The Serotonin-Mediated Anti-Allodynic Effect of Yokukansan on Paclitaxel-Induced Neuropathic Pain.

Refractory peripheral neuropathy can occur as a side effect in 60-70% of patients receiving Paclitaxel (PTX). Yokukansan (YKS) is a Japanese herbal medicine reported to have analgesic properties for entrapment nerve injuries. Therefore, we investigated the anti-allodynic effect of Yokukansan on Paclitaxel-induced neuropathic pain. All experiments used 6-week-old male Sprague Dawley rats. Mechanical allodynia was evaluated using a dynamic plantar aesthesiometer. A mobile touch-stimulator unit applied progressively increasing force to the mid-plantar region of the hind paw in a vertical direction until the animal withdrew its paw. This was carried out before the Paclitaxel administration and during the first, second, third, and fourth weeks. Using a rat model of PTX-induced neuropathic pain (PTX rat), we injected PTX (intraperitoneally, 2 mg/kg) five times every 2 days. Using the dynamic plantar test, we evaluated the anti-allodynic effect of YKS (orally administered, 1 g/kg). YKS administration on a daily basis significantly enhanced the withdrawal threshold in PTX rats and reduced the expression level of activated microglia immunostaining with Iba1, a specific marker for microglia. The intrathecal administration of WAY-100635 (5-hydroxytryptamine [5-HT]1A receptor antagonist) and Ketanserin (5-HT2A/2C receptor antagonist) inhibited the protective effects of YKS. YKS exhibited an anti-allodynic effect in a rodent model of PTX-induced neuropathic pain by reducing the sensitivity to pain stimuli. These results suggest that Yokukansan may activate 5-HT receptors in the spinal cord, mediating Paclitaxel-induced neuropathic pain.

Antinociceptive and anti-inflammatory activity of extracts and α-pyrones isolated from Cantinoa stricta.

This study evaluated the composition and the antinociceptive and anti-inflammatory activity of the crude extracts and two isolated compounds, anamarine (ANA) and 10-epi-olguine (eOL), obtained from the leaves of Cantinoa stricta (Lamiaceae). Crude ethanolic extract (EEt) and dichloromethane extract (DCM), selected based on NMR data, were submitted to pharmacological tests in male Swiss mice. The oral administration of EEt and DCM significantly reduced the second phase of formalin-induced nociception (60%), lipopolysaccharide (LPS)-induced mechanical hyperalgesia (90%), and carrageenan (Cg)-induced edema (25%). ANA and eOL, the major compounds in EEt and DCM extracts, administered orally or locally (in the paw), also reduced the LPS-induced mechanical hyperalgesia (Oral ID50 1.9 and 3.9 mg/kg; Local ID50 93.4 and 677.3 ng, respectively) without changing the thermal acute nociception or the motor performance of the animals. Local administration of ANA and eOL also reduced Cg-induced edema (40 and 23%, respectively). These isolated compounds did not change the mechanical hyperalgesia induced by tumor necrosis factor-α, interleukin-1ß, prostaglandin E2, dibutyryl cyclic AMP, or forskolin but reversed the hyperalgesia induced by dopamine, epinephrine, and phorbol 12-myristate 13-acetate. The hyperalgesia induced by epinephrine was reversed in male but not in female mice, in which this response is not dependent on protein kinase C (PKC). These results suggest that C. stricta extracts possess antinociceptive and anti-inflammatory activity which is related to the presence of ANA and eOL. Differently from the known analgesics, these substances seem to exert their action mainly interfering with the sympathetic component of pain, possibly with PKC.

Massage ameliorates lumbar disc herniation-related radicular pain in rats by suppressing TLR4/NLRP3 inflammasome signaling transduction.

