in the TRPV4 ion channel. We used the TRPV4 antagonist GSK2193874 (GSK219) to verify the involvement of TRPV4 in the therapeutic effects of EA. RESULTS: EA effectively improved neurological impairments and reduced cerebral infarction volume in MCAO mice. It suppressed activated microglia/macrophages and inhibited their polarization toward the M1 phenotype post-MCAO. EA also downregulated the expression of pro-inflammatory cytokines, including Tnf-α, Il-6, Il-1ß, and Ccl-2 mRNA. Furthermore, EA reduced the elevated expression of TRPV4 following MCAO. Treatment with the TRPV4 antagonist GSK219 mirrored the effects of EA in MCAO mice. Notably, the combination of EA and GSK219 did not demonstrate an additive or synergistic effect. CONCLUSION: EA may inhibit neuroinflammation and exhibit a protective effect against ischemic brain injury by suppressing TRPV4 and the subsequent M1 polarization of microglia/macrophages.

Puerarin inhibits cisplatin-induced ototoxicity in mice through regulation of TRPV1-dependent calcium overload.

Puerarin (PUE), a flavonoid derivative with vasodilatory effects found in the traditional Chinese medicine kudzu, has anti-sensorineural hearing loss properties. However, the mechanism of its protective effect against ototoxicity is not well understood. In this study, we used in vitro and in vivo methods to investigate the protective mechanism of puerarin against cisplatin (CDDP)-induced ototoxicity. We established an ototoxicity model of CDDP in BALB/c mice and assessed the degree of hearing loss and cochlear cell damage. We used bioinformatics analysis, molecular docking, histological analysis, and biochemical and molecular biology to detect the expression of relevant factors. Our results show that puerarin improved CDDP-induced hearing loss and reduced hair cell loss. It also blocked CDDP-induced activation of TRPV1 and inhibited activation of IP3R1 to prevent intracellular calcium overload. Additionally, puerarin blocked CDDP-stimulated p65 activation, reduced excessive ROS production, and alleviated cochlear cell apoptosis. Our study provides new evidence and potential targets for the protective effect of puerarin against drug-induced hearing loss. Puerarin ameliorates cisplatin-induced ototoxicity and blocks cellular apoptosis by inhibiting CDDP activated TRPV1/IP3R1/p65 pathway, blocking induction of calcium overload and excessive ROS expression.

Borneol exerts its antipruritic effects by inhibiting TRPA1 and activating TRPM8.

ETHNOPHARMACOLOGICAL RELEVANCE: Borneol is a long-established traditional Chinese medicine that has been found to be effective in treating pain and itchy skin. However, whether borneol has a therapeutic effect on chronic itch and its related mechanisms remain unclear. AIM OF THE STUDY: To investigate the antipruritic effect of borneol and its molecular mechanism. MATERIALS AND METHODS: DrugBAN framework and molecular docking were applied to predict the targets of borneol, and the calcium imaging or patch-clamp recording analysis were used to detect the effects of borneol on TRPA1, TRPM8 or TRPV3 channels in HEK293T cells. In addition, various mouse models of acute itch and chronic itch were established to evaluate the antipruritic effects of borneol on C57BL/6J mice. Then, the borneol-induced pruritic relief was further investigated in Trpa1-/-, Trpm8-/-, or Trpa1-/-/Trpm8-/- mice. The effects of borneol on the activation of TRPM8 and the inhibition of TRPA1 were also measured in dorsal root ganglia neurons of wild-type (WT), Trpm8-/- and Trpv1-/- mice. Lastly, a randomized, double-blind study of adult patients was conducted to evaluate the clinical antipruritic effect of borneol. RESULTS: TRPA1, TRPV3 and TRPM8 are the potential targets of borneol according to the results of DrugBAN algorithm and molecular docking. Calcium imaging and patch-clamp recording analysis demonstrated that borneol activates TRPM8 channel-induced cell excitability and inhibits TRPA1 channel-mediated cell excitability in transfected HEK293T cells. Animal behavior analysis showed that borneol can significantly reduce acute and chronic itch behavior in C57BL/6J mice, but this effect was eliminated in Trpa1-/-, Trpm8-/- mice, or at least in Trpa1-/-/Trpm8-/- mice. Borneol elicits TRPM8 channel induced [Ca2+]i responses but inhibits AITC or SADBE-induced activation of TRPA1 channels in dorsal root ganglia neurons of WT and Trpv1-/- mice, respectively. Furthermore, the clinical results indicated that borneol could reduce itching symptoms in patients and its efficacy is similar to that of menthol. CONCLUSION: Borneol has therapeutic effects on multiple pruritus models in mice and patients with chronic itch, and the mechanism may be through inhibiting TRPA1 and activating TRPM8.

