ALIX and TSG101 are essential for cellular entry and replication of two porcine alphacoronaviruses.

Alphacoronaviruses are the primary coronaviruses responsible for causing severe economic losses in the pig industry with the potential to cause human outbreaks. Currently, extensive studies have reported the essential role of endosomal sorting and transport complexes (ESCRT) in the life cycle of enveloped viruses. However, very little information is available about which ESCRT components are crucial for alphacoronaviruses infection. By using RNA interference in combination with Co-immunoprecipitation, as well as fluorescence and electron microscopy approaches, we have dissected the role of ALIX and TSG101 for two porcine alphacoronavirus cellular entry and replication. Results show that infection by two porcine alphacoronaviruses, including porcine epidemic diarrhea virus (PEDV) and porcine enteric alphacoronavirus (PEAV), is dramatically decreased in ALIX- or TSG101-depleted cells. Furthermore, PEDV entry significantly increases the interaction of ALIX with caveolin-1 (CAV1) and RAB7, which are crucial for viral endocytosis and lysosomal transport, however, does not require TSG101. Interestingly, PEAV not only relies on ALIX to regulate viral endocytosis and lysosomal transport, but also requires TSG101 to regulate macropinocytosis. Besides, ALIX and TSG101 are recruited to the replication sites of PEDV and PEAV where they become localized within the endoplasmic reticulum and virus-induced double-membrane vesicles. PEDV and PEAV replication were significantly inhibited by depletion of ALIX and TSG101 in Vero cells or primary jejunal epithelial cells, indicating that ALIX and TSG101 are crucial for PEDV and PEAV replication. Collectively, these data highlight the dual role of ALIX and TSG101 in the entry and replication of two porcine alphacoronaviruses. Thus, ESCRT proteins could serve as therapeutic targets against two porcine alphacoronaviruses infection.

KPNA2 suppresses porcine epidemic diarrhea virus replication by targeting and degrading virus envelope protein through selective autophagy.

IMPORTANCE: Porcine epidemic diarrhea, characterized by vomiting, dehydration, and diarrhea, is an acute and highly contagious enteric disease caused by porcine epidemic diarrhea virus (PEDV) in neonatal piglets. This disease has caused large economic losses to the porcine industry worldwide. Thus, identifying the host factors involved in PEDV infection is important to develop novel strategies to control PEDV transmission. This study shows that PEDV infection upregulates karyopherin α 2 (KPNA2) expression in Vero and intestinal epithelial (IEC) cells. KPNA2 binds to and degrades the PEDV E protein via autophagy to suppress PEDV replication. These results suggest that KPNA2 plays an antiviral role against PEDV. Specifically, knockdown of endogenous KPNA2 enhances PEDV replication, whereas its overexpression inhibits PEDV replication. Our data provide novel KPNA2-mediated viral restriction mechanisms in which KPNA2 suppresses PEDV replication by targeting and degrading the viral E protein through autophagy. These mechanisms can be targeted in future studies to develop novel strategies to control PEDV infection.

Porcine Enteric Alphacoronavirus Entry through Multiple Pathways (Caveolae, Clathrin, and Macropinocytosis) Requires Rab GTPases for Endosomal Transport.

Porcine enteric alphacoronavirus (PEAV) is a new bat HKU2-like porcine coronavirus, and its endemic outbreak has caused severe economic losses to the pig industry. Its broad cellular tropism suggests a potential risk of cross-species transmission. A limited understanding of PEAV entry mechanisms may hinder a rapid response to potential outbreaks. This study analyzed PEAV entry events using chemical inhibitors, RNA interference, and dominant-negative mutants. PEAV entry into Vero cells depended on three endocytic pathways: caveolae, clathrin, and macropinocytosis. Endocytosis requires dynamin, cholesterol, and a low pH. Rab5, Rab7, and Rab9 GTPases (but not Rab11) regulate PEAV endocytosis. PEAV particles colocalize with EEA1, Rab5, Rab7, Rab9, and Lamp-1, suggesting that PEAV translocates into early endosomes after internalization, and Rab5, Rab7, and Rab9 regulate trafficking to lysosomes before viral genome release. PEAV enters porcine intestinal cells (IPI-2I) through the same endocytic pathway, suggesting that PEAV may enter various cells through multiple endocytic pathways. This study provides new insights into the PEAV life cycle. IMPORTANCE Emerging and reemerging coronaviruses cause severe human and animal epidemics worldwide. PEAV is the first bat-like coronavirus to cause infection in domestic animals. However, the PEAV entry mechanism into host cells remains unknown. This study demonstrates that PEAV enters into Vero or IPI-2I cells through caveola/clathrin-mediated endocytosis and macropinocytosis, which does not require a specific receptor. Subsequently, Rab5, Rab7, and Rab9 regulate PEAV trafficking from early endosomes to lysosomes, which is pH dependent. The results advance our understanding of the disease and help to develop potential new drug targets against PEAV.

