The effect and mechanism of Huangqin-Baishao herb pair in the treatment of dextran sulfate sodium-induced ulcerative colitis.

Background: The haungqing (Scutellariae Radix) and baishao (Paeoniae Radix Alba) herb pair (HBHP) is a common prescribed herbal formula or is added to other traditional Chinese medicine (TCM) prescriptions to treat ulcerative colitis (UC). However, the underlying mechanism is unclear. Purpose: Elucidate the efficacy and potential mechanism of HBHP against UC. Methods: First, The UC model of mice induced by dextran sulfate sodium (DSS) was established. The mice were randomly divided into Control group, DSS group, SASP group (390 mg/kg), and HPHP group (1.95 g/kg), with 8 mice per group. Drugs were administrated via oral gavage for 7 days. Then, Disease activity index (DAI), length of the colon, histopathology, and changes in inflammatory cytokines in colonic tissues were analyzed to assess the effect of HBHP on UC. Besides, Network pharmacology was applied to identify the active compounds, core targets of HBHP in the treatment of UC, and the corresponding signaling pathways to explore the underlying mechanisms. Finally, Western blot (WB), immunohistochemistry (IHC) and molecular docking were performed to validate the results. Results: HBHP significantly reduced DAI score and decreased colon length shortening in DSS-induced UC mice. The administration of HBHP was able to effectively alleviated mucosal ulceration and epithelial destruction. In addition, HBHP treatment obviously - reduced the expressions of TNF-α, IL-6, and IL-1ß in colon tissues (p < 0.05 or p < 0.01). 35 bioactive compounds and 290 HBHP targets related to UC were obtained. Among them 3 key active compounds (baicalein, panicolin, and norwogonin) with higher degree values in the drug-compound-target network and 21 hub genes (STAT3, JAK2, SRC, AKT1, PIK3CA, and VEGFA, etc.) were identified. KEGG enrichment analysis suggested that HBHP's mechanisms mainly involve the JAK-STAT pathway. Abnormal activation of JAK/STAT signaling is believed to be involved in the pathogeneses of UC. Notably, WB and IHC showed that HBHP significantly down-regulated the protein expression levels of p-JAK2 (p < 0.05) and p-STAT3 (p < 0.05 or p < 0.01). JAK2 and STAT3 might be core targets for the action of HBHP; this possibility was also supported by molecular docking. Conclusions: HBHP could alleviate DSS-induced UC, reduce tissue inflammation, and its mechanism might primarily be achieved by inhibiting JAK2/STAT3 signaling pathway. Meanwhile, our work revealed that network pharmacology combined with experimental verification is a cogent means of studying the mechanism of TCM.

[Electroacupuncture pretreatment alleviates apoptosis of renal tubular epithelia cells by down-regulating expression of TRPC6 in hyperglycemic mice].

OBJECTIVE: To observe the effect of electroacupuncture (EA) pretreatment on apoptosis of renal tubular epithelial cells in mice with hyperglycemia, so as to explore its mechanisms underlying protecting the kidney from hyperglycemia-induced injury. METHODS: Eighty male C57BL/6 mice were equally and randomly divided into control, model, EA and sham EA groups. The hyperglycemia model was established by intraperitoneal injection of streptozocin (STZ, 50 mg·kg-1·d-1) for 5 consecutive days. Before modeling, EA (2 Hz/15 Hz, 0.3-0.5 mA) was applied to bilateral "Zusanli" (ST36) and "Shenshu" (BL23) for 30 min, once daily for 7 days, while mice in the sham EA group were treated with the same acupoints but without electrical stimulation. The blood glucose values were measured after fasting for 6 hours after 3 days of modeling. The degree of renal tissue injury was observed by microscope after H.E. staining, and the apoptosis level of renal tubular epithelial cells observed by TUNEL staining. The expression levels of transient receptor potential channel 6 (TRPC6) and related apoptotic proteins Caspase-3, Bax and Bcl-2 in the renal tissue were detected by Western blot and immunohistochemistry, separately. RESULTS: Compared with the control group, the blood glucose content and the expression levels of TRPC6, Caspase-3 and Bax proteins, as well as the level of the renal apoptotic cells were significantly increased (P<0.001, P<0.000 1), while the expression level of Bcl-2 protein and the ratio of Bcl-2/Bax were remarkably decreased in the model group (P<0.000 1). In comparison with the model and sham EA groups, the blood glucose content, percentage of apoptotic cells and the expression levels of TRPC6, Caspase-3 and Bax were significantly decreased (P<0.01, P<0.000 1, P<0.05, P<0.001), and the expression level of Bcl-2 and the ratio of Bcl-2/Bax were apparently increased in the EA group (P<0.01, P<0.05, P<0.001). HE statin showed abnormal dilation of the capillary lumen and disappearance of the proximal tubules in the model group, which was relatively milder in the EA group. CONCLUSION: EA pretreatment can lower blood glucose level and reduce renal apoptosis in hyperglycemia mice, which may be related to its effects in down-regulating the expression of TRPC6 and Caspase-3 and up-regulating the ratio of Bcl-2/Bax.