Effect of Yiyuan moxibustion on urodynamics and TRPV4/ATP/P2X5 signal pathway of bladder tissue in rats with neurogenic bladder after sacral cord injury. / ç›Šå ƒç¸å¯¹éª¶é«“æŸä¼¤åŽç¥žç»æºæ€§è†€èƒ±å¤§é¼ å°¿æµåŠ¨åŠ›å­¦åŠè†€èƒ±ç»„ç»‡TRPV4/ATP/P2X5信号通路的影响.

OBJECTIVES: To observe the effect of Yiyuan moxibustion on urodynamics and the expressions of transient receptor potential vanilloid 4 (TRPV4), adenosine triphosphate (ATP), tyrosine protein kinase KIT (C-Kit) and adenosine triphosphate receptor P2X5 in bladder tissue of rats with detrusor reflex-free neurogenic bladder (NB) after sacral cord injury (SCI), so as to explore its mechanism in promoting the recovery of urination function of NB rats. METHODS: Female SD rats were randomly divided into sham operation, model, Yiyuan moxibustion, Yiyuan moxibustion+inhibitor (combination) and inhibitor groups, with 12 rats in each group. The model of detruser reflex-free NB after sacral SCI was established by modified Hassan Shaker spinal cord transection method. The behavioral score of Basso Beasttie Bresnahan (BBB) and urodynamic indexes were used to evaluate the model of rats after operation. Fifteen days after modeling, Yiyuan moxibustion was applied to "Shenque" (CV8) and "Guanyuan" (CV4) for 20 min, once daily for 14 days. Rats of the inhibitor and combination groups were given intravesical instillation of HC067047 (1 mL, 1 µmol/L, 30 min). After the interventions, urodynamics was used to evaluate the bladder function of rats. HE staining was used to observe the morphology of bladder tissue. ATP content in bladder tissue was detected by colorimetric method. The positive expression rates of C-Kit and their receptor P2X5 in bladder tissue were observed by immunofluorescence double labeling method, and TRPV4, C-Kit, and P2X5 protein expression levels in bladder tissue were detected by Western blot. RESULTS: Compared with the sham operation group, the maximum bladder capacity and bladder compliance of rats in the model group were increased (P<0.01), the leak point pressure, ATP content, the possitive expression rates of C-Kit and P2X5, and the protein expression levels of TRPV4, C-Kit, P2X5 in bladder tissue were decreased (P<0.01). In comparison with the model and combination groups, the Yiyuan moxibustion group showed a decrease in maximum bladder capacity and bladder compliance (P<0.01), an increase in leakage point pressure, ATP content, the possitive expression rates of C-Kit and P2X5, and TRPV4, C-Kit, and P2X5 protein expression levels (P<0.01, P<0.05)；However, these indicators showed opposite trends in the inhibitor group (P<0.01, P<0.05). CONCLUSIONS: Yiyuan moxibustion can improve the urodynamics and bladder function in rats with bladder detrusor nonreflective after SCI, which may be related to its effect in activating the TRPV4 channel in bladder tissue, promoting the release of ATP from bladder epithelium, thus increasing the expression of bladder Cajal interstitial cells and their purinergic P2X5 receptors.

Acupoint Catgut Embedding Diminishes Fibromyalgia Pain through TRPV1 in the Mouse Brain.

BACKGROUND: Chronic pain refers to pain that persists for over three months. Chronic pain may restrict activities of daily living, including work, learning, social life, and can lead to anxiety, depression, and sleep disturbance. Imaging data have demonstrated that central sensitization often occurs in the brain of patients with chronic pain, which arises from imbalanced neurotransmission in the central nervous system. Transient receptor potential vanilloid 1 (TRPV1) is an ion channel to serve as an inflammatory detector in the brain. We aim to determine the properties of acupoint catgut embedding (ACE) on cold stress-induced mice fibromyalgia (FM) and surveyed the character of TRPV1 and linked molecules in chronic FM pain. METHODS: Intermittent cold stress (ICS) was used to induce mice FM model. Mice were subgrouped into normal mice, ICS-induced FM group, FM mice with ACE, and FM in Trpv1-⁣/- group. ACE is a novel acupuncture technique that provides convenience and continuous nerve stimulation that has been reported effective on pain management. RESULTS: Our behavioral experiments showed similar levels of pain response among all groups before treatment. After ICS, prolonged mechanical and thermal pain was initiated (mechanical threshold: 1.96 ± 0.12 g; thermal latency: 4.86 ± 0.21 s) and were alleviated by ACE treatment and TRPV1 gene deletion. Inflammatory mediators were increased in the plasma of FM mice, while TRPV1 and related kinases were amplified in the hypothalamus and cerebellum. These changes were ameliorated in the ACE-treated and Trpv1-⁣/- groups. CONCLUSIONS: These novel findings suggest that chronic FM pain can be modulated by ACE or TRPV1 gene deletion. The analgesic effect of ACE through the TRPV1 pathway may reflect its potential as a therapeutic target for FM treatment.

