Folic acid attenuates chronic visceral pain by reducing clostridiales abundance and hydrogen sulfide production.

Irritable bowel syndrome (IBS) related chronic visceral pain affects 20% of people worldwide. The treatment options are very limited. Although the scholarly reviews have appraised the potential effects of the intestinal microbiota on intestinal motility and sensation, the exact mechanism of intestinal microbiota in IBS-like chronic visceral pain remains largely unclear. The purpose of this study is to investigate whether Folic Acid (FA) attenuated visceral pain and its possible mechanisms. Chronic visceral hyperalgesia was induced in rats by neonatal colonic inflammation (NCI). 16S rDNA analysis of fecal samples from human subjects and rats was performed. Patch clamp recording was used to determine synaptic transmission of colonic-related spinal dorsal horn. Alpha diversity of intestinal flora was increased in patients with IBS, as well as the obviously increased abundance of Clostridiales order (a main bacteria producing hydrogen sulfide). The hydrogen sulfide content was positive correlation with visceral pain score in patients with IBS. Consistently, NCI increased Clostridiales frequency and hydrogen sulfide content in feces of adult rats. Notably, the concentration of FA was markedly decreased in peripheral blood of IBS patients compared with non-IBS human subjects. FA supplement alleviated chronic visceral pain and normalized the Clostridiales frequency in NCI rats. In addition, FA supplement significantly reduced the frequency of sEPSCs of neurons in the spinal dorsal horn of NCI rats. Folic Acid treatment attenuated chronic visceral pain of NCI rats through reducing hydrogen sulfide production from Clostridiales in intestine.

Effects of moxibustion on pain behaviors in patients with rheumatoid arthritis: A meta-analysis.

BACKGROUND: Pain is the main symptom of patients with rheumatoid arthritis (RA). Reports of the effects of moxibustion on patients with rheumatoid arthritis have reached various conclusions. The aim of this meta-analysis was to evaluate the effect of moxibustion on pain in patients with RA. METHODS: A systematic search of MEDLINE, EMBASE, the Cochrane Library, and the Chinese databases Wan Fang Med Database, CNKI, and VIP (until November, 2018) was used to identify studies reporting pain (on a visual analogue scale (VAS)), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) and rheumatoid factor (RF) levels, response rate, and the ACR50 rate in patients with RA. Results were expressed as mean difference (MD) and 95% confidence intervals (CI). RESULTS: Six studies involving 281 participants were included. Moxibustion had significant effects on pain (VAS: MD = -0.53, 95% CI [-0.94, -0.12], P =.01). Moreover, moxibustion had effects on CRP (MD = -2.84, 95% CI [-5.13, -0.55], P =.01), ESR (MD = -8.44, 95% CI ([-13.19, -3.68], P =.0005), and RF (MD = -6.39, 95% CI [-18.57, 5.79], P =.30). Additionally, it had effects on response rate (n = 249, RR = 1.26, 95% CI [1.11, 1.43], P =.0004) and ACR50 rate (n = 140, RR = 1.44, 95% CI [1.11, 1.88], P =.007). CONCLUSION: We found that moxibustion with Western medicine therapy is superior to Western medicine therapy alone for pain in patients with RA. Moxibustion had significant effects on pain in patients with RA, but the effects of moxibustion on inflammatory factors in RA were unclear.

A new central post-stroke pain rat model: autologous blood injected thalamic hemorrhage involved increased expression of P2X4 receptor.

Stroke is the leading cause of disability and death in the world. Central post-stroke pain (CPSP), a central neuropathic pain syndrome occurring after cerebral stroke, is a serious problem. But on account of the lack of reliable animal models, the mechanisms underlying CPSP remains poorly understood. To better understand of the pathophysiological basis of CPSP, we developed and characterized a new rat model of CPSP. This model is based on a hemorrhagic stroke lesion with intra-thalamic autologous blood (ITAB) injection in the ventral posterolateral nucleus of the thalamus. Behavioral analysis demonstrated that the animals displayed a significant decrease in mechanical allodynia threshold. We found a significant increase in P2 × 4 receptor expression in microglia in thalamic peri-lesion tissues post-hemorrhage. The mechanical allodynia in rats with CPSP were reversed by blocking P2 × 4 receptors. A significant alleviation of mechanical allodynia was achieved following the administration of adrenergic antidepressants and antiepileptics. Meanwhile, we found a significant decrease in P2 × 4 receptor expression after treatment with these drugs. Taken together, our results suggest that targeting P2 × 4 receptor may be effective in the treatment of CPSP.