Electroacupuncture Pretreatment Attenuates Cerebral Ischemia-Reperfusion Injury in Rats Through Transient Receptor Potential Vanilloid 1-Mediated Anti-apoptosis via Inhibiting NF-κB Signaling Pathway.

Our previous study showed that electroacupuncture (EA) pretreatment elicited protective effect on cerebral ischemia-reperfusion injury (CIRI) in rats, at least partly, which was associated with transient receptor potential vanilloid 1 (TRPV1)-regulated anti-oxidant stress and anti-inflammation. In this study, we further investigated the possible contribution of TRPV1-mediated anti-apoptosis in EA pretreatment-evoked neuroprotection in CIRI. After EA pretreatment at Baihui (GV20), bilateral Shenshu (BL23) and Sanyinjiao (SP6) acupoints, transient focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) for 2 h followed by reperfusion for 6 h in rats. Then, infarct volume, nerve cell injury, neuronal apoptosis, NF-κB signaling activation, and expression of TRPV1 were evaluated by TTC staining, Hematoxylin-Eosin staining, transmission electron microscopy, immunochemistry, immunofluorescence, and Western blot, respectively. The presented data showed that EA pretreatment significantly reduced infarct volume, relieved nerve cell injury, decreased the expression of pro-apoptotic proteins Bax and cleaved caspase-3, increased the level of anti-apoptotic protein Bcl-2, inhibited NF-κB (p65) transcriptional activity, and curbed TRPV1 expression in MCAO rats. By contrast, enhancement of TRPV1 expression accompanying capsaicin application, the specific TRPV1 agonists, markedly accelerated nerve cell damage, aggravated neuronal apoptosis, prompted nuclear translocation of NF-κB (p65), resulting in the reversion of EA pretreatment-evoked neuroprotective effect in MCAO rats. Thus, we conclude that EA pretreatment-induced downregulation of neuronal TRPV1 expression plays an anti-apoptosis role through inhibiting NF-κB signaling pathway, thereby protecting MCAO rats from cerebral ischemia-reperfusion injury.

[Electroacupuncture preconditioning alleviates kidney injury in hyperglycemic mice].

