Moxibustion ameliorates visceral hypersensitivity by regulating hypothalamus-spinal cord-colon axis in rats with irritable bowel syndrome with diarrhea. / 基于下丘脑-脊髓-ç»“è‚ è½´æŽ¢è®¨è‰¾ç¸æ”¹å–„è ¹æ³»åž‹è‚ æ˜“æ¿€ç»¼åˆå¾å¤§é¼ å† è„é«˜æ•æ„Ÿçš„ä½œç”¨æœºåˆ¶.

OBJECTIVES: To observe the effect of moxibustion intervention on the hypothalamus-spinal cord-colon axis of rats with irritable bowel syndrome with diarrhea (IBS-D) and explore the mechanism of moxibustion in improving visceral hypersensitivity in rats with IBS-D. METHODS: A total of 36 SD rats were randomly divided into normal, model, and moxibustion groups, with 12 rats in each group. The IBS-D model was established by maternal separation + acetic acid stimulation + chronic restraint. Rats of the moxibustion group received bilateral moxibustion on "Tianshu" (ST25) and "Shangjuxu" (ST37) for 15 min, once a day for 7 consecutive days. The body weight, loose stool rate, and minimum threshold volume of abdominal withdrawal reflex (AWR) were measured before and after moxibustion intervention, respectively. The histopathological changes in the colon tissue were observed after HE staining. The number of colonic mucosal mast cells (MCs) was measured by toluidine blue staining. The activation of MCs was determined by tryptase positive expression level and examined by immunohistochemical staining. The content, protein and mRNA expression levels and positive expression levels of corticotropin releasing factor (CRF), substance P (SP), and calcitonin gene-related peptide (CGRP) in the hypothalamus, spinal cord and colon tissues were measured by ELISA, Western blot, real-time fluorescent quantitative PCR and immunofluorescence staining, respectively. RESULTS: Compared with the normal group, the loose stool rate was increased (P<0.01)；the body weight and minimum threshold volume of AWR were decreased (P<0.01)；the inflammatory infiltration of colon tissues was obvious；the number of MCs and positive expression level of tryptase in the colon tissue were increased (P<0.01)；the contents, positive expression le-vels, protein and mRNA expression levels of CRF, SP and CGRP in the hypothalamus, spinal cord and colon tissues were increased (P<0.01, P<0.05) in the model group. After the intervention, compared with the model group, all these indicators showed opposite trends (P<0.01, P<0.05) in the moxibustion group. CONCLUSIONS: Moxibustion can improve visceral hypersensitivity in rats with IBS-D, and its mechanism may be related to regulating the hypothalamic-spinal-colon axis to reduce the release of CRF, SP and CGRP, and thus to inhibite MC in colon tissue.

Electroacupuncture at Sensitized Acupoints Relieves Somatic Referred Pain in Colitis Rats by Inhibiting Sympathetic-Sensory Coupling to Interfere with 5-HT Signaling Pathway.

OBJECTIVE: To investigate whether electroacupuncture (EA) at sensitized acupoints could reduce sympathetic-sensory coupling (SSC) and neurogenic inflammatory response by interfering with 5-hydroxytryptamine (5-HT)ergic neural pathways to relieve colitis and somatic referred pain, and explore the underlying mechanisms. METHODS: Rats were treated with 5% dextran sodium sulfate (DSS) solution for 7 days to establish a colitis model. Twelve rats were randomly divided into the control and model groups according to a random number table (n=6). According to the "Research on Rat Acupoint Atlas", sensitized acupoints and non-sensitized acupoints were determined. Rats were randomly divided into the control, model, Zusanli-EA (ST 36), Dachangshu-EA (BL 25), and Xinshu (BL 15) groups (n=6), as well as the control, model, EA, and EA + GR113808 (a 5-HT inhibitor) groups (n=6). The rats in the control group received no treatment. Acupuncture was administered on 2 days after modeling using the stimulation pavameters: 1 mA, 2 Hz, for 30 min, with sparse and dense waves, for 14 consecutive days. GR113808 was injected into the tail vein at 5 mg/kg before EA for 10 min for 7 consecutive days. Mechanical sensitivity was assessed with von Frey filaments. Body weight and disease activity index (DAI) scores of rats were determined. Hematoxylin and eosin staining was performed to observe colon histopathology. SSC was analyzed by immunofluorescence staining. Immunohistochemical staining was performed to detect 5-HT and substance P (SP) expressions. The calcitonin gene-related peptide (CGRP) in skin tissue and tyrosine hydroxylase (TH) protein levels in DRG were detected by Western blot. The levels of hyaluronic acid (HA), bradykinin (BK), prostaglandin I2 (PGI2) in skin tissue, 5-HT, tryptophan hydroxylase 1 (TPH1), serotonin transporters (SERT), 5-HT 3 receptor (5-HT3R), and 5-HT 4 receptor (5-HT4R) in colon tissue were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: BL 25 and ST 36 acupoints were determined as sensitized acupoints, and BL 15 acupoint was used as a non-sensitized acupoint. EA at sensitized acupoints improved the DAI score, increased mechanical withdrawal thresholds, and alleviated colonic pathological damage of rats. EA at sensitized acupoints reduced SSC structures and decreased TH and CGRP expression levels (P<0.05). Furthermore, EA at sensitized acupoints reduced BK, PGI2, 5-HT, 5-HT3R and TPH1 levels, and increased HA, 5-HT4R and SERT levels in colitis rats (P<0.05). GR113808 treatment diminished the protective effect of EA at sensitized acupoints in colitis rats (P<0.05). CONCLUSION: EA at sensitized acupoints alleviated DSS-induced somatic referred pain in colitis rats by interfering with 5-HTergic neural pathway, and reducing SSC inflammatory response.

[Protective effect of Liujing Toutong Tablets on rats with permanent cerebral ischemia via NF-κB signaling pathway].