Phosphorylation of NR2B NMDA subunits by protein kinase C in arcuate nucleus contributes to inflammatory pain in rats.

The arcuate nucleus (ARC) of the hypothalamus plays a key role in pain processing. Although it is well known that inhibition of NMDA receptor (NMDAR) in ARC attenuates hyperalgesia induced by peripheral inflammation, the underlying mechanism of NMDAR activation in ARC remains unclear. Protein kinase C (PKC) is involved in several signalling cascades activated in physiological and pathological conditions. Therefore, we hypothesised that upregulation of PKC activates NMDARs in the ARC, thus contributing to inflammatory hyperalgesia. Intra-ARC injection of chelerythrine (CC), a specific PKC inhibitor, attenuated complete Freund's adjuvant (CFA) induced thermal and mechanical hyperalgesia in a dose-dependent manner. In vivo extracellular recordings showed that microelectrophoresis of CC or MK-801 (a NMDAR antagonist) significantly reduced the enhancement of spontaneous discharges and pain-evoked discharges of ARC neurons. In addition, CFA injection greatly enhanced the expression of total and phosphorylated PKCγ in the ARC. Interestingly, CFA injection also remarkably elevated the level of phosphorylated NR2B (Tyr1472) without affecting the expression of total NR2B. Importantly, intra-ARC injection of CC reversed the upregulation of phosphorylated NR2B subunits in the ARC. Taken together, peripheral inflammation leads to an activation of NMDARs mediated by PKC activation in the ARC, thus producing thermal and mechanical hyperalgesia.

Upregulation of cystathionine-ß-synthetase expression contributes to inflammatory pain in rat temporomandibular joint.

BACKGROUND: Hydrogen sulfide (H2S), an endogenous gaseotransmitter/modulator, is becoming appreciated that it may be involved in a wide variety of processes including inflammation and nociception. However, the role for H2S in nociceptive processing in trigeminal ganglion (TG) neuron remains unknown. The aim of this study was designed to investigate whether endogenous H2S synthesizing enzyme cystathionine-ß-synthetase (CBS) plays a role in inflammatory pain in temporomandibular joint (TMJ). METHODS: TMJ inflammatory pain was induced by injection of complete Freund's adjuvant (CFA) into TMJ of adult male rats. Von Frey filaments were used to examine pain behavioral responses in rats following injection of CFA or normal saline (NS). Whole cell patch clamp recordings were employed on acutely isolated TG neurons from rats 2 days after CFA injection. Western blot analysis was carried out to measure protein expression in TGs. RESULTS: Injection of CFA into TMJ produced a time dependent hyperalgesia as evidenced by reduced escape threshold in rats responding to VFF stimulation. The reduced escape threshold was partially reversed by injection of O-(Carboxymethyl) hydroxylamine hemihydrochloride (AOAA), an inhibitor for CBS, in a dose-dependent manner. CFA injection led to a marked upregulation of CBS expression when compared with age-matched controls. CFA injection enhanced neuronal excitability as evidenced by depolarization of resting membrane potentials, reduction in rheobase, and an increase in number of action potentials evoked by 2 and 3 times rheobase current stimulation and by a ramp current stimulation of TG neurons innervating the TMJ area. CFA injection also led to a reduction of IK but not IA current density of TG neurons. Application of AOAA in TMJ area reduced the production of H2S in TGs and reversed the enhanced neural hyperexcitability and increased the IK currents of TG neurons. CONCLUSION: These data together with our previous report indicate that endogenous H2S generating enzyme CBS plays an important role in TMJ inflammation, which is likely mediated by inhibition of IK currents, thus identifying a specific molecular mechanism underlying pain and sensitization in TMJ inflammation.

Promoter demethylation of cystathionine-ß-synthetase gene contributes to inflammatory pain in rats.