BACKGROUND: Lumbar disc herniation (LDH) is a common spinal disease that can cause severe radicular pain. Massage, also known as Tuina in Chinese, has been indicated to exert an analgesic effect in patients with LDH. Nonetheless, the mechanism underlying this effect of massage on LDH remains unclarified. METHODS: Forty Sprague-Dawley rats were randomly divided into four groups. A rat LDH model was established by autologous nucleus pulpous (NP) implantation, followed by treatment with or without massage. A toll-like receptor 4 (TLR4) antagonist TAK-242 was administrated to rats for blocking TLR4. Behavioral tests were conducted to examine rat mechanical and thermal sensitivities. Western blotting was employed for determining TLR4 and NLRP3 inflammasome-associated protein levels in the spinal dorsal horn (SDH). Immunofluorescence staining was implemented for estimating the microglial marker Iba-1 expression in rat SDH tissue. RESULTS: NP implantation induced mechanical allodynia and thermal hyperalgesia in rat ipsilateral hindpaws and activated TLR4/NLRP3 inflammasome signaling transduction in the ipsilateral SDH. Massage therapy or TAK-242 administration relieved NP implantation-triggered pain behaviors in rats. Massage or TAK-242 hindered microglia activation and blocked TLR4/NLRP3 inflammasome activation in ipsilateral SDH of LDH rats. CONCLUSION: Massage ameliorates LDH-related radicular pain in rats by suppressing microglia activation and TLR4/NLRP3 inflammasome signaling transduction.

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 alleviates streptozotocin-induced diabetic neuropathic pain via suppressing phosphorylated CaMKIIα in rats.

Diabetic neuropathic pain (DNP) is a frequent complication of diabetes. Calcium/calmodulin-dependent protein kinase II α (CaMKIIα), a multi-functional serine/threonine kinase subunit, is mainly located in the surface layer of the spinal cord dorsal horn (SCDH) and the primary sensory neurons in dorsal root ganglion (DRG). Numerous studies have indicated electroacupuncture (EA) takes effect in various kinds of pain. In this research, we explored whether CaMKIIα on rats' SCDH and DRG participated in DNP and further explored the mechanisms underlying the analgesic effects of EA. The DNP model in rats was successfully established by intraperitoneal injection of streptozotocin. Certain DNP rats were treated with intrathecal injections of KN93, a CaMKII antagonist, and some of the DNP rats received EA intervention. The general conditions, behaviors, the expressions of CaMKIIα and phosphorylated CaMKIIα (p-CaMKIIα) were evaluated. DNP rats' paw withdrawal threshold was reduced and the expressions of p-CaMKIIα in SCDH and DRG were upregulated compared with the Normal group, while the level of CaMKIIα showed no significance. KN93 attenuated DNP rats' hyperalgesia and reduced the expressions of p-CaMKIIα. We also found EA attenuated the hyperalgesia of DNP rats and reduced the expressions of p-CaMKIIα. The above findings suggest that p-CaMKIIα in SCDH and DRG is involved in DNP. The analgesic effect of EA in DNP might be related to the downregulation of p-CaMKIIα expression level. Our study further supports that EA can be an effective clinical treatment for DNP.

Tibetan medicine Ruyi Zhenbao Pill ameliorates neuropathic pain by inhibiting the CXCL10-CXCR3 pathway in spinal cord of spinal nerve ligation model.

ETHNOPHARMACOLOGICAL RELEVANCE: Ruyi Zhenbao Pill (RYZBP) is a traditional Tibetan medicine that has been used for over 300 years in China to treat neurological diseases, specifically neuropathic pain (NP). However, its characteristics and mechanism of action in treating NP remains unclear. AIM OF THE STUDY: Based on animal experiments and transcriptomics to evaluate the characteristics and mechanism of RYZBP in treating NP. METHODS: Mice were divided into six groups using random assignment: sham-operation group, spinal nerve ligation (SNL) group, RYZBP low (0.65 g kg-1), medium (1.30 g kg-1), high (2.60 g kg-1) doses groups, and positive drug pregabalin (PGB, 0.05 g kg-1) group. Mice received intragastrical administered for 14 consecutive days. SNL and intrathecal injection models were employed. The analgesic effects were assessed using the Von Frey test, Acetone test, and Hot Plate test. L5 spinal dorsal horns were collected for transcriptomics on day 15. The potential signaling pathways and Hub genes of RYZBP to ameliorate NP were obtained through transcriptomics and network pharmacology. Molecular docking was utilized to evaluate the binding ability of candidate active ingredients with the Hub genes. Finally, western blot (WB) and immunofluorescence (IF) were used to validate the predicted targets. RESULTS: RYZBP demonstrated a dose-dependent alleviation of mechanical allodynia, cold and heat stimulus-induced pain in SNL mice. Transcriptomics analysis identified 24 differentially expressed genes, and pathway enrichment analysis revealed that the CXCL10-CXCR3 signal axis may be the primary biological pathway through which RYZBP relieve NP. Molecular docking test indicated that the active ingredient in RYZBP exhibit a strong affinity for the target protein CXCL10. WB and IF tests showed that RYZBP can significantly inhibit CXCL10 and CXCR3 and its downstream molecules expression in the spinal dorsal horn of SNL mice. Additionally, intrathecal injection of rmCXCL10 worsened pain hypersensitivity, while RYZBP was able to suppress the pain hypersensitivity response induced by rmCXCL10 and reduce the expression levels of CXCL10 and CXCR3 and its downstream molecules. CONCLUSION: RYZBP had a significant analgesic effect on NP model, and this effect may be related to inhibiting the CXCL10-CXCR3 pathway in the spinal dorsal horn.

