Withaferin A inhibits ferroptosis and protects against intracerebral hemorrhage.

Recent studies have indicated that suppressing oxidative stress and ferroptosis can considerably improve the prognosis of intracerebral hemorrhage (ICH). Withaferin A (WFA), a natural compound, exhibits a positive effect on a number of neurological diseases. However, the effects of WFA on oxidative stress and ferroptosis-mediated signaling pathways to ICH remain unknown. In this study, we investigated the neuroprotective effects and underlying mechanism for WFA in the regulation of ICH-induced oxidative stress and ferroptosis. We established a mouse model of ICH by injection of autologous tail artery blood into the caudate nucleus and an in vitro cell model of hemin-induced ICH. WFA was injected intracerebroventricularly at 0.1, 1 or 5 µg/kg once daily for 7 days, starting immediately after ICH operation. WFA markedly reduced brain tissue injury and iron deposition and improved neurological function in a dose-dependent manner 7 days after cerebral hemorrhage. Through in vitro experiments, cell viability test showed that WFA protected SH-SY5Y neuronal cells against hemin-induced cell injury. Enzyme-linked immunosorbent assays in vitro and in vivo showed that WFA markedly decreased the level of malondialdehyde, an oxidative stress marker, and increased the activities of anti-oxidative stress markers superoxide dismutase and glutathione peroxidase after ICH. Western blot assay, quantitative polymerase chain reaction and immunofluorescence results demonstrated that WFA activated the nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling axis, promoted translocation of Nrf2 from the cytoplasm to nucleus, and increased HO-1 expression. Silencing Nrf2 with siRNA completely reversed HO-1 expression, oxidative stress and protective effects of WFA. Furthermore, WFA reduced hemin-induced ferroptosis. However, after treatment with an HO-1 inhibitor, the neuroprotective effects of WFA against hemin-induced ferroptosis were weakened. MTT test results showed that WFA combined with ferrostatin-1 reduced hemin-induced SH-SY5Y neuronal cell injury. Our findings reveal that WFA treatment alleviated ICH injury-induced ferroptosis and oxidative stress through activating the Nrf2/HO-1 pathway, which may highlight a potential role of WFA for the treatment of ICH.

[Effect of electroacupuncture on colonic motility and RhoA and ROCK protein expression in IBS-D rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) of "Tianshu"(ST25) and "Shangjuxu"(ST37)on gastrointestinal motility, psychological abnormality and expression of RhoA and ROCK protein in rats with diarrhea predominant irritable bowel syndrome (IBS-D), so as to explore its underlying mechanism in improving IBS-D. METHODS: Thirty-six male rats were randomly and equally divided into control, model, EA and medication groups (n=9 in each group). The IBS-D model with psychological abnormality was established by gavage of dinitrobenzene sulfonic acid (DNBS) + chronic restraint stress. EA (2 Hz/100 Hz, 0.3 mA) was applied to bilateral ST25 and ST37 for 20 min, once a day for 7 days. Rats of the medication group received gavage of pinaverium bromide solution (15 mg/kg), once a day for 7 days. The rats' food intake in 24 h, body mass and colonic contraction waves were recorded. The anhedonia-related behavior was measured using the sucrose consumption test. The elevated plus maze test (the open-arm residence time ratio) was used to assess the anxiety-like behavior. The small intestinal propulsion rate test was used to assess the intestinal motility. The expression levels of RhoA and ROCK proteins in the colonic tissue were measured by Western blot. RESULTS: After modeling, the body mass, food consumption, sucrose preference index, the open-arm residence time ratio andlatency of colonic contraction waves were significantly decreased (P<0.05), and the number of contraction waves, intestinal propulsive rate, and the expression levels of RhoA and ROCK proteins considerably increased (P<0.01，P<0.05) in the model group relevant to the control group. Following the interventions, the decrease of body mass, food consumption, sucrose preference index, open-arm residence time ratio and latency of contraction waves, and the increase of the contraction waves, intestinal propulsive rate, and the expression levels of RhoA and ROCK proteins were all reversed by both EA and medication (P<0.05，P<0.01). The effect of EA was significantly superior to that of medication in increasing the sucrose pre-ference index (P<0.05). CONCLUSION: EA can improve both colonic motility and psychological disorders in IBS-D rats with psychological disorder, which may be related to its function in down-regulating the expression of colonic RhoA and ROCK proteins.