Differential expressions of hypothalamic thermosensitive TRP ion channels may underlie the posthatching ontogeny of brain cooling capacity in broiler chickens.

Two trials were performed to evaluate the association of hypothalamic abundances of thermosensitive transient receptor potential (TRP) ion channels with thermoregulation in broiler chickens. In trial 1, temporal changes in body temperatures, and hypothalamic expression patterns of TRP channels and thermoregulatory neurotransmitter concentrations were assessed from 3 to 42 d of age. In trial 2, the same variables were compared at 2 age stages between 2 distinct types of birds with high or low rectal temperatures (HRT or LRT). The core-to-brain temperature difference exhibited a rapid increase after hatching, arriving at a steady state in the fourth week (P < 0.01). The hypothalamus saw a progressive decrease of TRPV4 protein expression through 28 d (P < 0.01), followed by a great increase in the abundance of other channels right up to the end (P < 0.05). Compared to LRT birds, a decline in hypothalamic content of TRPV4 (P < 0.05), together with a bigger core-to-brain temperature difference (P < 0.01), was evident in the HRT counterpart at 33 d. In both trials, the core-to-brain and core-to-surface temperature differences were controlled in a synchronous and coordinated manner. These results allow concluding that developmental changes in the thermal sensitivity of hypothalamic neurons, determined by brain cooling capacity, involve a neuro-genomic mechanism, which regulates the ratio between thermosensitive TRP ion channels to attain a lower proportion of TRPV4 in comparison with other channels.

The Vanilloid (Capsaicin) Receptor TRPV1 in Blood Pressure Regulation: A Novel Therapeutic Target in Hypertension?

Today's sedentary lifestyle with excess food and little exercise increases the number of people with hypertension, a major risk factor for stroke. New knowledge of treatments in this field is of utmost importance. In animal experiments, the activation by capsaicin of TRPV1-expressing sensory afferents evokes a drop in blood pressure by triggering the Bezold-Jarisch reflex. In hypertensive rats, capsaicin reduces blood pressure. Conversely, genetic ablation of the TRPV1 receptor results in elevated nocturnal (but not diurnal) blood pressure. These observations imply a therapeutic potential for TRPV1 activation in hypertensive patients. Indeed, in a major epidemiological study involving 9273 volunteers, dietary capsaicin was found to lower the risk for hypertension. New research indicates that the mechanism of action of capsaicin on blood pressure regulation is far more complex than previously thought. In addition to the well-recognized role of capsaicin-sensitive afferents in blood pressure regulation, TRPV1 seems to be expressed both in endothelial cells and vascular smooth muscle. This review aims to evaluate the therapeutic potential of TRPV1-targeting drugs in hypertensive patients.

Nanoparticle-mediated TRPV1 channel blockade amplifies cancer thermo-immunotherapy via heat shock factor 1 modulation.

The survival of malignant tumors is highly dependent on their intrinsic self-defense pathways such as heat shock protein (HSP) during cancer therapy. However, precisely dismantling self-defenses to amplify antitumor potency remains unexplored. Herein, we demonstrate that nanoparticle-mediated transient receptor potential vanilloid member 1 (TRPV1) channel blockade potentiates thermo-immunotherapy via suppressing heat shock factor 1 (HSF1)-mediated dual self-defense pathways. TRPV1 blockade inhibits hyperthermia-induced calcium influx and subsequent nuclear translocation of HSF1, which selectively suppresses stressfully overexpressed HSP70 for enhancing thermotherapeutic efficacy against a variety of primary, metastatic and recurrent tumor models. Particularly, the suppression of HSF1 translocation further restrains the transforming growth factor ß (TGFß) pathway to degrade the tumor stroma, which improves the infiltration of antitumor therapeutics (e.g. anti-PD-L1 antibody) and immune cells into highly fibrotic and immunosuppressive pancreatic cancers. As a result, TRPV1 blockade retrieves thermo-immunotherapy with tumor-eradicable and immune memory effects. The nanoparticle-mediated TRPV1 blockade represents as an effective approach to dismantle self-defenses for potent cancer therapy.

Expression of Trpa1 and Trpv1 Genes in the Hypothalamus and Blood Pressure in Normotensive and Hypertensive Rats. Effect of Losartan and Captopril.

Analysis of the role of genomic regulation of systolic BP (SBP) in normal and hypertensive rats showed the presence of an inverse relationship between the level of Trpa1 gene expression in the anterior hypothalamus and SBP. Losartan, an antagonist of angiotensin II type 1 receptors, shifts it to the region of lower SBP and greater expression of the Trpa1 gene, which can attest to interaction of the TRPA1 ion channel in the anterior hypothalamus with angiotensin II type 1 receptors. No association was found between the expression of the Trpv1 gene in the hypothalamus and SBP. We have previously shown that activation of the peripheral ion channel TRPA1 in the skin also contributes to SBP decrease in hypertensive animals. Hence, activation of the TRPA1 ion channel both in the brain and at the periphery has similar effects on SBP and leads to its decrease.