This study investigated the neuroprotective effects and underlying mechanism of Liujing Toutong Tablets(LJTT) on a rat model of permanent middle cerebral artery occlusion(pMCAO). The pMCAO model was established using the suture method. Eighty-four male SPF-grade SD rats were randomly divided into a sham operation group, a model group, a nimodipine group(0.020 g·kg~(-1)), and high-, medium-, and low-dose LJTT groups(2.8, 1.4, and 0.7 g·kg~(-1)). The Longa score, adhesive removal test and laser speckle contrast imaging technique were used to evaluate the degree of neurological functional impairment and changes in local cerebral blood flow. The survival and mortality of rats in each group were recorded daily. After seven days of continuous administration following the model induction, the rats in each group were euthanized, and brain tissue and blood samples were collected for corresponding parameter measurements. Nissl staining was used to examine pathological changes in brain tissue neurons. The levels of tumor necrosis factor-alpha(TNF-α), interleukin-6(IL-6), IL-1ß, vascular endothelial growth factor(VEGF), calcitonin gene-related peptide(CGRP), beta-endorphin(ß-EP), and endogenous nitric oxide(NO) in rat serum were measured using specific assay kits. The entropy weight method was used to analyze the weights of various indicators. The protein expression levels of nuclear factor kappa-B(NF-κB), inhibitor kappaB alpha(IκBα), phosphorylated IκBα(p-IκBα), and phosphorylated inhibitor of NF-κB kinase alpha(p-IKKα) in brain tissue were determined using Western blot. Immunohistochemistry was used to detect the protein expression of chemokine-like factor 1(CKLF1) and C-C chemokine receptor 5(CCR5) in rat brain tissue. Compared with the sham operation group, the model group showed significantly higher neurological functional impairment scores, prolonged adhesive removal time, decreased cerebral blood flow, increased neuronal damage, reduced survival rate, significantly increased levels of TNF-α, IL-1ß, IL-6, CGRP, and NO in serum, significantly decreased levels of VEGF and ß-EP, significantly increased expression levels of NF-κB p65, p-IκBα/IκBα, and p-IKKα in rat brain tissue, and significantly upregulated protein expression of CKLF1 and CCR5. Compared with the model group, the high-dose LJTT group significantly improved the neurological functional score of pMCAO rats after oral administration for 7 days. LJTT at all doses significantly reduced adhesive removal time and restored cerebral blood flow. The high-and medium-dose LJTT groups significantly improved neuronal damage. The LJTT groups at all doses showed reduced levels of TNF-α, IL-1ß, IL-6, CGRP, and NO in rat serum, increased VEGF and ß-EP levels, and significantly decreased expression levels of NF-κB p65, p-IκBα/IκBα, p-IKKα, and CCR5 protein in rat brain tissue. The entropy weight analysis revealed that CGRP and ß-EP were significantly affected during the model induction, and LJTT exhibited a strong effect in reducing the release of inflammatory factors such as TNF-α and IL-1ß. LJTT may exert a neuroprotective effect on rats with permanent cerebral ischemia by reducing neuroinflammatory damage, and its mechanism may be related to the inhibition of the NF-κB signaling pathway and the regulation of the CKLF1/CCR5 axis. Additionally, LJTT may exert certain analgesic effects by reducing CGRP and NO levels and increasing ß-EP levels.

[An experimental study of acute toxicity and pharmacology of fermented Platycodonis Radix].

This study investigated the acute toxicity of fermented Platycodonis Radix on mice and its effect on coughing in mice infected with Mycoplasma pneumoniae. The maximum dosage(MAD) was used in the acute toxicity experiment on mice to observe the signs of mice. After 14 days, dissection, blood biochemical examination, and pathological tissue section observation were conducted. In the pharmacological experiment of fermented Platycodonis Radix, 60 healthy BALB/c mice, 30 males and 30 females, were randomly divided into a blank group, a model group, a carbetapentane group(0.013 g·kg~(-1)·d~(-1)), and high-, medium-, and low-dose fermented Platycodonis Radix groups(5.2, 2.6, and 1.3 g·kg~(-1)·d~(-1)), with 10 mice in each group. Except for the blank group, the mice in the other five groups underwent model induction by intranasally instilling 20 µL of 1×10~6 CCU M. pneumoniae for 3 days, and the mice in each group were orally administered the corresponding drugs for 7 days. Cough induction experiment was conducted to observe and record the cough latency and total cough count within 3 min for each group. Hematoxylin-eosin(HE) staining and Masson staining were used to observe the pathological changes in lung tissues. Immunohistochemistry was performed to observe the protein expression of transient receptor potential A1(TRPA1), calcitonin gene-related peptide(CGRP), and substance P(SP) in the lung tissues of mice in each group. Real-time fluorescence-based quantitative polymerase chain reaction(qRT-PCR) was used to elucidate the changes in the mRNA levels of cough-related factors TRPA1, CGRP, and SP in mice treated with fermented Platycodonis Radix. No mice died in the acute toxicity experiment, and there were no changes in general behavior and major organ histopathological examinations. Compared with the blank group, there were no statistically significant differences in blood biochemical indexes. In the pharmacological experiment of fermented Platycodonis Radix, compared with the model group, the high-and medium-dose fermented Platycodonis Radix groups showed improved lung tissue structure of mice, with clear structure and regular tissue morphology. The qRT-PCR and immunohistochemical detection showed a decrease in the expression of TRPA1, CGRP, and SP in the fermented Platycodonis Radix groups. Fermented Platycodonis Radix can exert an inhibitory effect on cough by suppressing the expression of TRPA1, CGRP, and SP in lung tissues, thereby identifying the target of the drug.

Effects of electroacupuncture combined with acupoint catgut embedding on gastrointestinal motility and gastrointestinal hormones in rats with functional dyspepsia.