RTA-408 Regulates p-NF-κB/TSLP/STAT5 Signaling to Ameliorate Nociceptive Hypersensitivity in Chronic Constriction Injury Rats.

Neuropathic pain following nerve injury is a complex condition, which often puts a negative impact on life and remains a sustained problem. To make pain management better is of great significance and unmet need. RTA 408 (Omaveloxone) is a traditional Asian medicine with a valid anti-inflammatory property. Thus, we aim to investigate the therapeutic effect of RTA-408 on mechanical allodynia in chronic constriction injury (CCI) rats as well as the underlying mechanisms. Neuropathic pain was induced by using CCI of the rats' sciatic nerve (SN) and the behavior testing was measured by calibrated forceps testing. Activation of Nrf-2, the phosphorylation of nuclear factor-κB (NF-κB), and the inflammatory response were assessed by western blots. The number of apoptotic neurons and degree of glial cell reaction were examined by immunofluorescence assay. RTA-408 exerts an analgesic effect on CCI rats. RTA-408 reduces neuronal apoptosis and glial cell activation by increasing Nrf-2 expression and decreasing the inflammatory response (TNF-α/ p-NF-κB/ TSLP/ STAT5). These data suggest that RTA-408 is a candidate with potential to reduce nociceptive hypersensitivity after CCI by targeting TSLP/STAT5 signaling.

Metformin inhibits paclitaxel-induced mechanical allodynia by activating opioidergic pathways and reducing cytokines production in the dorsal root ganglia and thalamus.

It has been shown that AMP-activated protein kinase (AMPK) is involved in the nociceptive processing. This observation has prompted us to investigate the effects of the AMPK activator metformin on the paclitaxel-induced mechanical allodynia, a well-established model of neuropathic pain. Mechanical allodynia was induced by four intraperitoneal (i.p) injections of paclitaxel (2 mg/kg.day) in mice. Metformin was administered per os (p.o.). Naltrexoneandglibenclamide were used to investigate mechanisms mediating metformin activity. Concentrations of cytokines in the dorsal root ganglia (DRG) and thalamus were determined. After a single p.o. administration, the two highest doses of metformin (500 and 1000 mg/kg) attenuated the mechanical allodynia. This response was attenuated by all doses of metformin (250, 500 and 1000 mg/kg) when two administrations, 2 h apart, were carried out. Naltrexone (5 and 10 mg/kg, i.p.), but not glibenclamide (20 and 40 mg/kg, p.o.), attenuated metformin activity. Concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and CXCL-1 in the DRG were increased after administration of paclitaxel. Metformin (1000 mg/kg) reduced concentrations of TNF-α, IL-1ß and CXCL-1 in the DRG. Concentration of IL-6, but not TNF-α, in the thalamus was increased after administration of paclitaxel. Metformin (1000 mg/kg) reduced concentration of IL-6 in the thalamus. In summary, metformin exhibits activity in the model of neuropathic pain induced by paclitaxel. This activity may be mediated by activation of opioidergic pathways and reduced production of TNF-α, IL-1ß and CXCL-1 in the DRG and IL-6 in the thalamus.

Electroacupuncture prevents the development or establishment of chronic pain via IL-33/ST2 signaling in hyperalgesic priming model rats.