OBJECTIVE: To explore the protective effect and molecular mechanism of electroacupuncture (EA) preconditioning on renal injury induced by streptozotocin (STZ) in hyperglycemic mice. METHODS: Eighty male C57BL/6 mice were randomly divided into 4 groups: control, model, EA and sham EA groups, n=20 in each group. The hyperglycemic model was established by intraperitoneal injection of 0.1% STZ solution (50 mg/kg) for 5 days. EA preconditioning or sham EA was applied at "Zusanli" (ST36) and "Shenshu" (BL23), once daily for 7 successive days in the EA or sham EA group. Three days after mode-ling, the blood glucose was measured after fasting for 6 hours. The degree of renal injury was observed by HE staining and PAS staining; the expressions of transient receptor potential cation channel 6 (TRPC6) and Nephrin protein in glomerulus were observed by immunohistochemistry; the expressions of TRPC6 and Nephrin protein in renal cortex were detected by Western blot. RESULTS: Compared with the control group, the blood glucose level was significantly increased (P<0.01), the glomerular cross-sectional area was apparently increased (P<0.000 1), the glomerular capillaries dilated, the matrix proliferated, the brush edge of renal tubules disappeared, the proportion of abnormal renal tubules increased (P<0.000 1), and the expressions of TRPC6 and Nephrin in glomeruli and renal tissue were significantly increased (P<0.01) in the model group. Following EA treatment and compared with the model and sham EA groups, the blood glucose was significantly down-regulated (P<0.01), the renal tissue injury was apparently alleviate, the cross-sectional area of glomerulus was reduced (P<0.05), the brush edge of renal tubules changes were obviously improved, the proportion of abnormal renal tubules decreased (P<0.000 1), and the expressions of TRPC6 and Nephrin in glomerulus and renal tissue decreased (P<0.05) in the EA group. CONCLUSION: EA preconditioning can alleviate renal injury in hyperglycemic mice, which is closely related to its effects in reducing renal TRPC6 and Nephrin expressions and inhibiting podocyte activation.

Cortex Mori Radicis extract promotes neurite outgrowth in diabetic rats by activating PI3K/AKT signaling and inhibiting Ca2+ influx associated with the upregulation of transient receptor potential canonical channel 1.

Cortex Mori Radicis extract (CMR) has various pharmacological properties, such as anti­inflammatory, anti­allergic and anti­hyperglycemic effects. However, the effects and mechanisms of CMR in the neuroregeneration of diabetic peripheral neuropathy (DPN) are unclear. In the present study, the effects of CMR on neurite outgrowth of dorsal root ganglia (DRG) neurons in diabetic rats were investigated and its underlying mechanisms were explored. SD rats were subjected to a high­fat diet with low­dose streptozotocin to induce a Type II diabetes model with peripheral neuropathy. CMR was then applied for four weeks continuously with or without injection of small interfere (si)RNA targeting the transient receptor potential canonical channel 1 (TRPC1) via the tail vein. Blood glucose levels, the number of Nissl bodies, neurite outgrowth and growth cone turning in DRG neurons were evaluated. The expression of TRPC1 protein, Ca2+ influx and activation of the PI3K/AKT signaling pathway were also investigated. The results of the present study showed that CMR significantly lowered blood glucose levels, reversed the loss of Nissl bodies, induced neurite outgrowth and restored the response of the growth cone of DRG neurons in diabetic rats. CMR exerted neurite outgrowth­promoting effects by increasing TRPC1 expression, reducing Ca2+ influx and enhancing AKT phosphorylation. siRNA targeting TRPC1 in the CMR group abrogated its anti­diabetic and neuroregenerative effects, suggesting the involvement of TRPC1 in the biological effects of CMR on DPN.

Electroacupuncture Pretreatment Elicits Neuroprotection Against Cerebral Ischemia-Reperfusion Injury in Rats Associated with Transient Receptor Potential Vanilloid 1-Mediated Anti-Oxidant Stress and Anti-Inflammation.

Electroacupuncture (EA) pretreatment, electrical stimulation using metal needle at specific acupoints in advance, possesses the potential to prevent cerebral ischemia-reperfusion injury (CIRI). Transient receptor potential vanilloid 1 (TRPV-1) has been indicated to take part in cerebral protection of EA; however, the detailed mechanisms remain unclear. The aim of this study was to investigate whether neuroprotection of EA pretreatment against CIRI is associated with TRPV-1 and explore the underlying mechanisms. Middle cerebral artery occlusion (MCAO) was performed to induce CIRI after EA pretreatment at Baihui (GV20), bilateral Shenshu (BL23), and Sanyinjiao (SP6) acupoints in rats. Neurological deficit scores, infarct volumes, oxidative stress damage, inflammatory cytokine production, MAPK signaling activation, and the expression of TRPV-1 were assessed. EA pretreatment lowered neurological deficit scores, reduced infarct volumes, impeded oxidative stress injury, inhibited inflammatory cytokine production, curbed P38 phosphorylation, and suppressed TRPV-1 expression in MCAO rats. Attributing to inhibition of TRPV-1 expression, AMG-517 (TRPV-1 antagonist) showed the synergistic effect with EA pretreatment on the neuroprotection against ischemia-reperfusion injury. However, TRPV-1 agonists capsaicin significantly abrogated the neuroprotective effects of EA pretreatment in MCAO rats accompanying enhancement of TRPV-1 expression. These findings indicated EA pretreatment exerted neuroprotection in rats with cerebral ischemia-reperfusion injury, which at least partially were associated with TRPV1-mediated anti-oxidant stress and anti-inflammation via inhibiting P38 MAPK activation.