[Effect of electroacupuncture on visceral sensitivity and colonic NGF, TrkA, TRPV1 expression in IBS-D rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) at "Tianshu" (ST 25) and "Shangjuxu" (ST 37) on mental state, visceral sensitivity and protein expression of nerve growth factor (NGF), tyrosine kinase receptor A (TrkA) and transient receptor potential vanilloid 1 (TRPV1) of colonic tissue in diarrhea-predominant irritable bowel syndrome (IBS-D) rats, and to explore its possible mechanism on treating IBS-D. METHODS: A total of 36 male SD rats of SPF grade were randomized into a blank group, a model group, an EA group and a western medication group, 9 rats in each group. In the model group, the EA group and the western medication group, IBS-D model was established by enema of dinitrobenzene sulfonic acid (DNBS) combined with chronic restraint stress method. In the EA group, EA was applied at "Tianshu" (ST 25) and "Shangjuxu" (ST 37), with disperse-dense wave, in frequency of 2 Hz/100 Hz, 20 min each time, once a day for 7 days. In the western medication group, pinaverium bromide suspension was given by gavage (15 mg•kg-1•d-1) for 7 days. Before and after model establishment, and after intervention, the body mass, 24 h food intake and fecal water content were observed, the visceral sensitivity was detected by abdominal withdrawal reflex (AWR); after intervention, the mental state was evaluated by elevated plus maze (EPM) test, the protein expression of NGF, TrkA and TRPV1 was detected by immunohistochemistry and Western blot in the 4 groups. RESULTS: After model establishment, compared with the blank group, the body mass and 24 h food intake were decreased (P<0.05), first systolic latency of AWR was shortened and number of contraction wave of AWR was increased (P<0.05), and fecal water content was increased (P<0.05) in the model group, the EA group and the western medication group. After intervention, compared with the blank group, open arm residence time ratio (OT%) of EPM was decreased (P<0.05) and protein expression of NGF, TrkA, TRPV1 in colonic tissue was increased in the model group (P<0.05); compared with the model group, the body mass and 24 h food intake were increased (P<0.05), first systolic latency of AWR was lengthened and number of contraction wave of AWR was decreased (P<0.05), the fecal water content was decreased (P<0.05), OT% of EPM was increased (P<0.05), and protein expression of NGF, TrkA, TRPV1 in colonic tissue was decreased (P<0.05) in the EA group and the western medication group. CONCLUSION: Electroacupuncture at "Tianshu" (ST 25) and "Shangjuxu" (ST 37) can relieve the anxiety and depression-like behaviors in IBS-D rats, down-regulate the protein expression of NGF, TrkA, TRPV1 in colonic tissue, so as to reduce the visceral sensitivity and relieve symptoms.

[Acupuncture and moxibustion treatment for dry eye disease: a network Meta-analysis of rando-mized controlled trial].

OBJECTIVE: A network Meta-analysis based on Bayesian theory was used to evaluate efficacy and safety of acupuncture and moxibustion in the treatment of dry eye disease(DED), so as to provide evidence-based research basis for clinical application. METHODS: Randomized controlled trials (RCT) for acupuncture and moxibustion treatment of DED published from the inception of database to November 25, 2020 were searched from PubMed, Embase, Cochrane Library, Web of Science, Sinomed, CNKI, Wanfang and VIP Database. Two reviewers independently screened the literatures, extracted the data. The quality of the included literature was evaluated, and network Meta-analysis was performed by using Stata14.0 and R4.0.3 software. RESULTS: A total of 71 literatures were identified, including 5 536 patients with DED, covering 11 different interventions. Network Meta-analysis showed that acupuncture+traditional Chinese medicine+artificial tears was the best treatment option in terms of the clinical effective rate, breakup time of tear film (BUT), Schirmer I test (SIT) with surface under cumulative ranking area value. Acupuncture+traditional Chinese medicine+artificial tears was better than artificial tears in the clinical effective rate (odds ratio［OR］=12.34, 95% confidence interval［CI］［4.72, 36.89］), BUT(mean differenc［MD］=2.76, 95%CI［0.16, 5.40］), SIT(MD=4.76, 95%CI［1.23, 8.29］). CONCLUSION: Acupuncture and moxibustion in the treatment of DED are generally better than artificial tears, and acupuncture-moxibustion combined with other traditional Chinese medicine therapy has the best effect.

[Effect of acupuncture on intestinal flora and TLR4 in brain and intestinal tissues in rats with stress gastric ulcer].

OBJECTIVE: To observe the effect of acupuncture at "Guanyuan" (CV 4) and "Xiajuxu" (ST 39) on intestinal flora and Toll-like receptors-4 (TLR4) in brain and intestinal tissue in rats with stress gastric ulcer (SGU), and to explore the possible mechanism of acupuncture for SGU. METHODS: Thirty-one male SD rats were randomly divided into a blank group (n=7), a model group (n=8), an acupuncture group (n=8) and a drug group (n=8). The rats in the model group, acupuncture group and drug group were treated with modified restraint plus water-immersion stress method to establish SGU model. After modeling, the rats in the acupuncture group were treated with acupuncture at "Guanyuan" (CV 4) and "Xiajuxu" (ST 39), 20 min each time, and the needles were twirled for 30 s every 5 min. The rats in the drug group were treated with intragastric administration of 2 mL omeprazole enteric-coated tablets (20 mg/mL). Both the treatments were given once a day for 5 days. After the intervention, the gastric mucosal damage index was calculated by Guth method; the morphological changes of gastric mucosa were observed by HE staining; the diversity of intestinal flora was detected by 16S rDNA high-throughput sequencing; TLR4 contents in brain and intestinal tissues were determined by ELISA. RESULTS: Compared with the blank group, the gastric mucosal damage index was significantly increased in the model group (P<0.01); the morphological changes of gastric mucosa were obvious; the Observed Species index and Shannon index of α diversity index of intestinal flora were decreased (P<0.05); the ß diversity showed that the spatial distance between the model group and the blank group was far; the TLR4 contents in the brain and intestinal tissue were increased (P<0.05, P<0.01). Compared with the model group, the gastric mucosal damage index was decreased in the acupuncture group and the drug group (P<0.05); the morphology of gastric mucosa was improved; the Observed Species index and Shannon index of α diversity index of intestinal flora in the acupuncture group was increased (P<0.05), and the Shannon index in the drug group was increased (P<0.05); the ß diversity showed that the spatial distance between the acupuncture group and the blank group was close; the TLR4 contents in the brain and intestinal tissues of the acupuncture group were decreased (P<0.01). Compared with the drug group, the contents of TLR4 in the intestinal tissue of the acupuncture group were decreased (P<0.05). CONCLUSION: Acupuncture at "Guanyuan" (CV 4) and "Xiajuxu" (ST 39) could alleviate SGU in rats, and its mechanism may be related to increasing the diversity of intestinal flora, promoting the disorder of intestinal flora to normal, and reducing the overexpression of TLR4 in brain and intestinal tissues.