Hydrogen sulfide (H(2)S), an endogenous gas molecule synthesized by cystathionine-ß-synthetase (CBS), is involved in inflammation and nociceptive signaling. However, the molecular and epigenetic mechanisms of CBS-H(2)S signaling in peripheral nociceptive processing remain unknown. We demonstrated that peripheral inflammation induced by intraplantar injection of complete Freund adjuvant significantly up-regulated expression of CBS at both protein and mRNA levels in rat dorsal root ganglia (DRG). The CBS inhibitors hydroxylamine and aminooxyacetic acid attenuated mechanical hyperalgesia in a dose-dependent manner and reversed hyperexcitability of DRG neurons in inflamed rats. Intraplantar administration of NaHS (its addition mimics CBS production of H(2)S) or l-cysteine in healthy rats elicited mechanical hyperalgesia. Application of NaHS in vitro enhanced excitability and tetrodotoxin (TTX)-resistant sodium current of DRG neurons from healthy rats, which was attenuated by pretreatment of protein kinase A inhibitor H89. Methylation-specific PCR and bisulfite sequencing demonstrated that promoter region of cbs gene was less methylated in DRG samples from inflamed rats than that from controls. Peripheral inflammation did not alter expression of DNA methyltransferase 3a and 3b, the 2 major enzymes for DNA methylation, but led to a significant up-regulation of methyl-binding domain protein 4 and growth arrest and DNA damage inducible protein 45α, the enzymes involved in active DNA demethylation. Our findings suggest that epigenetic regulation of CBS expression may contribute to inflammatory hyperalgesia. H(2)S seems to increase TTX-resistant sodium channel current, which may be mediated by protein kinase A pathway, thus identifying a potential therapeutic target for the treatment of chronic pain.

Electroacupuncture suppresses mechanical allodynia and nuclear factor κ B signaling in streptozotocin-induced diabetic rats.

AIMS: To investigate whether electroacupuncture (EA) produced analgesic effect and whether nuclear factor kappa B (NF-κB) and cystathionine ß synthase (CBS) involved in EA-mediated analgesia in painful diabetic neuropathy in rats. METHODS: Diabetes was induced by an intraperitoneal injection of streptozotocin (STZ) in adult female rats. Mechanical pain threshold was measured by von Frey filaments. EA was applied at acupoint Zu-San-Li (ST-36) in both hindlimbs. Western blot analysis was employed to detect changes in protein levels of NF-κB and CBS in spinal dorsal root ganglion (DRGs). RESULTS: Mechanical allodynia was developed 2 weeks after STZ injection and lasted for another 4 weeks. STZ injection significantly enhanced expression of p65 and CBS in lumbar L4-6 DRGs when compared with age-matched controls. EA markedly attenuated mechanical allodynia. Importantly, EA treatment remarkably inhibited p65 and CBS expression in DRGs. Additionally, intrathecal injection of the p65 antagonist pyrrolidine dithiocarbamate attenuated mechanical allodynia and markedly inhibited CBS expression in DRGs in STZ rats. CONCLUSIONS: These data indicate that EA produced an analgesic effect, which might be mediated at least in a part by inhibition of NF-κB signaling pathway in primary sensory neurons in rats with diabetes.

Electroacupuncture alleviates stress-induced visceral hypersensitivity through an opioid system in rats.