Pharmacological Potential of cis-jasmone in Adult Zebrafish (Danio rerio).

This study evaluates the pharmacological potential of cis-jasmone (CJ) in adult zebrafish (Danio rerio; aZF). Initially, aZF (n = 6/group) were pretreated (20 µL; p. o.) with CJ (0.1 or 0.3 or 1.0 mg/mL) or vehicle (0.5% Tween 80). The animals were submitted to acute toxicity and locomotion tests, pentylenetetrazole-induced seizure, carrageenan-induced abdominal edema, and cinnamaldehyde-, capsaicin-, menthol-, glutamate-, and acid saline-induced orofacial nociception. The possible mechanisms of anticonvulsant, anxiolytic, and antinociceptive action were evaluated. The involvement of central afferent fibers sensitive to cinnamaldehyde and capsaicin and the effect of CJ on the relative gene expression of TRPA1 and TRPV1 in the brain of aZF were also analyzed, in addition to the study of molecular docking between CJ and TRPA1, TRPV1 channels, and GABAA receptors. CJ did not alter the locomotor behavior and showed pharmacological potential in all tested models with no toxicity. The anticonvulsant effect of CJ was prevented by flumazenil (GABAergic antagonist). The anxiolytic-like effect of CJ was prevented by flumazenil and serotonergic antagonists. The antinociceptive effect was prevented by TRPA1 and TRPV1 antagonists. Chemical ablation with capsaicin and cinnamaldehyde prevented the orofacial antinociceptive effect of CJ. Molecular docking studies indicate that CJ interacted with TRPA1, TRPV1, and GABAA receptors. CJ inhibited the relative gene expression of TRPA1 and TRPV1. CJ has pharmacological potential for the treatment of seizures, anxiety, inflammation, and acute orofacial nociception. These effects are obtained by modulating the GABAergic and serotonergic systems, as well as the TRPs and ASIC channels.

miRNA-203b-3p Induces Acute and Chronic Pruritus through 5-HTR2B and TRPV4.

Growing evidence indicates that transient receptor potential (TRP) channels contribute to different forms of pruritus. However, the endogenous mediators that cause itch through transient receptor potential channels signaling are poorly understood. In this study, we show that genetic deletion or pharmacological antagonism of TRPV4 attenuated itch in a mouse model of psoriasis induced by topical application of imiquimod. Human psoriatic lesions showed increased expression of several microRNAs, including the miR-203b-3p, which induced a calcium ion response in rodent dorsal root ganglion neurons and scratching behavior in mice through 5-HTR2B activation and the protein kinase C‒dependent phosphorylation of TRPV4. Computer simulation revealed that the miR-203b-3p core sequence (GUUAAGAA) that causes 5-HTR2B/TRPV4-dependent itch targets the extracellular side of 5-HTR2B by interacting with a portion of the receptor pocket consistent with its activation. Overall, we reveal the unconventional pathophysiological role of an extracellular microRNA that can behave as an itch promoter through 5-HTR2B and TRPV4.

Combination therapy of a TRPV2 agonist with a TNF inhibitor achieves sustained suppression of disease severity and reduced joint damage.

We aimed to compare a transient receptor potential vanilloid 2 (TRPV2) agonist with a TNF inhibitor, and to test the potential of their combination in collagen-induced arthritis (CIA) as a potential future strategy for rheumatoid arthritis (RA). Following the onset of CIA DBA1/j mice were started on treatment with either vehicle, etanercept (8 mg/kg three times a week), the TRPV2 agonist O1821 (20-30 mg/kg/day), or a combination of both. Mice were scored over a 61-day period. Synovial tissues were obtained for RNA sequencing. Mice on monotherapy with either O1821 or etanercept developed milder clinical disease. The O1821 protection was observed at an earlier time-point than in the etanercept group. The combination therapy group achieved a more robust and sustained reduction in disease severity than either monotherapy group. All treatment groups had reduced scores for synovial inflammation, synovial hyperplasia, and erosive changes, compared with controls, with the combination group achieving the most significant protection. RNA sequencing and pathway analyses of synovial tissues identified pathways and processes regulated by the TRPV2 agonist, such as chemotaxis and cytokine receptor signaling, including IL6R. The combination therapy affected additional pathways not seen in the monotherapy groups. In conclusion, the TRPV2 agonist achieved an overall similar reduction in arthritis severity and histology scores as etanercept, but the combination therapy achieved a more sustained disease control and more pronounced reduction in joint damage, suggesting a potential future option for improving disease control in RA. RNA sequencing analyses identified new pathways regulated by TRPV2, and also by the combination treatment.