BACKGROUND: Chronic pain is acomplexhealth issue. Compared to acute pain, which has a protective value, chronic pain is defined as persistent pain after tissue injury. Few clinical advances have been made to prevent the transition from acute to chronic pain. Electroacupuncture (EA), the most common form of acupuncture, is widely used in clinical practice to relieve pain. METHODS: The hyperalgesic priming model, established via a carrageenan injection followed by a prostaglandin E2 injection, was used to investigate the development or establishment of chronic pain. We observed the hyperalgesic effect of EA on rats and investigated the expression p38 mitogen-activated protein kinase, interleukin-33 (IL-33), and its receptor ST2 in astrocytes in the L4-L6 spinal cord dorsal horns (SDHs) after EA. The IL-33/ST2 signaling pathway in SDH is associated with the development of chronic pain. RESULTS: EA can reverse the pain threshold in hyperalgesic priming model rats and regulates the expression of phosphorylated p38, IL-33, and ST2 in astrocytes in the L4-L6 SDHs. We discovered that EA raises the pain threshold. This suggests that EA can prevent the development or establishment of chronic pain by inhibiting IL-33/ST2 signaling in the lower central nervous system. CONCLUSIONS: EA can alleviate the development or establishment of chronic pain by modulating IL-33/ST2 signaling in SDHs. Our findings will help clinicians understand the mechanisms of EA analgesia.

Electroacupuncture attenuates inflammatory pain via peripheral cannabinoid receptor type 1 signaling pathway in mice.

Pain is strongly associated with neuro-immune activation. Thus, the emerging role of the endocannabinoid system in neuro-inflammation is important. Acupuncture has been used for over 2500 years and is widely accepted for the management of pain. Our study aimed to investigate the effects of electroacupuncture on the regulation of cannabinoid receptor type 1 within the peripheral nervous system. Inflammatory pain was induced by injecting Complete Freund's adjuvant to induce mechanical and thermal hyperalgesia. Electroacupuncture significantly attenuated the mechanical and thermal sensitivities, and AM251, a cannabinoid receptor type 1 antagonist, eliminated these effects. Dual immunofluorescence staining demonstrated that electroacupuncture elevated expression of cannabinoid receptor type 1, co-localized with Nav 1.8. Furthermore, electroacupuncture significantly reduced levels of Nav 1.8 and COX-2 by western blot analysis, but not vice versa as AM251 treatment. Our data indicate that electroacupuncture mediates antinociceptive effects through peripheral endocannabinoid system signaling pathway and provide evidence that electroacupuncture is beneficial for pain treatment.

Photobiomodulation Activates Microglia/Astrocytes and Relieves Neuropathic Pain in Inferior Alveolar Nerve Injury.

Objective: This study aimed to determine microglial/astrocyte changes and their associated analgesic effect in inferior alveolar nerve injury (IANI) model rats treated with photobiomodulation therapy (PBMT) using a 940-nm diode laser. Background: Very few basic studies have investigated microglial/astrocyte dynamics following PBMT aimed at relieving neuropathic pain caused by IANI. Methods: Rats were divided into an IANI-PBM group, IANI+PBM group, and sham+PBM group. Observations were made on the day before IANI or the sham operation and on postoperative days 3, 5, 7, 14, and 28. PBMT was delivered for 7 consecutive days, with an energy density of 8 J/cm2. Behavioral analysis was performed to determine pain thresholds, and immunohistological staining was performed for the microglia marker Iba1 and astrocyte marker glial fibrillary acidic protein, which are observed in the spinal trigeminal nucleus. Results: Behavioral analysis showed that the pain threshold returned to the preoperative level on postoperative day 14 in the IANI+PBM group, but decreased starting from postoperative day 1 and did not improve thereafter in the IANI-PBM group (p ≤ 0.001). Immunological analysis showed that microglial and astrocyte cell counts were similar in the IANI+PBM group and IANI-PBM group shortly after IANI (day 3), but the expression area was larger (p ≤ 0.001) and hypertrophy of microglia and astrocyte cell bodies and end-feet extension (i.e., indicators of activation) were more prominent in the IANI+PBM group. Conclusions: PBMT after IANI prevented hyperalgesia and allodynia by promoting glial cell activation shortly after injury.

Projections from anteromedial thalamus nucleus to the midcingulate cortex mediate pain and anxiety-like behaviors in mice.