Electroacupuncture (EA) or acupoint catgut embedding (ACE) plays a therapeutic role in functional dyspepsia (FD). Herein, we aimed to elucidate the influences of EA combined with ACE on gastrointestinal motility and gastrointestinal hormones in rats with FD. Sprague-Dawley rats were randomized into the control group, model group, EA group, ACE group, and EA + ACE group (n = 10). Except for the control group, the rats in all groups were modeled by combining neonatal iodoacetamide gastrogavage and modified tail-clamping stimulation. The rats were treated with different treatments according to their groups. The rats were observed for changes in general behavior, body weight, food intake, and paw mechanical pain threshold. Gastric emptying rate (GER) and intestinal propulsive ratio (IPR) were measured in each group, and serum gastrointestinal hormone (motilin [MTL], leptin, gastrin [GAS], vasoactive intestinal peptide [VIP], calcitonin gene-related peptide [CGRP], and somatostatin [SS]) levels, oxidative stress factors (superoxide dismutase [SOD] and malondialdehyde [MDA]) and 5-hydroxytryptamine (5-HT) levels were also measured. Decreased mean body weight, paw mechanical pain thresholds, food intake, and GER and IPR were found in rats of the model group in comparison to the control group. Serum MTL, GAS, SS, and SOD levels were reduced, and serum leptin, VIP, CGRP, MDA, and 5-HT levels were increased in rats of the model group in comparison to the control group. Elevated mean body weight, paw mechanical pain threshold, food intake, GER and IPR, and serum MTL, GAS, SS, and SOD levels, and reduced serum leptin, VIP, CGRP, MDA, and 5-HT levels were observed in rats of the EA, ACE, and EA + ACE groups relative to the model group. EA combined with ACE treatment was more effective than the EA or ACE treatment alone. EA combined with ACE treatment improves gastrointestinal motility and gastrointestinal hormone levels, promotes food intake, and reduces visceral hypersensitivity in FD rats.

Effect of acupuncture on the expression of neuropeptides and related inflammatory factors in rats with diarrhea-predominant irritable bowel syndrome. / é’ˆåˆºå¯¹è ¹æ³»åž‹è‚ æ˜“æ¿€ç»¼åˆå¾å¤§é¼ è„‘è‚ è‚½åŠç›¸å ³ç‚Žæ€§å› å­è¡¨è¾¾çš„å½±å“.

OBJECTIVES: To observe the effect of acupuncture on the expressions of neuropeptides and related inflammatory factors in rats with diarrhea-predominant irritable bowel syndrome(IBS-D), so as to explore the mechanism of acupuncture in the treatment of IBS-D. METHODS: Male Wistar rats were randomly divided into blank group, model group, medication group, and acupuncture group, with 6 rats in each group. Except for the blank group, the other groups were subjected to 14-day "acetic acid enema + restraint stress" to establish the IBS-D rat model. After successful modeling, the medication group received gavage of pinaverium bromide(15 mg/kg) once a day, and the acupuncture group received acupuncture at "Baihui"(GV20) and bilateral "Tianshu"(ST25), "Shangjuxu"(ST37), "Zusanli"(ST36), and "Taichong"(LR3) for 20 min every day, both groups were treated continuously for 14 days. The general state of the rats in each group was observed, and the body weight of the rats was measured. The open-field experiment was conducted to measure the horizontal and vertical movements, and the number of fecal pellets of rats. The histopathological morphology of hypothalamus and colon of rats was observed by HE staining. Toluidine blue staining was used to observe and count the mast cells(MCs) in the colon tissue of rats. ELISA was used to detect the serum contents of tumor necrosis factor-α(TNF-α) and interleukin(IL)-10. Real-time fluorescence quantitative PCR was performed to detect the mRNA expressions of calcitonin gene-related peptide(CGRP) in the hypothalamus and colon tissue. Western blot was used to detect the expressions of corticotropin-releasing factor(CRF) in the hypothalamus and colon tissue. RESULTS: HE staining showed that there was inflammatory cell infiltration in the lamina propria of colon in the model group, and it was reduced in the other groups. Compared with the blank group, the model group showed significantly decreased body weight, decreased walking distance and upright times in open field experiment, decreased serum IL-10 contents(P<0.05, P<0.01), increased fecal pellet number (P<0.01), increased MC number in the colon tissue, serum TNF-α contents, and CGRP mRNA expressions and CRF expressions in the hypothalamus and colon tissue(P<0.01). Compared with the model group, both medication and acupuncture groups showed significantly increased body weight, walking distance and upright times in the open-field experiment, and serum IL-10 contents(P<0.01, P<0.05), significantly decreased fecal pellet number (P<0.05), significantly decreased MC number in the colon tissue, serum TNF-α contents, and CGRP mRNA expressions in the hypothalamus and colon tissue(P<0.01)；at the same time, the acupuncture group showed significantly decreased CRF expressions in the hypothalamus and colon tissue(P<0.01, P<0.05). There was no significant difference in the above indicators between the medication group and the acupuncture group. CONCLUSIONS: Acupuncture can improve the general and emotional state, inflammatory response, and neuropeptide expression in rats with IBS-D, and alleviate the symptoms of IBS-D, which may be related to the regulation of neuropeptides and inflammatory factors levels.

The potent analgesia of intrathecal 2R, 6R-HNK via TRPA1 inhibition in LF-PENS-induced chronic primary pain model.