[Improvement effect of Shegan Mahuang Decoction on rats with cold-induced asthma based on TRPV1/NRF-1/mtTFA pathway].

This study investigated the therapeutic effect of Shegan Mahuang Decoction(SGMHD) on cold-induced asthma in rats and explored its underlying mechanism. Seventy-two healthy male SD rats of specific pathogen free(SPF) grade were randomly divided into a blank group, a model group, a positive control group(dexamethasone, 0.4 mg·kg~(-1)), and low-, medium-, and high-dose SGMHD groups(3.2, 6.4, and 12.8 g·kg~(-1)). The blank group received saline, while the other groups were sensitized by intraperitoneal injection of ovalbumin(OVA) solution. Subsequently, the rats were placed in a cold chamber adjustable to 0-2 ℃, and OVA solution was ultrasonically nebulized to induce cold-induced asthma in rats. After three weeks of treatment, the general behaviors of rats were observed. Hematoxylin-eosin(HE) staining was used to evaluate pathological changes in lung tissues, periodic acid-Schiff(PAS) staining assessed mucin changes, and Masson staining was performed to examine collagen deposition. Enzyme-linked immunosorbent assay(ELISA) was used to measure the levels of the inflammatory factors interleukin-4(IL-4) and vascular endothelial growth factor(VEGF) in serum and bronchoalveolar lavage fluid(BALF). Real-time quantitative polymerase chain reaction(RT-PCR) was employed to assess the mRNA expression levels of transient receptor potential vanilloid subfamily member 1(TRPV1), nuclear respiratory factor 1(NRF-1), and mitochondrial transcription factor A(mtTFA) in lung tissues. Western blot was used to measure the protein expression levels of TRPV1, NRF-1, and mtTFA in lung tissues. Compared with the blank group, the model group exhibited signs of rapid respiration, increased frequency of defecation with looser stools, and disheveled and dull fur. Pathological results showed significant infiltration of inflammatory cells in lung tissues, narrowing of bronchial lumens, increased mucin secretion, and enhanced collagen deposition in the model group. Additionally, the levels of IL-4 and VEGF in serum and BALF were significantly elevated, and the mRNA and protein expression levels of TRPV1, NRF-1, and mtTFA in lung tissues were significantly increased. Compared with the model group, SGMHD improved the behaviors of rats, alleviated pathological changes in lung tissues, mucin production, and collagen deposition, significantly decreased the levels of IL-4 and VEGF in serum and BALF, and reduced the mRNA expression levels of TRPV1, NRF-1, and mtTFA in lung tissues, with the medium-dose SGMHD group showing the most significant effect. Moreover, the protein expression levels of TRPV1, NRF-1, and mtTFA in lung tissues were also reduced, with the medium-dose SGMHD group exhibiting the most significant effect. In conclusion, this study demonstrates that SGMHD can alleviate airway inflammation and inhibit airway remodeling in cold-induced asthma rats. These effects may be associated with the modulation of the TRPV1/NRF-1/mtTFA signaling pathway.

[Effect of electroacupuncture on visceral sensitivity and colonic NGF, TrkA, TRPV1 expression in IBS-D rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) at "Tianshu" (ST 25) and "Shangjuxu" (ST 37) on mental state, visceral sensitivity and protein expression of nerve growth factor (NGF), tyrosine kinase receptor A (TrkA) and transient receptor potential vanilloid 1 (TRPV1) of colonic tissue in diarrhea-predominant irritable bowel syndrome (IBS-D) rats, and to explore its possible mechanism on treating IBS-D. METHODS: A total of 36 male SD rats of SPF grade were randomized into a blank group, a model group, an EA group and a western medication group, 9 rats in each group. In the model group, the EA group and the western medication group, IBS-D model was established by enema of dinitrobenzene sulfonic acid (DNBS) combined with chronic restraint stress method. In the EA group, EA was applied at "Tianshu" (ST 25) and "Shangjuxu" (ST 37), with disperse-dense wave, in frequency of 2 Hz/100 Hz, 20 min each time, once a day for 7 days. In the western medication group, pinaverium bromide suspension was given by gavage (15 mg•kg-1•d-1) for 7 days. Before and after model establishment, and after intervention, the body mass, 24 h food intake and fecal water content were observed, the visceral sensitivity was detected by abdominal withdrawal reflex (AWR); after intervention, the mental state was evaluated by elevated plus maze (EPM) test, the protein expression of NGF, TrkA and TRPV1 was detected by immunohistochemistry and Western blot in the 4 groups. RESULTS: After model establishment, compared with the blank group, the body mass and 24 h food intake were decreased (P<0.05), first systolic latency of AWR was shortened and number of contraction wave of AWR was increased (P<0.05), and fecal water content was increased (P<0.05) in the model group, the EA group and the western medication group. After intervention, compared with the blank group, open arm residence time ratio (OT%) of EPM was decreased (P<0.05) and protein expression of NGF, TrkA, TRPV1 in colonic tissue was increased in the model group (P<0.05); compared with the model group, the body mass and 24 h food intake were increased (P<0.05), first systolic latency of AWR was lengthened and number of contraction wave of AWR was decreased (P<0.05), the fecal water content was decreased (P<0.05), OT% of EPM was increased (P<0.05), and protein expression of NGF, TrkA, TRPV1 in colonic tissue was decreased (P<0.05) in the EA group and the western medication group. CONCLUSION: Electroacupuncture at "Tianshu" (ST 25) and "Shangjuxu" (ST 37) can relieve the anxiety and depression-like behaviors in IBS-D rats, down-regulate the protein expression of NGF, TrkA, TRPV1 in colonic tissue, so as to reduce the visceral sensitivity and relieve symptoms.