Prior research has demonstrated the involvement of the midcingulate cortex (MCC) and its downstream pathway in pain regulation. However, the mechanism via which pain information is conveyed to the MCC remains unclear. The present study utilized immunohistochemistry, chemogenetics, optogenetics, and behavior detection methods to explore the involvement of MCC, anteromedial thalamus nucleus (AM), and AM-MCC pathway in pain and emotional regulation. Chemogenetics or optogenetics methods were employed to activate/inhibit MCCCaMKIIα, AMCaMKIIα, AMCaMKIIα-MCC pathway. This manipulation evokes/relieves mechanical and partial heat hyperalgesia, as well as anxiety-like behaviors. In the complete Freund,s adjuvant (CFA) inflammatory pain model, chemogenetic inhibition of the AMCaMKIIα-MCCCaMKIIα pathway contributed to pain relief. Notably, this study presented the first evidence implicating the AM in the regulation of nociception and negative emotions. Additionally, it was observed that the MCC primarily receives projections from the AM, highlighting the crucial role of this pathway in the transmission of pain and emotional information.

Upregulation of Calhm2 in the anterior cingulate cortex contributes to the maintenance of bilateral mechanical allodynia and comorbid anxiety symptoms in inflammatory pain conditions.

Peripheral inflammation-induced chronic pain tends to evoke concomitant anxiety disorders. It's common knowledge that the anterior cingulate cortex (ACC) plays a vital role in maintaining pain modulation and negative emotions. However, the potential mechanisms of chronic inflammation pain and pain-related anxiety remain elusive. Here, it was reported that injecting complete Freund's adjuvant (CFA) unilaterally resulted in bilateral mechanical allodynia and anxiety-like symptoms in mice via behavioral tests. In addition, CFA induced the bilateral upregulation and activation of calcium homeostasis modulator 2 (Calhm2) in ACC pyramidal neurons by quantitative analysis and double immunofluorescence staining. The knockdown of Calhm2 in the bilateral ACC by a lentiviral vector harboring ribonucleic acid (RNA) interference sequence reversed CFA-induced pain behaviors and neuronal sensitization. Furthermore, the modulating of ACC pyramidal neuronal activities via a designer receptor exclusively activated by designer drugs (DREADD)-hM4D(Gi) greatly changed Calhm2 expression, mechanical paw withdrawal thresholds (PWTs) and comorbid anxiety symptoms. Moreover, it was found that Calhm2 regulates inflammation pain promoting the upregulation of N-methyl-D-aspartic acid (NMDA) receptor 2B (NR2B) subunits. Calhm2 knockdown in ACC exhibited a significant decrease in NR2B expression. These results demonstrated that Calhm2 in ACC pyramidal neurons modulates chronic inflammation pain and pain-related anxiety symptoms, which provides a novel underlying mechanism for the development of inflammation pain.

[Mechanism of Mongolian drug Naru-3 in initiation of neuroinflammation of neuropathic pain from MMP9/IL-1ß signaling pathway].

Neuropathic pain(NP) has similar phenotypes but different sequential neuroinflammatory mechanisms in the pathological process. It is of great significance to inhibit the initiation of neuroinflammation, which has become a new direction of NP treatment and drug development in recent years. Mongolian drug Naru-3 is clinically effective in the treatment of trigeminal neuralgia, sciatica, and other NPs in a short time, but its pharmacodynamic characteristics and mechanism of analgesia are still unclear. In this study, a spinal nerve ligation(SNL) model simulating clinical peripheral nerve injury was established and the efficacy and mechanism of Naru-3 in the treatment of NPs was discussed by means of behavioral detection, side effect evaluation, network analysis, and experimental verification. Pharmacodynamic results showed that Naru-3 increased the basic pain sensitivity threshold(mechanical hyperalgesia and thermal radiation hyperalgesia) in the initiation of SNL in animals and relieved spontaneous pain, however, there was no significant effect on the basic pain sensitivity threshold and motor coordination function of normal animals under physiological and pathological conditions. Meanwhile, the results of primary screening of target tissues showed that Naru-3 inhibited the second phase of injury-induced nociceptive response of formalin test in mice and reduced the expression of inflammatory factors in the spinal cord. Network analysis discovered that Naru-3 had synergy in the treatment of NP, and its mechanism was associated with core targets such as matrix metalloproteinase-9(MMP9) and interleukin-1ß(IL-1ß). The experiment further took the dorsal root ganglion(DRG) and the stage of patho-logical spinal cord as the research objects, focusing on the core targets of inducing microglial neuroinflammation. By means of Western blot, immunofluorescence, agonists, antagonists, behavior, etc., the mechanism of Naru-3 in exerting NP analgesia may be related to the negative regulation of the MMP9/IL-1ß signaling pathway-mediated microglia p38/IL-1ß inflammatory loop in the activation phase. The relevant research enriches the biological connotation of Naru-3 in the treatment of NP and provides references for clinical rational drug use.