P2X7 and P2Y1 receptors in DRG mediate electroacupuncture to inhibit peripheral sensitization in rats with IBS visceral pain.

Although multiple purinergic receptors mediate the analgesic effects of acupuncture, it remains unclear whether there is mutual interaction between purinergic receptors to jointly mediate the electroacupuncture inhibition of peripheral sensitization in visceral pain. Visceral hypersensitivity was induced by intracolonic 2,4,6-trinitrobenzene sulfonic acid (TNBS) in rat. The antinociception effect of electroacupuncture on visceral pain was evaluated by morphology, behaviors, neuroelectrophysiology and molecular biology techniques. After labeling the colon-related primary sensory neurons with neural retrograde tracer and employing neuropharmacology, neuroelectrophysiology, and molecular biotechnology, the mechanisms of P2X7R, P2Y1R, and P2X3R in colon-related dorsal root ganglion (DRG) neurons alleviating visceral hypersensitivity of irritable bowel syndrome (IBS) by electroacupuncture at Zusanli and Sanyinjiao acupoints.were elucidated from the perspective of peripheral sensitization. Electroacupuncture significantly inhibited TNBS-induced colonic hypersensitivity in rats with IBS, and Satellite Glial Cells (SGCs) in DRG were found to be involved in electroacupuncture-mediated regulation of the electrophysiological properties of neurons. P2X7R was found to play a pain-inducing role in IBS visceral hypersensitivity by affecting P2X3R, and electroacupuncture exerted an analgesic effect by inhibiting P2X7R activation. P2Y1R was found to play an analgesic role in the process of visceral pain, mediating electroacupuncture to relieve visceral hypersensitivity. P2Y1R relieved visceral pain by inhibiting P2X3R in neurons associated with nociception, with P2X7R identified as upstream of P2Y1R up-regulation by electroacupuncture. Our study suggests that the P2X7R → P2Y1R → P2X3R inhibitory pathway in DRG mediates the inhibition of peripheral sensitization by electroacupuncture in rats with IBS visceral hypersensitivity.

Spinal cord astrocyte P2X7Rs mediate the inhibitory effect of electroacupuncture on visceral hypersensitivity of rat with irritable bowel syndrome.

This study explored the role of P2X7 receptors in spinal cord astrocytes in the electroacupuncture-induced inhibition of visceral hypersensitivity (VH) in rats with irritable bowel syndrome (IBS). Visceral hypersensitivity of IBS was intracolonically induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS). Visceromotor responses to colorectal distension (CRD-20,40,60,80 mmHg) and abdominal withdrawal reflex scoring (AWRs) were recorded after electroacupuncture at bilateral Zusanli (ST36) and Sanyinjiao (SP6) acupoints to evaluate the analgesic effect of electroacupuncture on visceral pain in rats with IBS. Fluorocitric acid (FCA), an astrocyte activity inhibitor, was injected intrathecally before electroacupuncture intervention and AWRs were recorded. Western blot and real-time qPCR were used to detect the expression of NMDA and P2X7 receptor to observe the regulation effect of electroacupuncture on NMDA receptor in the spinal cord of rats with visceral hypersensitivity. Intrathecal injection of P2X7 agonist or antagonist was administered before electroacupuncture treatment. To observe the effect of P2X7 receptor in spinal astrocytes on the inhibition of visceral hyperalgesia by electroacupuncture, the changes of AWR score, NMDA receptor in the spinal cord, and GFAP expression in astrocytes were detected. Inflammation of the colon had basically subsided at day 21 post-TNBS; persistent visceral hypersensitivity could be suppressed by electroacupuncture. This analgesic effect could be inhibited by FCA. The analgesic effect, downregulation of NMDA receptor NR1 subunit, and P2X7 protein of electroacupuncture were all reversed by FCA. P2X7 receptor antagonist A740003 can cooperate with EA to carry out analgesic effect in rats with visceral pain and downregulate the expression of NR1, NR2B, and GFAP in spinal dorsal horn. However, the P2X7 receptor agonist BzATP could partially reverse the analgesic effect of EA, inhibiting the downregulatory effect of EA on the expression of NR1, NR2B, and GFAP. These results indicate that EA may downregulate the expression of the NMDA receptor by inhibiting the P2X7 receptor in the spinal cord, thereby inhibiting spinal cord sensitization in IBS rats with visceral pain, in which astrocytes are an important medium.