[Effect of Maxing Shigan Decoction and dissembled prescriptions against airway inflammation in RSV-aggravated asthma and mechanism of regulating TRPV1].

This study investigated the effect of Maxing Shigan Decoction(MXSGD) and its disassembled prescriptions against the airway inflammation in respiratory syncytial virus(RSV)-aggravated asthma and the regulation of transient receptor potential vanilloid-1(TRPV1). To be specific, ovalbumin(OVA) and RSV were used to induce aggravated asthma in mice(female, C57BL/6). Then the model mice were intervened by MXSGD and the disassembled prescriptions. The eosinophil(EOS) in peripheral blood, inflammatory cells in bronchoalveolar lavage fluid(BALF), enhanced pause(Penh) variation, and lung pathological damage in each group were observed, and the changes of interleukin(IL)-4, IL-13, substance P(SP), and prostaglandin E2(PGE2) in BALF were mea-sured by enzyme-linked immunosorbent assay(ELISA). Quantitative real time polymerase chain reaction(qPCR) and Western blot were used to detect mRNA and protein of TRPV1 in mouse lung tissue. In the in vitro experiment, 16 HBE cells were stimulated with IL-4 and RSV. Then the changes of TRPV1 expression after the intervention with the serum containing MXSGD and its disassembled prescriptions were observed. Besides, the intracellular Ca~(2+) level after the stimulation with TRPV1 agonist was evaluated. The results showed that the mice in the model group had obvious asthma phenotype, the levels of various inflammatory cells in the peripheral blood and BALF and Penh were significantly increased(P<0.05, P<0.01), and the lung tissue was severely damaged compared with the control group. Compared with the model group, the levels of EOS in the peripheral blood and BALF were significantly decreased in the MXSGD group, the SG group and the MXC group(P<0.05, P<0.01). The levels of WBC and neutrophils in BALF were significantly decreased in the MXSGD group and SG group(P<0.01), the levels of neutrophils in BALF were decreased in the MXC group(P<0.05). The improvement effect of the MXGSD on the level of inflammatory cells in peripheral blood and BALF was better than that of two disassembled groups(P<0.05, P<0.01). After 50 mg·mL~(-1) acetylcholine chloride stimulation, the Penh values of the MXSGD group and the MXC group significantly decreased(P<0.01), and the Penh value of the SG group decreased(P<0.05). The levels of IL-4, IL-13, PGE2 and SP in BALF could be significantly decreased in the MXSGD group(P<0.05, P<0.01), the levels of IL-13 and PGE2 in BALF could be decreased in the MXC group(P<0.05, P<0.01), and the levels of IL-13, PGE2 and SP in BALF could be decreased in the SG group(P<0.05, P<0.01). MXSGD could down-regulate the protein and mRNA expression of TRPV1 in lung tissue(P<0.05, P<0.01). The serum containing MXSGD and its disassembled prescriptions could down-regulate TRPV1 expression in 16 HBE cells stimulated by IL-4 combined with RSV and inhibit the inward flow of Ca~(2+) induced by TRPV1 agonist, especially the serum containing MXSGD which showed better effect than the serum containing disassembled ones(P<0.05). In vivo and in vitro experiments verified the protective effect of MXSGD and its disassembled prescriptions against airway inflammation in RSV-exacerbated asthma, the whole decoction thus possessed synergy in treating asthma, with better performance than the dissembled prescriptions. Different groups of prescription had made contributions in improving airway hyperresponsiveness, anti-allergy and anti-inflammation. The mechanism is the likelihood that it regulates TRPV1 channel and levels of related inflammatory mediators.

[Effect of electroacupuncture on the expressions of TRPV1, P2X3 receptors in bladder of rats with interstitial cystitis].