NGF/PI3K/TRPV1 pathway mediates the regulation of visceral pain by acupuncture at "Shangjuxu" (ST37) in irritable bowel syndrome rats with chronic visceral hyperalgesia. / NGF/PI3K/TRPV1通路介导针刺"上巨虚"è°ƒèŠ‚è‚ æ˜“æ¿€ç»¼åˆå¾æ ¢æ€§å† è„ç—›æ•æ¨¡åž‹å¤§é¼ å† è„ç—›.

OBJECTIVES: To investigate the effect of manual acupuncture at "Shangjuxu"(ST37) on nerve growth factor(NGF)/phosphatidylinositol 3-kinase(PI3K)/transient receptor potential vanilloid 1(TRPV1) signaling pathway in rats with chronic visceral hyperalgesia of irritable bowel syndrome (IBS), so as to explore its underlying mechanism in treating IBS chronic visceral hyperalgesia. METHODS: IBS chronic visceral hyperalgesia model was established by colorectal dilation stimulation for 2 weeks for SD pups at 8 d after birth, which were fed until 8-week age after the stimulation. Then the verified successfully modeled adult rats were randomly divided into model, Shangjuxu, and non-acupoint groups, with 6 rats in each group, and 6 unmodeled rats were selected as normal group. On the next day of model evaluation, rats in the Shangjuxu group received acupuncture at right ST37 while rats in the non-acupoint group received acupuncture at the non-meridian and non-acupoint point in the right hypochondrium, both for 15 min, with manual twisting of mild reinforcing and reducing performed for 30 s at an interval of 5 min, once a day, for a total of 7 d. Abdominal withdrawal reflex(AWR) score was used to evaluate the degree of chronic visceral pain in rats. Western blot and real-time fluorescence quantitative PCR were used to detect the colonic protein and mRNA expressions of NGF, tropomyosin receptor kinase A (TrkA), PI3K and TRPV1. The positive expressions of PI3K and TRPV1 proteins in the colon of rats were detected by immunohistochemistry method. RESULTS: Compared with the normal group, AWR scores corresponding to 4 pressure levels of 20, 40, 60 and 80 mm Hg, mRNA and protein expressions of NGF, TrkA, PI3K and TRPV1 in colon tissue, and positive expressions of PI3K and TRPV1 in colon tissue were significantly increased(P<0.05) in the model group. After intervention, compared with the model group, rats in the Shangjuxu group had reduced AWR scores corresponding to 4 pressure levels of 20, 40, 60 and 80 mm Hg, lower colonic mRNA and protein expressions of NGF, TrkA, PI3K and TRPV1, and decreased positive expressions of PI3K and TRPV1 in colon tissue(P<0.05), while there were no significant differences in the above indexes of the non-acupoint group. CONCLUSIONS: Manual acupuncture at ST37 can alleviate IBS chronic visceral hyperalgesia in rat and its analgesic effect may be related to regulating NGF/PI3K/TRPV1 signaling pathway.

Mechanisms involved in the antinociceptive and anti-inflammatory effects of xanthotoxin.

Xanthotoxin (XAT) is a natural furanocoumarin clinically used in the treatment of skin diseases such as vitiligo and psoriasis. Recent studies have also investigated its effects on anti-inflammatory, anti-cognitive dysfunction, and anti-amnesia as a guideline for clinic application. However, little is known about its effects on pain relief. Here, we tested the analgesic effects of XAT in serious acute pain and chronic pain models. For acute pain, we used hot-, capsaicin- and formalin-induced paw licking. Nociceptive threshold was measured by mechanical stimuli with von Frey filaments. For chronic pain, we injected complete Freund's adjuvant (CFA) into the mice's plantar surface of the hind paw to induce inflammatory pain. Heat and mechanical hyperalgesia were evaluated by radiant heat and von Frey filament tests, respectively. To investigate the mechanisms underlying the analgesic effect of XAT, we used calcium imaging and western blot to assess transient receptor potential vanilloid 1 (TRPV1) activity and expression in isolated L4-L6 dorsal root ganglion (DRG) neurons. Haematoxylin and eosin (HE) staining, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) were used to examine immune cell recruitment and proinflammatory factor release from skin tissue from paw injection sites. Our results demonstrated that XAT not only reduced acute pain behaviors generated by hot, capsaicin, and formalin but also attenuated CFA-induced heat and mechanical hyperalgesia. The analgesic activity of XAT may be achieved by controlling peripheral inflammation, lowering immune cell infiltration at the site of inflammatory tissue, reducing inflammatory factor production, and therefore inhibiting TRPV1 channel sensitization and expression.