Electro-acupuncture Pretreatment at Zusanli (ST36) Acupoint Attenuates Lipopolysaccharide-Induced Inflammation in Rats by Inhibiting Ca2+ Influx Associated with Cannabinoid CB2 Receptors.

In this study, we aimed to investigate the effect of electro-acupuncture (EA) pretreatment at zusanli (ST36) acupoint on lipopolysaccharide (LPS)-induced endotoxemic rat model and explore the underlying molecular mechanisms. Rats were treated with EA at ST36 for 7 days before being subjected to LPS. Two hours post-LPS, samples such as serum, local acupoint tissues, and spleens were collected and processed for investigations including cytokine production, cytosolic calcium (Ca2+) concentration, Ca2+ influx, cannabinoid CB2 receptor (CB2R) expression, and TLR4/NF-κB signaling. Our results showed EA pretreatment significantly attenuated LPS-induced inflammatory cytokine production, such as TNF-α, IL-1ß, and IL-6. EA also enhanced CB2R expression, inhibited Ca2+ influx, and inactivated TLR4/NF-κB signaling, subsequently resulting in a substantial reduction of Ca2+ concentration. Importantly, CB2R antagonist AM630 effectively abrogated the suppressive effect of EA at ST36 on the endotoxemic rats, suggesting CB2R was involved in the anti-inflammatory effect of EA. EA pretreatment could enhance CB2R expression, inhibit Ca2+ influx, and inactivate TLR4/NF-κB signaling, which contributes to the alleviation of LPS-induced inflammation in rats.

Berberine suppresses IL-33-induced inflammatory responses in mast cells by inactivating NF-κB and p38 signaling.

Berberine (BBR) possesses many pharmacological characteristics including anti-inflammation, anti-allergy, anti-angiogenesis and anti-tumor. However, the effects and mechanisms of BBR on IL-33-induced mast cell inflammatory responses are kept unknown. To investigate these, rat peritoneal mast cells (RPMCs) were isolated from the peritoneal cavity and cultured with BBR treatment in combination IL-33 stimulation. Firstly, cytotoxic effect of BBR on RPMCs was detected by MTT assay. Then, IL-33-induced cytokine production and the expression of ST2 receptor, were evaluated by ELISA and real-time PCR, respectively. In addition, NF-κB and MAPK signaling involved in IL-33-mediated mast cell activation were assessed by Western blot, which also was confirmed using the signal transduction inhibitors. Simultaneously, the effect of BBR on IL-33-activated enhancement of IgE-mediated mast cell responses was analyzed. Lastly, SD rats were used to explore the effect of BBR on IL-33-induced inflammation in vivo. BBR treatment significantly reduced IL-33-stimulated cytokine production in RPMCs, such as IL-6, TNF-α, IL-13 and MCP-1, but had little effect in ST2 expression. BBR modulated IL-33 signaling via suppressing IL-33-induced NF-ΚB transcription and p38 phosphorylation, but not ERK and JNK. Additionally, BBR also hampered the combined effects of IL-33 and IgE-mediated mast cell activation. Decreased cytokine production followed BBR treatment in vitro was consistent with that in vivo, where BBR injection i.p. into rats obviously inhibited IL-33-induced plasma cytokine levels. These findings demonstrated that BBR suppressed IL-33-mediated inflammation in mast cells by inactivating NF-κB and p38 signaling, suggesting its potential application for the treatment of allergic inflammation.