OBJECTIVE: To observe the effect of electroacupuncture (EA) at "Ciliao" (BL 32) and "Huiyang" (BL 35) on the pain, urodynamic and the expressions of transient receptor poteintial vanilloid 1 (TRPV1) and P2X3 receptors in bladder of rats with interstitial bladder (IC), and to explore the possible mechanism on EA for IC. METHODS: A total of 24 Wistar female rats were randomly divided into a blank group, a model group and an EA group, 8 rats in each group. In the model group and the EA group, IC model was established by intraperitoneal injection of cyclophosphamide by 150 mg/kg at once. EA was applied at "Ciliao" (BL 32) and "Huiyang" (BL 35) in the EA group for 20 min, with continuous wave, 30 Hz in frequency, once a day for 3 consecutive days. Mechanical pain threshold of bladder and urodynamic indexes (first urination time, bladder effective volume and urination pressure) were observed after model establishment and after intervention, the expressions of TRPV1 and P2X3 receptors in the bladder were detected by Western blot. RESULTS: After model establishment, the mechanical pain threshold of bladder was decreased in the model group and the EA group compared with that in the blank group (P<0.01). After intervention, the mechanical pain threshold of bladder in the model group was lower than the blank group (P<0.01), and that in the EA group was higher than the model group (P<0.01). The urodynamic of the rats in the blank group was normal, obvious abnormal contraction during the filling period of bladder was found in the rats of the model group, while no abnormal contraction during the filling period was found in the rats of the EA group. After model establishment, in the model group and the EA group, the first urination time was earlier than the blank group (P<0.01), while bladder effective volume and urination pressure were lower than the blank group (P<0.01). After intervention, in the model group, the first urination time was earlier than the blank group (P<0.01), while bladder effective volume and urination pressure were lower than the blank group (P<0.05); in the EA group, the first urination time was later than the model group (P<0.05), while bladder effective volume and urination pressure were higher than the model group (P<0.05). Compared with the blank group, the protein expressions of TRPV1 and P2X3 receptors in bladder were up-regulated in the model group (P<0.01); compared with the model group, the protein expressions of TRPV1 and P2X3 receptors in bladder were down-regulated in the EA group (P<0.05). CONCLUSION: EA can relieve bladder pain and improve urodynamic in IC rats. The mechanism may be related to the down-regulation on the expressions of TRPV1 and P2X3 receptors and the further inhibition on the abnormal input of bladder signal.

[Effect of Jinzhen Oral Liquid on cough after lipopolysaccharide-induced infection in rats and mechanism].

This study aims to explore the effect of Jinzhen Oral Liquid(JOL) on cough after infection in rats and the mechanism. To be specific, a total of 60 male SD rats were classified into 6 groups: normal group(equivalent volume of distilled water, ig), model group(equivalent volume of distilled water, ig), Dextromethorphan Hydrobromide Oral Solution group(3.67 mL·kg~(-1), ig), high-, medium-, and low-dose JOL groups(11.34, 5.67, and 2.84 mL·kg~(-1), respectively, ig). Lipopolysaccharide(LPS, nasal drip), smoking, and capsaicin(nebulization) were employed to induce cough after infection in rats except the normal group. Administration began on the 19 th day and lasted 7 days. Capsaicin(nebulization) was used to stimulate cough 1 h after the last administration and the cough frequency and cough incubation period in rats were recorded. The pathological morphology of lung tissue was observed based on hematoxylin-eosin(HE) staining. Immunohistochemistry(IHC) was used to detect the specific expression of transient receptor potential vanilloid 1(Trpv1), nerve growth factor(NGF), tropomyosin receptor kinase A(TrkA), and phosphorylated-p38 mitogen-activated protein kinase(p-p38 MAPK) in lung tissue, Western blot the protein expression of Trpv1, NGF, TrkA, and p-p38 MAPK in lung tissue, and real-time fluorescent quantitative polymerase chain reaction(real-time PCR) the mRNA expression of Trpv1, NGF, and TrkA. The results showed that model group demonstrated significantly high cough frequency, obvious proliferation and inflammatory cell infiltration in lung tissue, significantly enhanced positive protein expression of Trpv1, NGF, TrkA, and p-p38 MAPK in lung tissue and significant increase in the mRNA expression of Trpv1, NGF, and TrkA compared with the normal group. Compared with the model group, JOL can significantly reduce the cough frequency, alleviate the pathological changes of lung tissue, and decrease the protein expression of Trpv1, NGF, TrkA, and p-p38 MAPK in lung tissue, and high-dose and medium-dose JOL can significantly lower the mRNA expression of Trpv1, NGF, and TrkA. This study revealed that JOL can effectively inhibit Trpv1 pathway-related proteins and improve cough after infection. The mechanism is that it reduces the expression of NGF, TrkA, and p-p38 MAPK in lung tissue, thereby decreasing the expression of Trpv1 and cough sensitivity.