BACKGROUND: Chronic primary pain (CPP) is an intractable pain of unknown cause with significant emotional distress and/or dysfunction that is a leading factor of disability globally. The lack of a suitable animal model that mimic CPP in humans has frustrated efforts to curb disease progression. 2R, 6R-hydroxynorketamine (2R, 6R-HNK) is the major antidepressant metabolite of ketamine and also exerts antinociceptive action. However, the analgesic mechanism and whether it is effective for CPP are still unknown. METHODS: Based on nociplastic pain is evoked by long-term potentiation (LTP)-inducible high- or low-frequency electrical stimulation (HFS/LFS), we wanted to develop a novel CPP mouse model with mood and cognitive comorbidities by noninvasive low-frequency percutaneous electrical nerve stimulation (LF-PENS). Single/repeated 2R, 6R-HNK or other drug was intraperitoneally (i.p.) or intrathecally (i.t.) injected into naïve or CPP mice to investigate their analgesic effect in CPP model. A variety of behavioral tests were used to detect the changes in pain, mood and memory. Immunofluorescent staining, western blot, reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and calcium imaging of in cultured dorsal root ganglia (DRG) neurons by Fluo-8-AM were used to elucidate the role and mechanisms of 2R, 6R-HNK in vivo or in vitro. RESULTS: Intrathecal 2R, 6R-HNK, rather than intraperitoneal 2R, 6R-HNK or intrathecal S-Ketamine, successfully mitigated HFS-induced pain. Importantly, intrathecal 2R, 6R-HNK displayed effective relief of bilateral pain hypersensitivity and depressive and cognitive comorbidities in a dose-dependent manner in LF-PENS-induced CPP model. Mechanically, 2R, 6R-HNK markedly attenuated neuronal hyperexcitability and the upregulation of calcitonin gene-related peptide (CGRP), transient receptor potential ankyrin 1 (TRPA1) or vanilloid-1 (TRPV1), and vesicular glutamate transporter-2 (VGLUT2) in peripheral nociceptive pathway. In addition, 2R, 6R-HNK suppressed calcium responses and CGRP overexpression in cultured DRG neurons elicited by the agonists of TRPA1 or/and TRPV1. Strikingly, the inhibitory effects of 2R, 6R-HNK on these pain-related molecules and mechanical allodynia were substantially occluded by TRPA1 antagonist menthol. CONCLUSIONS: In the newly designed CPP model, our findings highlighted the potential utility of intrathecal 2R, 6R-HNK for preventing and therapeutic modality of CPP. TRPA1-mediated uprgulation of CGRP and neuronal hyperexcitability in nociceptive pathways may undertake both unique characteristics and solving process of CPP.

Cannabidiol tempers alcohol intake and neuroendocrine and behavioural correlates in alcohol binge drinking adolescent rats. Focus on calcitonin gene-related peptide's brain levels.

Alcohol binge drinking is common among adolescents and may challenge the signalling systems that process affective stimuli, including calcitonin gene-related peptide (CGRP) signalling. Here, we employed a rat model of adolescent binge drinking to evaluate reward-, social- and aversion-related behaviour, glucocorticoid output and CGRP levels in affect-related brain regions. As a potential rescue, the effect of the phytocannabinoid cannabidiol was explored. Adolescent male rats underwent the intermittent 20% alcohol two-bottle choice paradigm; at the binge day (BD) and the 24 h withdrawal day (WD), we assessed CGRP expression in medial prefrontal cortex (mPFC), nucleus accumbens (NAc), amygdala, hypothalamus and brainstem; in addition, we evaluated sucrose preference, social motivation and drive, nociceptive response, and serum corticosterone levels. Cannabidiol (40 mg/kg, i.p.) was administered before each drinking session, and its effect was measured on the above-mentioned readouts. At BD and WD, rats displayed decreased CGRP expression in mPFC, NAc and amygdala; increased CGRP levels in the brainstem; increased response to rewarding- and nociceptive stimuli and decreased social drive; reduced serum corticosterone levels. Cannabidiol reduced alcohol consumption and preference; normalised the abnormal corticolimbic CGRP expression, and the reward and aversion-related hyper-responsivity, as well as glucocorticoid levels in alcohol binge-like drinking rats. Overall, CGRP can represent both a mediator and a target of alcohol binge-like drinking and provides a further piece in the intricate puzzle of alcohol-induced behavioural and neuroendocrine sequelae. CBD shows promising effects in limiting adolescent alcohol binge drinking and rebalancing the bio-behavioural abnormalities.

[Effect of acupuncture-moxibustion stimulation of combined "Biao-Ben" acupoints on autonomic nervous activity and related factors in rats with irritable bowel syndrome diarrhea].