AIM: To investigate whether stress-induced visceral hypersensitivity could be alleviated by electroacupuncture (EA) and whether EA effect was mediated by endogenous opiates. METHODS: Six to nine week-old male Sprague-Dawley rats were used in this study. Visceral hypersensitivity was induced by a 9-d heterotypic intermittent stress (HIS) protocol composed of 3 randomly stressors, which included cold restraint stress at 4°C for 45 min, water avoidance stress for 60 min, and forced swimming stress for 20 min, in adult male rats. The extent of visceral hypersensitivity was quantified by electromyography or by abdominal withdrawal reflex (AWR) scores of colorectal distension at different distention pressures (20 mmHg, 40 mmHg, 60 mmHg and 80 mmHg). AWR scores either 0, 1, 2, 3 or 4 were obtained by a blinded observer. EA or sham EA was performed at classical acupoint ST-36 (Zu-San-Li) or BL-43 (Gao-Huang) in both hindlimbs of rats for 30 min. Naloxone (NLX) or NLX methiodide (m-NLX) was administered intraperitoneally to HIS rats in some experiments. RESULTS: HIS rats displayed an increased sensitivity to colorectal distention, which started from 6 h (the first measurement), maintained for 24 h, and AWR scores returned to basal levels at 48 h and 7 d after HIS compared to pre-HIS baseline at different distention pressures. The AWR scores before HIS were 0.6 ± 0.2, 1.3 ± 0.2, 1.9 ± 0.2 and 2.3 ± 0.2 for 20 mmHg, 40 mmHg, 60 mmHg and 80 mmHg distention pressures, respectively. Six hours after termination of the last stressor, the AWR scores were 2.0 ± 0.1, 2.5 ± 0.1, 2.8 ± 0.2 and 3.5 ± 0.2 for 20 mmHg, 40 mmHg, 60 mmHg and 80 mmHg distention pressures, respectively. EA given at classical acupoint ST-36 in both hindlimbs for 30 min significantly attenuated the hypersensitive responses to colorectal distention in HIS rats compared with sham EA treatment [AWRs at 20 mmHg: 2.0 ± 0.2 vs 0.7 ± 0.1, P = 4.23,711 E-4; AWRs at 40 mmHg: 2.6 ± 0.2 vs 1.5 ± 0.2, P = 0.00,163; AWRs at 60 mmHg: 3.1 ± 0.2 vs 1.9 ± 0.1, P = 0.003; AWRs at 80 mmHg: 3.6 ± 0.1 vs 2.4 ± 0.2, P = 0.0023; electromyographic (EMG) at 20 mmHg: 24 ± 4.7 vs 13.8 ± 3.5; EMG at 40 mmHg: 60.2 ± 6.6 vs 30 ± 4.9, P = 0.00,523; EMG at 60 mmHg: 83 ± 10 vs 39.8 ± 5.9, P = 0.00,029; EMG at 80 mmHg: 94.3 ± 10.8 vs 49.6 ± 5.9, P = 0.00,021]. In addition, EA at the acupuncture point BL-43 with same parameters did not alleviate visceral hypersensitivity in HIS rats. EA in healthy rats also did not have any effect on AWR scores to colorectal distention at distention pressures of 20 and 40 mmHg. The EA-mediated analgesic effect was blocked by pretreatment with NLX in HIS rats [AWR scores pretreated with NLX vs normal saline (NS) were 2.0 vs 0.70 ± 0.20, 2.80 ± 0.12 vs 1.50 ± 0.27, 3 vs 2.00 ± 0.15 and 3.60 ± 0.18 vs 2.60 ± 0.18 for 20 mmHg, 40 mmHg, 60 mmHg and 80 mmHg; P = 0.0087, 0.0104, 0.0117 and 0.0188 for 20, 40, 60 and 80 mmHg, respectively]. Furthermore, EA-mediated analgesic effect was completely reversed by administration of m-NLX, a peripherally restricted opioid antagonist (EMG pretreated with m-NLX vs NS were 30.84 ± 4.39 vs 13.33 ± 3.88, 74.16 ± 9.04 vs 36.28 ± 8.01, 96.45 ± 11.80 vs 50.19 ± 8.28, and 111.59 ± 13.79 vs 56.42 ± 8.43 for 20 mmHg, 40 mmHg, 60 mmHg and 80 mmHg; P = 0.05,026, 0.00,034, 0.00,005, 0.000,007 for 20 mmHg, 40 mmHg, 60 mmHg and 80 mmHg, respectively). CONCLUSION: EA given at classical acupoint ST-36 alleviates stress-induced visceral pain, which is most likely mediated by opioid pathways in the periphery.

Peripheral inflammation sensitizes P2X receptor-mediated responses in rat dorsal root ganglion neurons.

ATP-gated P2X receptors in nociceptive sensory neurons participate in transmission of pain signals from the periphery to the spinal cord. To determine the role of P2X receptors under injurious conditions, we examined ATP-evoked responses in dorsal root ganglion (DRG) neurons isolated from rats with peripheral inflammation, induced by injections of complete Freund's adjuvant (CFA) into the hindpaw. Application of ATP induced both fast- and slow-inactivating currents in control and inflamed neurons. CFA treatment had no effect on the affinity of ATP for its receptors or receptor phenotypes. On the other hand, inflammation caused a twofold to threefold increase in both ATP-activated currents, altered the voltage dependence of P2X receptors, and enhanced the expression of P2X2 and P2X3 receptors. The increase in ATP responses gave rise to large depolarizations that exceeded the threshold of action potentials in inflamed DRG neurons. Thus, P2X receptor upregulation could account for neuronal hypersensitivity and contribute to abnormal pain responses associated with inflammatory injuries. These results suggest that P2X receptors are useful targets for inflammatory pain therapy.