Zhiqiao Gancao Decoction Ameliorates Hyperalgesia in Lumbar Disc Herniation via the CCL2/CCR2 Signaling Pathway.

Purpose: The aim of this study was to investigate the effect of Zhiqiao Gancao decoction (ZQGCD) on hyperalgesia in lumbar disc herniation (LDH) and its mechanism. Methods: The potential mechanism of ZQGCD's therapeutic effect on LDH was investigated through network pharmacology, which involved screening the targets of eight components that were absorbed into the bloodstream. The effects of CCR2 inhibitors and ZQGCD-containing serum on the excitability of the CCL2/CCR2 signaling pathway and dorsal root ganglion neurons (DRGn) were investigated in vitro. The effects of CCR2 inhibitors and ZQGCD on the expression of the CCL2/CCR2 signaling pathway and ASIC3 in the rat intervertebral disc and dorsal root ganglion (DRG), the degree of disc degeneration, the threshold of foot retreat, and the latency of foot retreat in LDH rats were examined in vivo. The binding affinities and interaction modes between CCR2 and the components absorbed into the blood were analyzed using the AutodockVina 1.2.2 software. Results: Network pharmacology revealed that ZQGCD could treat LDH through a mechanism involving the chemokine signaling pathway. It was observed that the CCR2 inhibitor and ZQGCD-containing serum downregulated CCR2 and ASIC3 expression and decreased cell excitability in DRGn. The CCL2/CCR2 signaling pathway was activated in the degenerated intervertebral disc and DRG of LDH rats, increased the expression of ASIC3, and decreased the mechanical allodynia domain and thermal hyperalgesia domain. However, a CCR2 inhibitor or ZQGCD could ameliorate the above changes in LDH rats. The target proteins, CCL2 and CCR2, exhibited a robust affinity for the eight components that were absorbed into the bloodstream. Conclusion: The CCL2/CCR2 pathway was activated in the intervertebral disc and DRG of LDH rats. This was accompanied by upregulation of ASIC3 expression, increased excitability of DRGn, and the occurrence of hyperalgesia. ZQGCD improves hyperalgesia in LDH rats by inhibiting the CCL2/CCR2 pathway and downregulating ASIC3 expression.

Peripheral Activation of Formyl Peptide Receptor 2/ALX by Electroacupuncture Alleviates Inflammatory Pain by Increasing Interleukin-10 Levels and Catalase Activity in Mice.

In the context of the electroacupuncture (EA) neurobiological mechanisms, we have previously demonstrated the involvement of formyl peptide receptor 2 (FPR2/ALX) in the antihyperalgesic effect of EA. The present study investigated the involvement of peripheral FPR2/ALX in the antihyperalgesic effect of EA on inflammatory cytokines levels, oxidative stress markers and antioxidant enzymes in an animal model of persistent inflammatory pain. Male Swiss mice underwent intraplantar (i.pl.) injection with complete Freund's adjuvant (CFA). Mechanical hyperalgesia was assessed with von Frey monofilaments. Animals were treated with EA (2/10 Hz, ST36-SP6, 20 minutes) for 4 consecutive days. From the first to the fourth day after CFA injection, animals received i.pl. WRW4 (FPR2/ALX antagonist) or saline before EA. Levels of inflammatory cytokines (TNF, IL-6, IL-4 and IL-10), antioxidant enzymes (catalase and superoxide dismutase), oxidative stress markers (TBARS, protein carbonyl, nitrite/nitrate ratio), and myeloperoxidase activity were measured in paw tissue samples. As previously demonstrated, i.pl. injection of the FPR2/ALX antagonist prevented the antihyperalgesic effect induced by EA. Furthermore, animals treated with EA showed higher levels of IL-10 and catalase activity in the inflamed paw, and these effects were prevented by the antagonist WRW4. EA did not change levels of TNF and IL-6, SOD and MPO activity, and oxidative stress markers. Our work demonstrates that the antihyperalgesic effect of EA on CFA-induced inflammatory pain could be partially associated with higher IL-10 levels and catalase activity, and that these effects may be dependent, at least in part, on the activation of peripheral FPR2/ALX.