OBJECTIVE: To observe the effect of acupuncture and moxibustion of the combined "Biao-Ben" acupoints on autonomic nervous function and related factors in rats with irritable bowel syndrome diarrhea (IBS-D). METHODS: Female SD rats were randomly divided into normal control, IBS-D model, "Biao-Ben" acupoint combination, and conventional acupoint combination groups, with 10 rats in each group. The IBS-D model was established by repeated chronic stress stimulation (water or food deprivation, painful tail pinching, exposure to a 43 ℃ environment, forced swimming in 4 ℃ water, day-night inversion and horizontal vibration) for 28 d, and followed by acute restraint stress (wrapping of shoulders, forelimbs and trunk) for 1 h and gavage of senna fluid, once daily for 28 d. For rats of the "Biao-Ben" acupoint combination group, acupuncture and moxibustion were applied to "Guanyuan" (CV4), bilateral "Zusanli" (ST36), and "Neiguan" (PC6), and for rats of the conventional acupoint combination group, acupuncture and moxibustion were applied to bilateral ST36, "Tianshu"(ST25), and "Shangjuxu"(ST37). Both acupuncture and suspension moxibustion treatment were conduced for 15 min, once daily for 21 days. The fecal water content was calculated, and the spontaneous activity behaviors (total distance of crossing and the number of squares crossed in 5 min) were evaluated by open-field tests. The abdominal withdrawal reflex (AWR) was detected. H.E. staining was used to observe the histopathological changes of colon tissue. The domains of heart rate variability (HRV) including low frequency (LF), high frequency (HF), and LF/HF were analyzed using an electrophysiological recorder. The contents of calcitonin gene-related peptide (CGRP) in serum and noradrenaline (NE) in plasma were detected using ELISA. The expression level and immunoactivity of 5-hydroxytryptamine (5-HT) in colon tissues were detected by Western blot and immunofluorescence histochemistry, separately. RESULTS: Compared with the normal group, the model group had a striking increase in fecal water content, AWR scores at 20, 40, 60, and 80 mm Hg, NE and CGRP contents, LF, LF/HF, and 5-HT protein expression and immunoactivity (P<0.01), and an obvious decrease in the total distance of crossing, the number of squares crossed, and HF of HRV (P<0.01). In comparison with the model group, the fecal water content, AWR scores at 20, 40, 60, and 80 mm Hg, NE and CGRP contents, LF, LF/HF, and 5-HT protein expression and immunoactivity were significantly decreased (P<0.01,P<0.05), while the total distance of cros-sing, number of squares crossed, HF of HRV were considerably increased (P<0.01, P<0.05) in both "Biao-Ben" acupoint combination and conventional acupoint combination groups. The effects of the "Biao-Ben" acupoint combination were apparently superior to those of conventional acupoint combination in down-regulating the fecal water content, AWR score at 20, 40, 60 and 80 mm Hg, NE and CGRP contents, LF, LF/HF, and 5-HT expression and immunoactivity, and in increasing the number of squares crossed, and HF of HRV (P<0.01, P<0.05). HE staining showed no pathological changes in colonic mucosa in all groups. CONCLUSION: Acupuncture and moxibustion stimulation of combined "Biao-Ben" acupoints can effectively improve the symptoms (spontaneous activities, visceral hypersensitivity) of IBS-D model rats, which may be related to its functions in regulating autonomic nervous activities, and down-regulating blood NE and CGRP contents and colonic 5-HT protein expression and immunoactivity, and the effects of "Biao-Ben" acupoint combination are superior to those of conventional acupoint combination.

Shaoyao Gancao decoction alleviates the central hyperalgesia of recurrent NTG-induced migraine in rats by regulating the NGF/TRPV1/COX-2 signal pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Shaoyao Gancao Decoction (SGD) is well known as an effective prescription for analgesia composed of two herbs, and is noted as traditional Chinese medicine morphine. It is widely used in various conditions causing pain, including migraine. However, there is currently no research exploring the mechanism of action in the treatment of migraines. AIM OF THE STUDY: The current research was devised to determine the underlying regulatory mechanism of SGD, by verifying its role in the NGF/TRPV1/COX-2 signal pathway. MATERIALS AND METHODS: The active components in SGD were identified by UHPLC-MS. A migraine model was prepared by subcutaneous (s.c.) injection of nitroglycerin (NTG) into the neck to detect migraine-like behavior, orbital hyperalgesia threshold changes, and the therapeutic effect of SGD. The mechanism of SGD in remedying migraine was studied through transcriptome sequencing (RNA-seq), which was further validated utilizing Elisa, Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting (WB) experiments. RESULTS: In the SGD chemical composition analysis, 45 components were identified including gallic acid, paeoniflorin and albiforin. In the behavioral experiments, SGD treatment significantly decreased the score of migraine-like head scratching in the NTG-induced migraine model (Mod) rats, while the hyperalgesia threshold increased outstandingly on days 10, 12, and 14 (P < 0.01, P < 0.001 or P < 0.0001). In migraine biomarkers experiment, compared with the Mod group, the 5-hydroxytryptamine (5-HT) contents were outstandingly enhanced by SGD treatment, while nitric oxide (NO) contents were markedly declined (P < 0.01). In the RNA-seq test, the down-regulated genes of SGD inhibiting hyperalgesia migraine included the neurotrophic factor (NGF) and transient receptor potential vanillic acid subfamily protein 1 receptor (TRPV1). The down-regulation pathway is the inflammatory mediator regulation of TRP channels. In gene set enrichment analysis (GSEA), SGD decreased the over-expression of protooncogene tyrosine-protein kinase Src (SRC) and TRPV1 in this pathway, and the two genes clustered at its lower end, with similar functions. PPI network results show that NGF interacts with TRPV1. Further verification shows that when compared with Mod group, the plasma cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2) protein expression levels and the dura mater calcitonin gene-related peptide (CGRP), extracellular signal-regulated kinase (ERK), p-ERK, SRC and NGF protein expression levels in the SGD group were remarkably decreased (P < 0.01, P < 0.001 or P < 0.0001), and the expression level of TRPV1 protein showed a downward trend (P = 0.06). The expression levels of COX-2, NO, CGRP, TRPV1, SRC and NGF mRNA in the dura mater was overtly down-regulated (P < 0.05, P < 0.01 or P < 0.001). CONCLUSIONS: SGD has a significant inhibitory effect on the NGF/TRPV1/COX-2 signaling pathway that mediates central hyperalgesia migraine, thus suggesting the molecular mechanism of SGD in improving the symptoms of migraine may be related to the central hyperalgesia neurotransmitter that regulates the pathogenesis of migraine.

Future targets for migraine treatment beyond CGRP.