Electro-Acupuncture at Zusanli Acupoint (ST36) Suppresses Inflammation in Allergic Contact Dermatitis Via Triggering Local IL-10 Production and Inhibiting p38 MAPK Activation.

Acupuncture has shown beneficial effect in the treatment of multiple dermatologic conditions including dermatitis, pruritus, urticaria, and hyperhidrosis; however, the detailed mechanisms are still kept unclear. This study aimed to investigate if electro-acupuncture (EA) treatment prevents 2,4-dinitrofluorobenzene (DNFB)-induced allergic contact dermatitis (ACD) in rats and explore its underlying mechanisms. ACD was induced by sensitizing and challenging with DNFB topically. Rats were treated daily following bilateral subcutaneous stimulation of EA at Zusanli acupoint (ST36) for 1 week. Ear swelling and serum IgE levels were measured. The ear biopsies were obtained for histology. Inflammatory cytokines on the dermatological ear and local acupoint tissue were assayed. Spleen lymphocytes and the homogenized supernatant of local acupuncture area were used to co-culture for flow cytology and immune analysis, respectively. EA treatment at ST36 notably inhibited ear swelling and inflammatory cell infiltration on DNFB-induced ACD. EA also decreased serum IgE concentrations and alleviated the production of inflammatory cytokines in dermatological ear. Additionally, EA treatment attenuated the percentage of CD4+IFN-γ+ and CD4+IL-4+ T cells associated with ACD. Interestingly, secretion of interleukin (IL)-10 in the local acupoint tissue following EA stimulation was increased and showed suppressive function when co-cultured with the spleen lymphocytes from DNFB group. Lastly, EA treatment demonstrably suppressed p38 MAPK activation in DNFB-treated rats. Our findings suggest that EA treatment at ST36 may ameliorate inflammation associated with DNFB-induced ACD via triggering local IL-10 production and inhibiting p38 MAPK activation, which provide an alternative and promising therapy for ACD.

Enhancement of immune cytokines and splenic CD4+ T cells by electroacupuncture at ST36 acupoint of SD rats.

Electroacupuncture at the ST36 acupoint can enhance the body's immune function. However, the mechanism for this enhancement has not been fully described. Our study was designed to investigate the effect of electroacupuncture on the immune function of Sprague-Dawley (SD) rats. The rats were randomly divided into three groups: a control group, a non-acupoint group (abdominal muscle acupuntured) and a ST36 acupoint group. Our results showed that successive electroacupuncture at the ST36 acupoint for 3 d significantly enhanced the interferon-γ (IFN-γ) level in the serum of SD rats. The results also showed that the serum and extracts from spleen cells of the ST36 acupoint group contained higher levels of interleukin (IL)-2 and IL-17 compared to those of the other two groups. Immunohistochemical analysis showed that electroacupuncture applied to the ST36 acupoint enhanced the expression level of CD4 in spleen cells. Furthermore, it was observed that CD4 co-localized with transient receptor potential vanilloid (TRPV) channels at the membrane of splenic CD4+ T cells and the expression level of CD4 was related to TRPV channels in the electroacupuncture treatment. These observations indicated that electroacupuncture stimulation at the ST36 acupoint enhanced the level of immune cytokines and splenic CD4+ T cells through TRPV channels in this system.

Electro-acupuncture at Acupoint ST36 Ameliorates Inflammation and Regulates Th1/Th2 Balance in Delayed-Type Hypersensitivity.