Porcine alveolar macrophages host proteins interacting with African swine fever virus p72.

Introduction: African swine fever virus (ASFV) is a highly contagious virus that spreads rapidly and has a mortality rate of up to 100% in domestic pigs, leading to significant economic losses in the pig industry. The major capsid protein p72 of ASFV plays a critical role in viral invasion and immune evasion. Methods: In this study, we used yeast two-hybrid screening to identify host proteins interacting with p72 in porcine alveolar macrophages (PAMs) and verified these proteins using confocal microscopy and immunoprecipitation techniques. Results and Discussion: We validated 13 proteins that interact with p72, including CD63, B2M, YTHDF2, FTH1, SHFL, CDK5RAP3, VIM, PELO, TIMP2, PHYH, C1QC, CMAS, and ERCC1. Enrichment analysis and protein-protein interaction network analysis of these interacting proteins revealed their involvement in virus attachment, invasion, replication, assembly, and immune regulation. These findings provide new insights into the function of p72 and valuable information for future research on the interaction between ASFV and host proteins.

Subcellular localization of viral proteins after porcine epidemic diarrhea virus infection and their roles in the viral life cycle.

Porcine epidemic diarrhea virus (PEDV) is one of the most devastating diseases affecting the pig industry globally. Due to the emergence of novel strains, no effective vaccines are available for prevention and control. Investigating the pathogenic mechanisms of PEDV may provide insights for creating clinical interventions. This study constructed and expressed eukaryotic expression vectors containing PEDV proteins (except NSP11) with a 3' HA tag in Vero cells. The subcellular localization of PEDV proteins was examined using endogenous protein antibodies to investigate their involvement in the viral life cycle, including endocytosis, intracellular trafficking, genome replication, energy metabolism, budding, and release. We systematically analyzed the potential roles of all PEDV viral proteins in the virus life cycle. We found that the endosome sorting complex required for transport (ESCRT) machinery may be involved in the replication and budding processes of PEDV. Our study provides insight into the molecular mechanisms underlying PEDV infection. IMPORTANCE: The global swine industry has suffered immense losses due to the spread of PEDV. Currently, there are no effective vaccines available for clinical protection. Exploring the pathogenic mechanisms of PEDV may provide valuable insights for clinical interventions. This study investigated the involvement of viral proteins in various stages of the PEDV lifecycle in the state of viral infection and identified several previously unreported interactions between viral and host proteins. These findings contribute to a better understanding of the pathogenic mechanisms underlying PEDV infection and may serve as a basis for further research and development of therapeutic strategies.

Secondary hand infection with Eikenella corrodens and Staphylococcus aureus in a patient with Behcet's disease: a case report.

We report the first case of a woman with Behcet's disease (BD) with multiple hand ulcers secondary to coninfection by Eikenella corrodens and Staphylococcus aureus resulting in necrotizing fasciitis. She had a long history of BD including long courses of prednisone and immunosuppressants. The patient was hospitalized for multiple superficial ulcers, swelling, and infection of the hands. After admission, pus culture examination revealed rare coinfection by E. corrodens and S. aureus. We administered moxifloxacin and vancomycin to control infection and methylprednisolone to control BD. We performed incision, drainage, and debridement of the ulcer surface on the hands to reduce the pus on the wound surface. E. corrodens infections occur in immunosuppressed patients and contribute to coinfections, particularly in patients with BD in whom destruction of the skin immune barrier increases risk to secondary infections. For severe and complicated hand infections, efforts should be made to identify pathogenic microorganisms so appropriate antibiotics and other interventions can be given to control the infection.

Interaction network of African swine fever virus structural protein p30 with host proteins.