BACKGROUND: Migraine is a disabling and chronic neurovascular headache disorder. Trigeminal vascular activation and release of calcitonin gene-related peptide (CGRP) play a pivotal role in the pathogenesis of migraine. This knowledge has led to the development of CGRP(-receptor) therapies. Yet, a substantial proportion of patients do not respond to these treatments. Therefore, alternative targets for future therapies are warranted. The current narrative review provides a comprehensive overview of the pathophysiological role of these possible non-CGRP targets in migraine. FINDINGS: We covered targets of the metabotropic receptors (pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal peptide (VIP), amylin, and adrenomedullin), intracellular targets (nitric oxide (NO), phosphodiesterase-3 (PDE3) and -5 (PDE5)), and ion channels (potassium, calcium, transient receptor potential (TRP), and acid-sensing ion channels (ASIC)). The majority of non-CGRP targets were able to induce migraine-like attacks, except for (i) calcium channels, as it is not yet possible to directly target channels to elucidate their precise involvement in migraine; (ii) TRP channels, activation of which can induce non-migraine headache; and (iii) ASICs, as their potential in inducing migraine attacks has not been investigated thus far. Drugs that target its receptors exist for PACAP, NO, and the potassium, TRP, and ASIC channels. No selective drugs exist for the other targets, however, some existing (migraine) treatments appear to indirectly antagonize responses to amylin, adrenomedullin, and calcium channels. Drugs against PACAP, NO, potassium channels, TRP channels, and only a PAC1 antibody have been tested for migraine treatment, albeit with ambiguous results. CONCLUSION: While current research on these non-CGRP drug targets has not yet led to the development of efficacious therapies, human provocation studies using these targets have provided valuable insight into underlying mechanisms of migraine headaches and auras. Further studies are needed on these alternative therapies in non-responders of CGRP(-receptor) targeted therapies with the ultimate aim to pave the way towards a headache-free future for all migraine patients.

[Effect of moxibustion and scraping on bioactive substances changes of acupoints in knee osteoarthritis rats].

OBJECTIVE: To compare the effects of moxibustion and scraping of "Yanglingquan" (GB34) and "Xuehai" (SP10) area on changes of bioactive substances in the region of acupoints in rats with knee osteoarthritis (KOA). METHODS: SD rats were randomly divided into blank, model, moxibustion, scraping, and moxibustion + scraping (combination) groups, with 8 rats in each group. The KOA model was established by injecting 50 µL 0.9% sodium chloride solution into the right knee cavity. Fourteen days after modeling, GB34 and SP10 on the right limb were stimulated by moxibustion (10 min) or scraping (till regional flush) once every other day for 7 times. The mechanical paw withdrawal threshold (PWT) and thermal withdrawal latency (TWL) were tested by Von Frey and hot stabbing instrument, separately. The pathological changes of the right knee joint were observed by HE staining. The serotonin (5-HT) contents of skin tissues in the region of acupoint GB34 and SP10 were detected by ELISA. The expression levels of substance P (SP) and calcitonin gene-related peptide (CGRP) in GB34 and SP10 region skin tissues were detected by Western blot. RESULTS: Compared with the blank group, the PWT and TWL of the rats in the model group were significantly decreased (P<0.001), while the contents of 5-HT and the expression levels of SP and CGRP in GB34 and SP10 region skin tissues were significantly increased (P<0.001, P<0.01). Following intervention and in comparison the with the model group, the TWL and PWT of rats in the three treatment groups were significantly increased (P<0.01), the content of 5-HT and the expression levels of SP and CGRP in GB34 and SP10 region skin tissues were significantly decreased (P<0.01, P<0.001, P<0.05). Except for the expression levels of CGRP, the above indexes of the combination group were significantly superior to those of the moxibustion and scraping groups (P<0.05, P<0.01). Findings of HE staining showed severe damaged cartilage, few chondrocytes on the surface, with subchondral neovascularization in the model group, which was relatively milder in the moxibustion, scraping, and combination groups. CONCLUSION: Moxibustion and scraping can relieve knee joint pain in KOA rats, which may be associated with its function in down-regulating the expression levels of SP and CGRP, and the content of 5-HT. The therapeutic effect of moxibustion plus scraping is better than that of moxibustion and scraping alone.

Electroacupuncture Zusanli (ST36) Relieves Somatic Pain in Colitis Rats by Inhibiting Dorsal Root Ganglion Sympathetic-Sensory Coupling and Neurogenic Inflammation.

Referred somatic pain triggered by hyperalgesia is common in patients with inflammatory bowel disease (IBD). It was reported that sprouting of sympathetic nerve fibers into the dorsal root ganglion (DGR) and neurogenic inflammation were related to neuropathic pain, the excitability of neurons, and afferents. The purpose of the study was to explore the potential and mechanism of electroacupuncture (EA) at Zusanli (ST36) for the intervention of colon inflammation and hyperalgesia. Sprague-Dawley (SD) was randomly divided into four groups, including control, model, EA, and sham-EA. Our results showed EA treatment significantly attenuated dextran sulfate sodium- (DSS-) induced colorectal lesions and inflammatory cytokine secretion, such as TNF-α, IL-1ß, PGE2, and IL-6. EA also inhibited mechanical and thermal pain hypersensitivities of colitis rats. Importantly, EA effectively abrogated the promotion effect of DSS on ipsilateral lumbar 6 (L6) DRG sympathetic-sensory coupling, manifested as the sprouting of tyrosine hydroxylase- (TH-) positive sympathetic fibers into sensory neurons and colocalization of and calcitonin gene-related peptide (CGRP). Furthermore, EA at Zusanli (ST36) activated neurogenic inflammation, characterized by decreased expression of substance P (SP), hyaluronic acid (HA), bradykinin (BK), and prostacyclin (PGI2) in colitis rat skin tissues corresponding to the L6 DRG. Mechanically, EA treatment reduced the activation of the TRPV1/CGRP, ERK, and TLR4 signaling pathways in L6 DRG of colitis rats. Taken together, we presumed that EA treatment improved colon inflammation and hyperalgesia, potentially by suppressing the sprouting of sympathetic nerve fibers into the L6 DGR and neurogenic inflammation via deactivating the TRPV1/CGRP, ERK, and TLR4 signaling pathways.

Network pharmacology and experimental verification to explore the anti-migraine mechanism of Yufeng Ningxin Tablet.