Increasing evidence indicates anti-allergic and anti-inflammatory effects of electro-acupuncture (EA) therapy. However, its underlying mechanism on delayed-type hypersensitivity (DTH), a classic allergic inflammatory disease, still remains unclear. In this study, we aimed to explore the immunomodulatory mechanism of EA intervention in a mouse model of ovalbumin (OVA)-induced DTH. Mice were randomly divided into four groups: Control, OVA-DTH, DTH + EA, DTH + Sham. "Zusanli" acupoint (ST36) was used for DTH + EA, whereas a non-acupoint (localized 5 mm below the "Zusanli" acupoint) was selected for DTH + Sham. Footpad thickness was checked, and the infiltration of inflammatory cells was estimated by hematoxylin and eosin staining. Levels of IgG and IgE in serum of different groups and inflammatory cytokines in the supernatants from homogenized footpads, including IFN-γ, TNF-α, IL-4, and IL-5, were determined by ELISA. Cell proliferation of spleen lymphocytes was assayed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT). The frequency of CD4+IFN-γ+ and CD4+IL-4+ T cells was analyzed with flow cytometry. In addition, the mRNA and protein expression of T-bet and GATA-3 were evaluated by real-time PCR and Western blotting, respectively. Our data showed EA treatment at acupoint ST36 relieved the pathological progression of DTH responses via reduction in footpad swelling, infiltration of inflammatory cells, levels of IgG and IgE as well as decreased production of IFN-γ and TNF-α in homogenized footpad tissue. Moreover, detailed studies were performed revealing that EA attenuated the percentage of CD4+IFN-γ+ T cells and prevented Th cells differentiation into Th1 cells, and this results from inhibiting secretion of IFN-γ and suppressing expression of T-bet, an IFN-γ transcription factor. The results indicated that EA treatment improved Th1-mediated allergic skin inflammation via restoring Th1/Th2 balance by curbing Th1 differentiation. These findings suggested that EA at acupoint ST36 might be a useful and promising therapeutic for allergic inflammatory as well as Th1-mediated inflammation response.

Cortex Mori Radicis Extract induces neurite outgrowth in PC12 cells activating ERK signaling pathway via inhibiting Ca(2+) influx.

Cortex Mori Radicis is a traditional Chinese herbal medicine which has a long history of use for the treatment of headaches, cough, edema and diabetes. However, its function and mode of action within nervous system remain largely unclear. In the present study, we have attempted to determine the effects of Cortex Mori Radicis Extract (CMRE) on neuronal differentiation. Here, we reported that CMRE induces the neurite outgrowth in pheochromocytoma PC12 cells and primary cortical neuron. Following the generation of neurite outgrowth, extracellular Ca(2+) influx was inhibited and intracellular Ca(2+) decreased. In addition, CMRE induced the extracellular signal-regulated kinase 1/2 (ERK1/2) activation and also stimulated the Rap1-GTP expression, which is closely linked to neuritogenesis. Moreover, the neurite outgrowth induced by CMRE was antagonized to a marked degree by suppressing activation of p-ERK1/2 with the specific ERK1/2 inhibitor (PD98059), suggesting the involvement of Rap1-GTP and ERK1/2 in CMRE-induced neurite outgrowth. Taken together, these results demonstrate that CMRE induces neurite outgrowth of PC12 cells through Rap1-ERK signaling pathway via inhibiting Ca(2+) influx, and provide a novel insight into the manner in which CMRE participates in neuritogenesis.

Fructus Corni extract-induced neuritogenesis in PC12 cells is associated with the suppression of stromal interaction molecule 1 expression and inhibition of Ca2+ influx.

Fructus Corni (Cornus officinalis Sieb. et Zucc.) is commonly prescribed as a traditional Chinese herbal medicine that possesses pharmacological actions against inflammation, diabetic nephropathy, tumors, oxidation and aging. However, its function and mode of action within the nervous system remain largely unclear. In this study, the effects of Fructus Corni extract (FCE) on neuronal differentiation were investigated. It was found that FCE significantly increased the percentage of PC12 cells bearing neurites (P<0.001). Following the generation of neurite outgrowth, FCE treatment decreased the mRNA expression of stromal interaction molecule 1 (STIM1; P<0.05) and suppressed the expression of STIM1 protein (P<0.001). In addition, extracellular calcium (Ca2+) influx was inhibited resulting in a reduction in the intracellular Ca2+ level, suggesting that the inhibition of Ca2+ influx may be involved in the FCE-promoted neurite outgrowth of PC12 cells. These results demonstrate that FCE induces neurite outgrowth in PC12 cells and that this is associated with the suppression of STIM1 expression and the inhibition of Ca2+ influx, which may partially explain the FCE-induced neuritogenesis.