African swine fever virus (ASFV) is a complex nucleocytoplasmic large DNA virus (NCLDV) that causes a lethal hemorrhagic disease that is currently threatening the global pig industry. ASFV structural protein p30 is a membrane phosphoprotein that suggests it may play a regulatory role, possibly in signal transduction. Despite its significance in internalization into host cells, the interaction between p30 and host proteins is relatively unknown. In this study, we describe the application of a DUALmembrane yeast two-hybrid assay to screen a primary porcine alveolar macrophages cDNA library and analyze the interactome of p30 protein. Our data identify seven host cellular proteins (DAB2, RPSA, OAS1, PARP9, CAPG, ARPC5, and VBP1) that putatively interact with the p30. We further verified the interaction between p30 and host proteins by laser confocal microscopy, co-immunoprecipitation, and GST-pulldown assay. To further understand the relationship between host proteins and p30, we drew the interaction network diagram and analyzed the functional enrichment of each host protein. Enrichment analysis of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes indicated that host proteins were mainly related to endocytosis, actin cytoskeleton regulation, and innate immunity. Collectively, we identified the interaction between p30 and host cell protein using a membrane protein yeast two-hybrid system, which increases our knowledge of the interaction between ASFV and the host and informs future research on antiviral strategies.

Acupuncture and Moxibustion Inhibited Intestinal Epithelial-Mesenchymal Transition in Patients with Crohn's Disease Induced by TGF- ß 1/Smad3/Snail Pathway: A Clinical Trial Study.

OBJECTIVE: To explore whether acupuncture combined with moxibustion could inhibit epithelialmesenchymal transition in Crohn's disease by affecting the transforming growth factor ß 1 (TGF- ß 1)/Smad3/Snail pathway. METHODS: Sixty-three patients with Crohn's disease were randomly divided into an observation group (31 cases) receiving moxibustion at 43 °C combined with acupuncture, and a control group (32 cases) receiving moxibustion at 37 °C combined with sham acupuncture using a random number table. Patients were treated for 12 weeks. Crohn's Disease Activity Index (CDAI) was used to evaluate disease activity. Hematoxylin-eosin staining and transmission electron microscopy were utilized to observe the morphological and ultrastructural changes. Immunohistochemistry was used to detect the expression of transforming growth factor ß 1 (TGF-ß 1), T ß R1, T ß R2, Smad3, Snail, E-cadherin and fibronectin in intestinal mucosal tissues. RESULTS: The decrease of the CDAI score, morphological and ultrastructural changes were more significant in observation group. The expression levels of TGF- ß 1, Tß R2, Smad3, and Snail in the observation group were significantly lower than those before the treatment (P<0.05 or P<0.01). After treatment, the expression levels of TGF-ß 1, TßR2, and Snail in the observation group were significantly lower than those in the control group (all P<0.05); compared with the control group, the expression of fibronectin in the observation group was significantly decreased, and the expression of E-cadherin was significantly increased (all P<0.05). CONCLUSIONS: Moxibustion at 43 °C combined with acupuncture may suppress TGF-ß 1/Smad3/Snail pathway-mediated epithelial-mesenchymal transition of intestinal epithelial cells in Crohn's disease patients by inhibiting the expression levels of TGF-ß 1, Tß R2, Smad3, and Snail. (Registration No. ChiCTR-IIR-16007751).

Long-term effect of moxibustion on irritable bowel syndrome with diarrhea: a randomized clinical trial.

BACKGROUND: Moxibustion is increasingly used for treatment of irritable bowel syndrome (IBS). This study investigated the long-term effects of moxibustion for IBS with diarrhea (IBS-D). METHODS: Patients with IBS-D were assigned to receive moxibustion or sham moxibustion (52 each, 3× per week, 6 weeks) and were followed up to 24 weeks. The acupoints were bilateral ST25 and ST36, body surface temperatures at acupoints were 43°C ± 1°C and 37°C ± 1°C for the moxibustion and sham groups, respectively. Primary outcome was changes in IBS Adequate Relief (IBS-AR) from baseline to 6 weeks. Secondary outcomes included the following: IBS symptom severity scale (IBS-SSS), Bristol stool form scale (BSS), IBS quality of life (IBS-QOL), and Hospital Anxiety and Depression Scale (HADS). RESULTS: Based on an intention-to-treat analysis, the rate of IBS-AR in the moxibustion group was significantly higher than the sham group at 6 weeks (76.9% versus 42.3%; p < 0.001); the mean decrease of total IBS-BSS score in the moxibustion group was lower than that of the sham group (-116.9 versus -61.5; p < 0.001), both of which maintained throughout the follow-up period. Five specific domains of the IBS-SSS were lower in the moxibustion group than the sham, throughout (p < 0.001). At week 6, the rate of reduction >50 points in IBS-SSS of the treatment group was significantly higher than that of the sham (p < 0.001), which persisted throughout the follow-up period. Similar long-lasting improvements were observed in BSS, stool frequency, and stool urgency (p < 0.001). Improvements of IBS-QOL and HADS were comparable between the groups. CONCLUSIONS: Moxibustion treatment benefits the long-term relief of symptoms in IBS-D patients. TRIAL REGISTRATION: Clinical trials.gov (NCT02421627). Registered on 20 April 2015.