Role of magnesium-L-Threonate in alleviating skin/muscle incision and retraction induced mechanical allodynia and anxiodepressive-like behaviors in male rats.

Chronic postsurgical pain (CPSP) and its emotional comorbidities poses health burden to patients who have received the surgical treatment. However, its underlying mechanism remains unclear. Emerging studies indicate that magnesium deficiency is associated with neurological diseases, and magnesium supplement confers protection under these disease conditions. In this study, we examined the role and mechanism of magnesium deficiency in the pathology of surgery-induced allodynia and negative emotion using a rat model of skin/muscle incision and retraction (SMIR) and investigated the therapeutic effects of magnesium supplementation by oral magnesium-L-Threonate (L-TAMS) in SMIR-injured rats. In the SMIR model, rats developed mechanical allodynia and anxiodepressive-like behaviors. Further, SMIR caused microglia and astrocyte activation and enhanced expression of pro-inflammatory cytokine (TNF-α, IL-1ß and IL-6) in the anterior cingulate cortex (ACC). Importantly, magnesium ion (Mg2+) levels decreased in the serum and cerebrospinal fluid (CSF) of SMIR-injured rats, which exhibited high correlation with pain and emotion behavioral phenotypes in these rats. Repeated oral administration of L-TAMS increased serum and CSF levels of Mg2+ in SMIR-injured rats. Notably, L-TAMS administration reversed SMIR-induced mechanical allodynia and anxiodepressive-like behaviors but did not affect pain and emotional behaviors in sham rats. Moreover, L-TAMS administration suppressed SMIR-caused glial activation and proinflammatory cytokine expression in the ACC but had no such effect in sham rats. Together, our study demonstrates the contributing role of magnesium deficiency in the pathology of surgery-induced chronic pain and negative emotion. Moreover, we suggest that L-TAMS might be a novel approach to treat CPSP and its emotional comorbidities.

Electroacupuncture relieves chronic pain by promoting microglia M2 polarization in lumbar disc herniation rats.

Electroacupuncture has an effective analgesia on chronic pain caused by lumbar disc herniation (LDH) clinically, however, the underlying mechanism is unclear. In this study, we investigated whether electroacupuncture alleviated pain in LDH model rats by inducing spinal microglia M2 polarization. We established a noncompression LDH rat model by implanting autologous caudal nucleus pulposus into L5/L6 nerve root. Electroacupuncture (30 min/day) treatment on the ipsilateral side was started on the 8th postoperative day, once a day for consecutive 7 days. Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were tested for pain behavior. Western blotting was used to detect the protein expression in lumbar enlargement (L5/L6). Immunofluorescence was used to detect iNOS+/Iba-1+ and Arg-1+/Iba-1+ and CB2R+/Iba-1+ in lumbar enlargement (L5/L6). We show that PWT and PWL decreased in the LDH group while Iba-1, iNOS, and TNF-α expression increased significantly in lumbar spinal dorsal horn (SDH) after LDH surgery, and revealing that microglia were activated and polarized towards proinflammatory M1 phenotype. Electroacupuncture treatment significantly increased PWT and PWL while reducing Iba-1, iNOS, and TNF-α expression, interestingly, Arg-1 and IL-10 expression were significantly increased. Moreover, electroacupuncture treatment led to CB2 receptors on microglia upregulation, while NF-κB and p-NF-κB expression in lumbar SDH downregulation. Our study indicated that electroacupuncture may reduce nociceptive hyperalgesia by inhibiting microglia activation and microglia M1 polarization and promoting microglia M2 polarization in lumbar SDH of LDH rats, which may be caused by the activation of CB2 receptors on microglia and inhibition of NF-κB pathway in lumbar SDH.