ETHNOPHARMACOLOGICAL RELEVANCE: Yufeng Ningxin Tablet (YNT) is a traditional Chinese medicine formula, that has been used clinically to treat migraine for many years. It is composed of one herb Pueraria lobata var. lobata (Willd.) Ohwi (Relevant Chinese name: Gegen). Previously, it has been recorded by traditional Chinese doctor that Gegen could be used as medicine to treat migraine. However, the underlying mechanism of action remains to be investigated. AIM OF THE STUDY: It was to explore the effect and mechanism of YNT on migraine based on network pharmacology and experimental verification. MATERIALS AND METHODS: First, with the network pharmacology, the effective chemical components and target genes of YNT were filtrated, the YNT-compound-migraine-targets network was constructed. The protein-protein interaction network (PPI) and literature reports were combined to identify potential targets of YNT in the treatment of migraine. Then, the representative compounds of YNT were characterized by LC-MS/MS and the major effect components were identified. Finally, the prediction results of network pharmacology were verified by animal and cell experiments. RESULTS: 7 bioactive components of YNT could act on 97 migraine potential targets. The 5 bioactive components could be characterized comprehensively of YNT. The key therapeutic targets and pathways were collected including 5-HT, CGRP, inflammation and nociceptive factors, and NF-κB signaling pathway. Animal experiments showed that YNT could increase the expression level of 5-HT and reduce the expression of CGRP, NF-κB, c-fos and IL-1ß. YNT could inhibit LPS-induced neuroinflammation by NF-κB in BV2 cells in vitro. Western blotting analysis results showed YNT inhibited the NF-κB and phospho-NF-κB levels. CONCLUSIONS: It is the first time to verify the consistency between the metabolic components of YNT by LC-MS/MS and the active components predicted by network pharmacology. Meanwhile, the potential mechanism of YNT in the treatment of migraine was studied by combining network pharmacology and in vitro and in vivo experiments.

Electroacupuncture Alleviates CFA-Induced Inflammatory Pain via PD-L1/PD-1-SHP-1 Pathway.

Inflammatory pain is difficult to treat clinically, but electroacupuncture (EA) has been demonstrated to be effective in alleviating inflammatory pain. Programmed cell death ligand-1 (PD-L1) and its downstream signal, Src homology region two domain-containing phosphatase-1 (SHP-1) have a critical role in relieving inflammatory pain. However, whether the PD-L1/PD-1-SHP-1 pathway mediates the analgesic and anti-inflammatory effects of EA in inflammatory pain remains unclear. Here, we observed that EA reversed the complete Freund's adjuvant (CFA)-induced hyperalgesia. EA reduced the expression of IL-6, iNOS, and NF-κB pathway in dorsal root ganglia (DRG) on day 7 after CFA injection but had no effect on the expression of IL-6, iNOS, and NF-κB PP65 on day 21 after CFA injection. Moreover, EA upregulated the protein levels of the PD-L1/PD-1-SHP-1 pathway on day 7 and day 21 after CFA injection. Furthermore, EA upregulated PD-L1 expression in calcitonin gene-related peptide (CGRP)+ but not in isohaemagglutinin B4 (IB4)+ and NF200+ neurons on day 7 and day 21 after CFA injection. Intrathecal injection of the PD-L1/PD-1 inhibitor BMS-1 (50 or 100 µg) blocked the EA-induced analgesic effect, significantly increased IL-6 and iNOS levels, and reduced the levels of PD-L1/PD-1-SHP-1. BMS-1 (50 or 100 µg) significantly reduced the expression of PD-L1 in IB4+, CGRP+, and NF200+ neurons. Our results show that EA's anti-inflammatory and analgesic effects are associated with activating the PD-L1/PD-1-SHP-1 pathway and suppressing its regulated neuroinflammation. This study provides a new potential therapeutic target for treating inflammatory pain.

Effectiveness of acupuncture in migraine rats: A systematic review.

OBJECTIVE: To systematically evaluate the effectiveness and potential underlying mechanisms of acupuncture in the treatment of experimental model of migraine in rats. METHODS: Nine electronic databases, including CNKI (China National Knowledge Infrastructure), WanFang, VIP (Chinese Scientific Journals Database), Sinomed, PubMed, Cochrane Library, Web of Science and EBSCO, were searched for randomized experimental studies on migraine in rats involving acupuncture intervention. The search period ranged from inception to June 2022. The methodological quality was assessed using the SYRCLE's risk of bias tool for animal studies. Data were analyzed using the Revman 5.3 software. RESULTS: A total of 13 studies were included in this analysis. Findings from the available experimental studies documented that acupuncture significantly reduced behavior scores of rats with migraine (MD = -15.01, 95%CI = [-18.01, -12.01], P<0.00001) and downregulated the expression of calcitonin gene-related peptide (CGRP) (MD = -16.14, 95%CI = [-21.45, -10.83], P<0.00001), substance P (SP) (MD = -11.47, 95%CI = [-15.97, -6.98], P<0.00001) and nitric oxide (NO) (MD = -3.02, 95%CI = [-3.79, -2.26], P<0.00001) in serum, and stimulatory G protein (Gsa) (MD = -62.90, 95%CI = [-69.88, -55.92], P<0.00001) in brainstem. Acupuncture also significantly increased the content of inhibitory G protein (Gia) (MD = 24.01, 95%CI = [20.10, 27.92], P<0.00001) in brainstem and 50% paw withdrawal threshold (50%PWT) (MD = 1.96, 95%CI = [1.15, 2.77], P<0.00001). CONCLUSION: Acupuncture can effectively improve the behavioral performance of rates with migraine, and its mechanism of action might involve the inhibition of meningeal vasodilation and inflammatory factors, and the reduction of neurogenic inflammation.

Neuromechanical Dimorphism of Hypoglycemic Effect of Electroacupuncture.