Herb-Partitioned Moxibustion Improves Crohn's Disease-Associated Intestinal Fibrosis by Suppressing the RhoA/ROCK1/MLC Pathway.

BACKGROUND AND AIMS: Intestinal fibrosis is one of the severe and common complications of Crohn's disease (CD), but the etiology and pathogenesis remain uncertain. The study intended to examine whether the effect of herb-partitioned moxibustion on rats with CD-associated intestinal fibrosis is associated with the RhoA/ROCK1/MLC pathway. METHODS: All experimental rats were randomly allocated into the normal control group (NC), model control group (MC), and herb-partitioned moxibustion group (HPM). Intestinal fibrosis was established in rats with CD by repeated rectal administrations of 2,4,6-trinitrobenzenesulfonic acid (TNBS). Herb-partitioned moxibustion was applied at the Qihai (CV6) and Tianshu (ST25) acupoints once daily for 10 days in the HPM group. In this study, histological changes were examined by hematoxylin and eosin (HE) staining; then, Masson's trichrome staining was used to assess the degree of fibrosis in each group. Experimental methods of immunohistochemistry, western blotting, and real-time PCR were applied to detect the levels of α-SMA, collagen III, RhoA, ROCK1, and p-MLC. Moreover, the double immunofluorescent staining for the colocalization of both α-SMA and ROCK1 was performed. RESULTS: Contrasted with the normal controls, the collagen deposition and fibrosis scores were increased in colonic tissue of model rats, and HPM decreased the collagen deposition and fibrosis scores. The protein of α-SMA and collagen III in the MC group exceeds that of the NC group; HPM decreased the expression of α-SMA and collagen III in rats with intestinal fibrosis. Similarly, the expression of RhoA, ROCK1, and p-MLC in model rats was obviously increased compared with normal controls; the expression of RhoA, ROCK1, and p-MLC was decreased after HPM. The coexpression of α-SMA and ROCK1 in rats with intestinal fibrosis was higher than normal rats. CONCLUSION: HPM improves CD-associated intestinal fibrosis by suppressing the RhoA/ROCK1/MLC pathway.

[Effect of acupuncture-moxibustion on negative emotions and plasma tryptophan metabolism in patients with Crohn's disease at active stage].

