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.

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.

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.

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).

[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.

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.

Randomized controlled trial: moxibustion and acupuncture for the treatment of Crohn's disease.

AIM: To evaluate the clinical efficacy and safety of acupuncture and moxibustion for the treatment of active Crohn's disease (CD). METHODS: Ninety-two patients were equally and randomly divided into the treatment group and received herb-partitioned moxibustion combined with acupuncture, and the control group received wheat bran-partitioned moxibustion combined with superficial acupuncture. The patients received three treatment sessions per week for 12 wk and were followed up for 24 wk. The main outcome was evaluated using the CD Activity Index (CDAI) score, and the secondary outcomes were evaluated using laboratory indicators such as hemoglobin (HGB), C-reactive protein (CRP), erythrocyte sedimentation rate, quality-of-life, endoscopic ratings, and intestinal histology scores. RESULTS: The CDAI scores of both the treatment and control groups were significantly reduced after treatment compared with those measured before treatment. However, the degree of improvement in the treatment group was significantly greater than that of the control group. The improvement in symptoms in patients of the treatment group was sustained at follow-up, whereas that of the control group was not. The overall efficacy of the treatment was significantly greater than that of the control. Both groups demonstrated significant improvements in quality-of-life ratings after treatment, but the improvement was significantly greater in the treatment group than in the control group. In addition, the patients in the treatment group showed significantly increased HGB and significantly decreased CRP levels and histopathological scores at the end of treatment, whereas the control group did not exhibit significant changes. CONCLUSION: Moxibustion with acupuncture provided significant therapeutic benefits in patients with active CD beyond the placebo effect and is therefore an effective and safe treatment for active CD.