INTRODUCTION: Electroacupuncture (EA) has a favorable impact on blood glucose stability. Blood glucose homeostasis is linked to sexual dimorphism. The majority of research has, however, focused on male participants, and sex differences have not been adequately taken into account. METHODS: Here, we investigated how EA intervention affected pancreatic metabolic stress and explored if there were any sex-related changes in the maintenance of pancreatic function following intraperitoneal injection of a 10 g/kg glucose solution. RESULTS: The transient receptor potential vanilloid 1 (TRPV1) calcitonin gene-related peptide (CGRP)-ß cell pathway of the male pancreas is vital to maintain glucose metabolism in mice. In contrast, there is a sex bias in TRPV1, which implies that female mice have additional routes for preserving glucose homeostasis. EA is ineffective on Trpv1-/- male mice. It also revealed that TRPV1 in male mice served as a crucial mediator for the EA control of blood glucose. Meanwhile, the sympathetic marker tyrosine hydroxylase showed higher expression in the male pancreas, while the cholinergic marker choline acetyltransferase is expressed predominantly in female mice. Injecting γ-aminobutyric acid into the paraventricular nucleus of male mice caused a disruption in blood glucose and a lack of response to EA. It verified that male mice had a more pronounced sympathetic innervation of the pancreas than female mice. CONCLUSION: Our research has demonstrated that the TRPV1 sensory afferent nerve and sympathetic efferent nerve are capable of maintaining glucose homeostasis, exhibiting a distinct sexual dimorphism. Furthermore, this regulation is contingent on the EA effect.

Ratanasampil is more effective than flunarizine in relieving migraine.

Aims: Migraine is a common neurological disorder with high incidence in population. This study aimed to investigate the therapeutic efficacy of Tibetan medicine Ratanasampil (RNSP) and to identify the serum biomarkers for diagnosis and response assessment.Materials and methods: We prospectively recruited 108 migraine patients living at high altitude (2,260 m), including 40 patients for RNSP group, 40 patients for flunarizine (FLZ) group, and 28 patients for placebo group. Serum levels of 5-hydroxytryptamine (5-HT), brain-derived neurotrophic factor (BDNF), calcitonin gene related peptide (CGRP), nerve growth factor (NGF) and ß-endorphin (ß-EP) before and after therapy were measured.Results: In comparison with placebo, both FLZ and RNSP significantly reduced the migraine days, HIT-6 score and verbal rating scale, headache intensity, duration, accompanying symptoms and headache score in four and eight weeks treatment. RNSP showed no significant difference to FLZ in the above parameters after four weeks treatment, but showed significantly better relief after eight weeks treatment. The overall effective rate of RNSP (92.5%) was also significantly higher than FLZ (74.4%, p < 0.05), mainly due to significantly higher ratio of patients with full recovery. The serum levels of biomarkers, including 5-HT, BDNF, NGF and ß-EP, significantly elevated after eight weeks of treatment with RNSP, whereas the level of CGRP significantly decreased. The serum level of 5-HT exhibited significantly bigger percentage changes than other markers.Conclusion: In conclusion, RNSP was more effective than FLZ in relieving migraine after eight weeks continuous treatment. Serum 5-HT, BDNF, CGRP, NGF and ß-EP were effective markers reflecting the response to RNSP and FLZ therapy.

Pitongshu Alleviates the Adverse Symptoms in Rats with Functional Dyspepsia Through Regulating Visceral Hypersensitivity Caused by 5-HT Overexpression.

AIM: The aim of the study was to explore the efficacy as well as the mechanism of action of Pitongshu (PTS) on rats with functional dyspepsia (FD) induced by iodoacetamide gavage and tail clamping. METHODS: The bioactive components of PTS were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), whereas the potential targets of PTS were obtained from the Similarity Ensemble Approach (SEA), TCMSP, and Swiss Target Prediction Database. The disease targets were obtained from the DisGeNET database, whereas Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed using the R Software. The method of iodoacetamide gavage combined with tail clamping was used to establish the FD rat model in this study. Body weight, food intake, gastrointestinal motility, gastric acidity and secretion, and the mechanical pain threshold of rats were measured. The open-field test was also performed. The stomach and duodenum were histologically observed. The levels of serotonin (5-HT), Calcitonin Gene-Related Peptide (CGRP), Motilin (MTL), and Gastrin (GAS) in gastric tissues were detected by ELISA. RESULTS: A total of 139 bioactive components and 17 potential targets of PTS were identified through a network pharmacology approach. The results of GO and KEGG enrichment analyses indicated that PTS could reduce the 5-HT secretion of gastric tissues through the serotonergic synaptic pathway and alleviate the symptoms of FD, indicating that PTS plays a therapeutic role. The results of animal experiments showed that PTS could increase body weight and food intake, improve autonomous activity, and decrease gastric acidity and secretion in FD rats. Furthermore, gastric sensitivity increased in FD rats, and PTS treatment could significantly decrease it. The results of ELISA showed that the overexpression of 5-HT and CGRP was decreased after PTS treatment in FD rats. Lastly, PTS could significantly improve gastrointestinal motility, as well as the levels of GAS and MTL in FD rats. CONCLUSION: PTS may reduce 5-HT secretion by regulating the serotonergic synaptic pathway, thereby reducing visceral sensitivity and alleviating the symptoms of FD.

Management of chronic migraine.

Chronic migraine is a neurologic disorder associated with considerable disability, lost productivity, and a profound economic burden worldwide. The past five years have seen a dramatic expansion in new treatments for this often challenging condition, among them calcitonin gene related peptide antagonists and neuromodulatory devices. This review outlines the epidemiology of and diagnostic criteria and risk factors for chronic migraine. It discusses evidence based drug and non-drug treatments, their advantages and disadvantages, and the principles of patient centered care for adults with chronic migraine, with attention to differential diagnosis and comorbidities, clinical reasoning, initiation and monitoring, cost, and availability. It discusses the international guidelines on drug treatment for chronic migraine and evaluates non-drug treatments including behavioral and complementary therapies and lifestyle modifications. Finally, it discusses the management of chronic migraine in special populations, including pediatrics, pregnancy, and older people, and considers future questions and emerging research in the field.