OBJECTIVE: To observe the effect of acupuncture-moxibustion on negative emotions and plasma tryptophan (Trip)-kynurenine (Kyn) metabolism in the patients with Crohn's disease (CD) at the mild and moderate active stage. METHODS: A total of 66 CD patients were randomized into an observation group (33 cases, 1 case dropped off) and a control group (33 cases, 2 cases dropped off). In the observation group, acupuncture was applied in combination with moxibustion. In the control group, the sham-acupuncture was used in combination with sham-moxibustion. In both of the observation group and the control group, acupuncture was applied to Zhongwan (CV 12), Shangjuxu (ST 37), Sanyinjiao (SP 6), Gongsun (SP 4), Hegu (LI 4), Quchi (LI 11), Taixi (KI 3) and Taichong (LR 3), and moxibustion was applied to Tianshu (ST 25) and Zusanli (ST 36). The treatment was given once every two days, 3 times a week, totally for 12 weeks. Separately, before and after treatment, the score of the hospital anxiety-depression scale (HADS) and the score of intestinal core symptoms (degree of abdominal pain and frequency of diarrhea) were observed in the patients of the two groups. The concentration of plasma indoleamine 2,3-dioxygenase 1 (IDO1) and the ratios of Kyn/Trp, QuinA/Kyn, KynA/Kyn and KynA/QuinA were compared between the two groups. RESULTS: Compared with before treatment, the scores of HADS-A and HADS-D in the observation group and the score of HADS-A in the control group were all reduced after treatment (P<0.01, P<0.05). The scores of abdominal pain degree in the two groups and score of diarrhea frequency in the observation group were all reduced after treatment (P<0.001). After treatment, the reducing ranges of the score of HADS-A and the scores of abdominal pain degree and diarrhea frequency in the observation group were all larger than the control group (P<0.01, P<0.05). Compared with before treatment, the plasma IDO1 concentration in the two groups and the ratios of plasma Kyn/Trp and QuinA/Kyn in the observation group were all reduced after treatment (P<0.001, P<0.05, P<0.01), the ratios of plasma KynA/Kyn and KynA/QuinA were increased after treatment in the observation group (P<0.05, P<0.01). After treatment, the changes in IDO1 concentration and the ratios of plasma QuinA/Kyn and KynA/QuinA in the observation were larger than the control group (P≤0.01, P<0.05). In the observation group, the difference in the ratio of plasma KynA/Kyn before and after treatment was negatively related to the improvement value of HADS-D (r =-0.67, P<0.05). After treatment, plasma IDO1 concentration was positively related to HADS-A in the observation group (r =0.65, P<0.05). CONCLUSION: Acupuncture and moxibustion relieve the negative emotions of anxiety and depression in CD patients at mild and moderate active stage, which is probably related to the regulation of plasma Trp-Kyn metabolic pathway.

Electroacupuncture Improves IBS Visceral Hypersensitivity by Inhibiting the Activation of Astrocytes in the Medial Thalamus and Anterior Cingulate Cortex.

OBJECTIVE: To explore whether the effect of electroacupuncture (EA) on visceral hypersensitivity (VH) in rats with irritable bowel syndrome (IBS) is related to the changes of astrocyte activation in the medial thalamus (MT) and anterior cingulate cortex (ACC). METHOD: Male Sprague-Dawley rats were randomly divided into the normal control (NC) group, model control (MC) group, electroacupuncture (EA) group, and fluorocitrate (FCA) group. A model of visceral hypersensitivity was established by neonatal colorectal irritation. In the EA group, needles were inserted into the skin at the Tianshu (ST25) and Shangjuxu (ST37) acupoints, once a day for 7 days. The FCA group received intrathecal injection of FCA on the 1st, 4th, and 7th days. Visceral hypersensitivity was evaluated by the abdominal withdrawal reflex (AWR), and glial fibrillary acidic protein (GFAP) mRNA and protein levels in the MT and ACC were detected by real-time PCR, immunohistochemistry, and western blots. RESULTS: The AWR score in the MC group was significantly higher than in the NC group, and EA and FCA reduced the AWR score of VH rats. GFAP mRNA and protein levels in the MT and ACC of rats in the MC group were significantly increased compared with the NC group. After either electroacupuncture or fluorocitrate, GFAP mRNA and protein levels in the MT and ACC were both clearly reduced. CONCLUSION: Electroacupuncture alleviates IBS visceral hypersensitivity by inhibiting the activation of astrocytes in the MT and ACC.

P2X3 Receptor in Primary Afferent Neurons Mediates the Relief of Visceral Hypersensitivity by Electroacupuncture in an Irritable Bowel Syndrome Rat Model.

BACKGROUND: Electroacupuncture (EA) has been confirmed effectiveness in the treatment of irritable bowel syndrome (IBS), and P2X3 receptors in the peripheral and central neurons participate in the acupuncture-mediated relief of the visceral pain in IBS. OBJECTIVE: To reveal the neurobiological mechanism that P2X3 receptor of colonic primary sensory neurons in the dorsal root ganglia of the lumbosacral segment is involved in the alleviation of visceral hypersensitivity by EA in an IBS rat model. METHODS: The IBS chronic visceral pain rat model was established according to the method of Al-Chaer et al. EA at the bilateral He-Mu points, including ST25 and ST37, was conducted for intervention. The behavioral studies, histopathology of colon, electrophysiology, immunofluorescence histochemistry, and real-time polymerase chain reaction assays were used to observe the role of P2X3 receptor in the colon and related DRG in relieving visceral hypersensitivity by EA. RESULTS: EA significantly reduced the behavior scores of the IBS rats under different levels (20, 40, 60, 80 mmHg) of colorectal distention stimulation and downregulated the expression levels of P2X3 receptor protein and mRNA in colon and related DRG of the IBS rats. EA also regulated the electrical properties of the membranes, including the resting membrane potential, rheobase, and action potential of colon-associated DRG neurons in the IBS rats. CONCLUSION: EA can regulate the P2X3 receptor protein and mRNA expression levels in the colon and related DRG of IBS rats with visceral pain and then regulate the excitatory properties of DRG neurons.