TRPV1-Mediated Sensing of Sodium and Osmotic Pressure in POMC Neurons in the Arcuate Nucleus of the Hypothalamus.

The central melanocortin system conducted by anorexigenic pro-opiomelanocortin (POMC) neurons and orexigenic agouti-related peptide (AgRP) neurons in the arcuate nucleus of the hypothalamus (ARC) not only regulates feeding behavior but also blood pressure. Excessive salt intake raises the Na+ concentration ([Na+]) in the cerebrospinal fluid (CSF) and worsens hypertension. The blood-brain barrier is immature in the ARC. Therefore, both AgRP and POMC neurons in the ARC have easy access to the electrolytes in the blood and can sense changes in their concentrations. However, the sensitivity of AgRP and POMC neurons to Na+ remains unclear. This study aimed to explore how the changes in the extracellular Na+ concentration ([Na+]) influence these neurons by measuring the cytosolic Ca2+ concentration ([Ca2+]i) in the single neurons isolated from the ARC that were subsequently immunocytochemically identified as AgRP or POMC neurons. Both AgRP and POMC neurons responded to increases in both [Na+] and osmolarity in C57BL/6 mice. In contrast, in transient receptor potential vanilloid 1 (TRPV1) knockout (KO) mice, POMC neurons failed to respond to increases in both [Na+] and osmolarity, while they responded to high glucose and angiotensin II levels with increases in [Ca2+]i. Moreover, in KO mice fed a high-salt diet, the expression of POMC was lower than that in wild-type mice. These results demonstrate that changes in [Na+] and osmolarity are sensed by the ARC POMC neurons via the TRPV1-dependent mechanism.

[Influence of electroacupuncture of"Zusanli"(ST36)on mast cells/TRPV1 signaling pathway in visceral hypersensitivity rats with functional dyspepsia].

OBJECTIVE: To explore the interventional mechanism of electroacupuncture (EA) of "Zusanli"(ST36)based on the involvement of mast cells/ transient receptor potential vanilloid type1 (TRPV1) signaling pathway in relieving visceral hypersensitivity in functional dyspepsia (FD) rats. METHODS: Sixty SD rats (half male and half female, 10 days in age) were randomly divided into normal control, model, medication (ketotifen) and EA groups, with 15 rats in each group. The FD model was established by gavage of iodoacetamide combined with tail clamping (stress stimulation). Rats of the medication group received intraperitoneal injection of ketotifen (1 mg·kg-1·d-1) for 14 d, and those of the EA group received EA of ST36 for 20 min, once a day for 14 d. An air-balloon was inserted into the rat's stomach for recording changes of the intragastric pressure (mL/mm Hg) via a pressure transducer. The visceral hypersensitivity was assessed using abdominal withdrawal reflex (AWR) score and the number and degranulation of mast cells of gastric mucosa were observed using toluidine blue staining. The expression levels of TRPV1 and proteinase activated receptor 2 (PAR2) in the stomach were observed using immunofluorescence histochemistry and Western blot, separately, and the contents of SP and CGRP in the stomach detected using ELISA. RESULTS: When the intragastric pressure was at 50, 60 and 70 mm Hg, the gastric compliance was significantly decreased (P<0.01), and the levels of visceral sensitivity increased in the model group （P<0.01）。 TRPV1 immunofluorescence tensity, expression of PAR2 and TRPV1 proteins, and contents of SP and CGRP in the stomach were considerably up-regulated in the model group compared with the normal control group (P<0.01). In comparison with the model group, under intragastric pressure of 50，60 and 70 mm Hg, the gastric compliance was obviously increased, and the visceral hypersensitivity decreased in the EA group （P<0.01,P<0.05）. TRPV1 immunofluorescence intensity, expression levels of PAR2 and TRPV1 proteins, and the contents of SP and CGRP in the stomach were considerably down-regulated in both medication and EA groups compared with the model group (P<0.01, P<0.05). The therapeutic effect of EA was significantly superior to that of medication in up-regulating the gastric compliance （at 70 mm Hg）, and down-regulating the contents of SP and CGRP (P<0.05). No significant differences were found between the EA and medication groups in up-regulating gastric compliance at intragastric pressure of 50 and 60 mm Hg, and in down-regulating the visceral sensitivity, TRPV1 fluorescence intensity, and expression of PAR2 and TRPV1 proteins (P>0.05). Toluidine blue staining showed an apparent increase of mast cell number with obvious degranulation in the gastric mucosa of rats in the model group, which was milder in the EA and medication groups. CONCLUSION: EA of ST36 can suppress visceral hypersensitivity and increase the gastric compliance in FD rats, which may be related with its effects in inhibiting the activation of gastric mast cells, and down-regulating the expression of gastric PAR2 and TRPV1 proteins and SP and CGRP contents.