Effect of herb-partitioned moxibustion on dopamine levels and dopamine receptor 1 expression in the colon and central nervous system in rats with Crohn's disease.

OBJECTIVE: To observe the effect of herb-partitioned moxibustion at the Tianshu (ST 25) and Qihai (CV 6) acupoints in rats with Crohn's disease, and explore the underlying mechanism from dopamine (DA) and dopamine receptor 1 (D1R) in the colon, spinal dorsal horn and hypothalamus. METHODS: The rats were randomly divided into the normal, model (CD), herb-partitioned moxibustion (Mox) and mesalazine (Mesa) groups. Damage in the colons was scored and observed by hematoxylin and eosin staining. DA and D1R protein expression in the colonic mucosa were detected by immunohistochemistry. The concentrations of DA and D1R in the spinal dorsal horn and hypothalamus were measured by enzyme-linked immunosorbent assay, and D1R mRNA expression was evaluated by quantitative real-time polymerase chain reaction. RESULTS: In the colon, compared with the normal group, DA, D1R protein expressions and D1R mRNA expression were significantly higher in the model group, while decreased in the Mox group and the Mesa group. In the spinal dorsal horn and hypothalamus, compared with the normal group, the concentrations of DA and D1R, and the D1R mRNA expressions were significantly higher in the model group, and decreased in the Mox group and the Mesa group. CONCLUSION: Herb-partitioned moxibustion at the Tianshu (ST 25) and Qihai (CV 6) acupoints relieved ulceration in CD rats, the underlying mechanism maybe relative with the regulation of DA and D1R in the colon, spinal dorsal horn and hypothalamus by moxibustion.

Neurobiological Mechanism of Acupuncture for Relieving Visceral Pain of Gastrointestinal Origin.

It is currently accepted that the neural transduction pathways of gastrointestinal (GI) visceral pain include the peripheral and central pathways. Existing research on the neurological mechanism of electroacupuncture (EA) in the treatment of GI visceral pain has primarily been concerned with the regulation of relevant transduction pathways. The generation of pain involves a series of processes, including energy transduction of stimulatory signals in the sensory nerve endings (signal transduction), subsequent conduction in primary afferent nerve fibers of dorsal root ganglia, and transmission to spinal dorsal horn neurons, the ascending transmission of sensory signals in the central nervous system, and the processing of sensory signals in the cerebral cortex. Numerous peripheral neurotransmitters, neuropeptides, and cytokines participate in the analgesic process of EA in visceral pain. Although EA has excellent efficacy in the treatment of GI visceral pain, the pathogenesis of the disease and the analgesic mechanism of the treatment have not been elucidated. In recent years, research has examined the pathogenesis of GI visceral pain and its influencing factors and has explored the neural transduction pathways of this disease.

Comparison of the analgesic effects between electro-acupuncture and moxibustion with visceral hypersensitivity rats in irritable bowel syndrome.

AIM: To observe whether there are differences in the effects of electro-acupuncture (EA) and moxibustion (Mox) in rats with visceral hypersensitivity. METHODS: EA at 1 mA and 3 mA and Mox at 43 °C and 46 °C were applied to the Shangjuxu (ST37, bilateral) acupoints in model rats with visceral hypersensitivity. Responses of wide dynamic range neurons in dorsal horns of the spinal cord were observed through the extracellular recordings. Mast cells (MC) activity in the colons of rats were assessed, and 5-hydroxytryptamine (5-HT), 5-hydroxytryptamine 3 receptor (5-HT3R) and 5-HT4R expressions in the colons were measured. RESULTS: Compared with normal control group, responses of wide dynamic range neurons in the dorsal horn of the spinal cord were increased in the EA at 1 mA and 3 mA groups (1 mA: 0.84 ± 0.74 vs 2.73 ± 0.65, P < 0.001; 3 mA: 1.91 ± 1.48 vs 6.44 ± 1.26, P < 0.001) and Mox at 43 °C and 46 °C groups (43 °C: 1.76 ± 0.81 vs 4.14 ± 1.83, P = 0.001; 46 °C: 5.19 ± 2.03 vs 7.91 ± 2.27, P = 0.01). MC degranulation rates and the expression of 5-HT, 5-HT3R and 5-HT4R in the colon of Mox 46 °C group were decreased compared with model group (MC degranulation rates: 0.47 ± 0.56 vs 0.28 ± 0.78, P < 0.001; 5-HT: 1.42 ± 0.65 vs 7.38 ± 1.12, P < 0.001; 5-HT3R: 6.62 ± 0.77 vs 2.86 ± 0.88, P < 0.001; 5-HT4R: 4.62 ± 0.65 vs 2.22 ± 0.97, P < 0.001). CONCLUSION: The analgesic effects of Mox at 46 °C are greater than those of Mox at 43 °C, EA 1 mA and EA 3 mA.

Effects of electroacupuncture on corticotropin-releasing hormone in rats with chronic visceral hypersensitivity.

AIM: To investigate the effect of electroacupuncture on corticotropin-releasing hormone (CRH) in the colon, spinal cord, and hypothalamus of rats with chronic visceral hypersensitivity. METHODS: A rat model of chronic visceral hypersensitivity was generated according to the internationally accepted method of colorectal balloon dilatation. In the 7(th) week after the procedure, rats were randomly divided into a model group (MG), electroacupuncture group (EA), and sham electroacupuncture group (S-EA). After treatment, the abdominal withdrawal reflex (AWR) score was used to assess the behavioral response of visceral hyperalgesia. Immunohistochemistry (EnVision method), ELISA, and fluorescence quantitative PCR methods were applied to detect the expression of CRH protein and mRNA in the colon, spinal cord, and hypothalamus. RESULTS: The sensitivity of the rats to the colorectal distension stimulus applied at different strengths (20-80 mmHg) increased with increasing stimulus strength, resulting in increasing AWR scores in each group. Compared with NG, the AWR score of MG was significantly increased (P < 0.01). After conducting EA, the AWR scores of the rats were decreased compared with MG rats. The relative expression of CRH mRNA in the colon, spinal cord, and hypothalamus of MG rats was significantly increased compared with NG rats (P < 0.01). CRH mRNA in the colon and spinal cord of EA and S-EA rats was decreased to varying degrees (P > 0.05) compared with normal rats (NG). However, the decrease in EA compared with MG rats was statistically significant (P < 0.01). The average optical density of CRH expression in the colon of the MG rats was significantly enhanced compared with NG (P < 0.05), while the average optical density of CRH expression in the EA and S-EA rats was significantly decreased compared with MG rats (P < 0.01, P < 0.05, respectively). Compared with MG rats, the CRH concentration in the spinal cord of EA rats was significantly reduced (P < 0.01), but there was no significant change in S-EA rats (P > 0.05). CONCLUSION: Electroacupuncture at the Shangjuxu acupoint was able to significantly reduce the visceral hypersensitivity in rats, and regulated the expression of CRH protein and mRNA in the colon, spinal cord and hypothalamus at different levels, playing a therapeutic role in this model of irritable bowel syndrome.

Mechanisms underlying the analgesic effect of moxibustion on visceral pain in irritable bowel syndrome: a review.

Irritable bowel syndrome (IBS) is a functional bowel disorder that causes recurrent abdominal (visceral) pain. Epidemiological data show that the incidence rate of IBS is as high as 25%. Most of the medications may lead to tolerance, addiction and toxic side effects. Moxibustion is an important component of traditional Chinese medicine and has been used to treat IBS-like abdominal pain for several thousand years in China. As a mild treatment, moxibustion has been widely applied in clinical treatment of visceral pain in IBS. In recent years, it has played an irreplaceable role in alternative medicine. Extensive clinical studies have demonstrated that moxibustion for treatment of visceral pain is simple, convenient, and inexpensive, and it is being accepted by an increasing number of patients. There have not been many studies investigating the analgesic mechanisms of moxibustion. Studies exploring the analgesic mechanisms have mainly focused on visceral hypersensitivity, brain-gut axis neuroendocrine system, and immune system. This paper reviews the latest developments in moxibustion use for treatment of visceral pain in IBS from these perspectives. It also evaluates potential problems in relevant studies on the mechanisms of moxibustion therapy to promote the application of moxibustion in the treatment of IBS.