[Effect of acupuncture on serum inflammatory cytokines and intestinal flora in rats with stress-induced gastric ulcer].

OBJECTIVE: To investigate the effect of acupuncture on serum inflammatory cytokines and intestinal flora in rats with stress-induced gastric ulcer (SGU), and to explore the mechanism of acupuncture in the treatment of SGU. METHODS: Sprague-Dawley rats were randomly divided into blank, model, acupuncture, and medication groups, with 7 rats in each group. Restraint water-immersion stress was used to establish the model of SGU. The rats in the acupuncture group were given acupuncture at "Zhongwan"(CV12) and bilateral "Zusanli"(ST36) for 20 min, once a day for 5 days, and those in the medication group were given 2 mL solution of Omeprazole enteric-coated tablets (0.2 mg/kg) by gavage, once a day for 5 days. The Guth method was used to calculate the gastric mucosa damage index, HE staining was used to observe the histopathological changes of the gastric mucosa, ELISA was used to measure the serum levels of interleukin-4 (IL-4) and interleukin-6 (IL-6), and 16S rDNA sequencing method was used to observe the change in intestinal flora. RESULTS: Compared with the blank group, the model group had a significant increase in gastric mucosa damage index (P<0.01), markedly pathological changes of the gastric mucosa shown by HE staining, a significant reduction in the content of serum IL-4 (P<0.01), and a significant increase in the content of serum IL-6 (P<0.01), as well as a significant reduction in Bacteroidetes and Firmicutes at the phylum level. Compared with the model group, the acupuncture group and the medication group had a significant reduction in gastric mucosa damage index (P<0.01, P<0.05). HE staining showed reduced pathological changes of the gastric mucosa, as well as a significant increase in the content of serum IL-4 (P<0.01, P<0.05) and a significant reduction in the content of serum IL-6 (P<0.01, P<0.05). As for the intestinal flora, there was a significant increase in Bacteroidetes. Compared with the medication group, the gastric mucosa damage index was decreased （P<0.05），the content of serum IL-4 was significantly increased （P<0.05）, while the content of serum IL-6 significantly decreased (P<0.05) in the acupuncture group. CONCLUSION: Acupuncture at CV12 and ST36 can down-regulate the content of serum IL-6, up-regulate the content of serum IL-4, maintain the relative homeostasis of inflammatory cytokines, and regulate the community structure of intestinal flora, and thus help to repair the damage of gastric mucosa.

[Effect of acupuncture on intestinal flora in rats with stress gastric ulcer].

OBJECTIVE: To observe the effect of acupuncture at "Baihui" (GV 20), "Zhongwan" (CV 12) and "Zusanli" (ST 36) on intestinal flora in rats with stress gastric ulcer (SGU) , and to explore the mechanism of acupuncture promoting SGU recovery. METHODS: Thirty-one SPF SD rats were randomly divided into a control group (7 rats), a model control group (8 rats), an acupuncture group (8 rats) and a medication group (8 rats). The rats in the model group, acupuncture group and medication group were selected to applied the improved restraint water-immersion stress method to establish the SGU model. After modeling, the rats in the control group and model group were fixed and restrained for 20 min every day for a total of 5 days; the rats in the acupuncture group were intervented with acupuncture at "Baihui" (GV 20), "Zhongwan" (CV 12) and "Zusanli" (ST 36), once a day, 20 min each time, and twisting needle for 30 s every 5 min for a total of 5 days; the rats in the medication group were gavaged by solution of omeprazole enteric-coated tablet (200 mg/mL), 2 mL for each rat, once a day. Guth method was used to calculate the gastric mucosal damage index (GMDI), HE staining was used to observe the pathological changes of gastric mucosa, and 16SrDNA identification was used to detect the structural abundance of intestinal flora. RESULTS: Compared with the control group, the GMDI of rats in the model group was increased (P<0.01), the gastric mucosal pathological changes were significant, and the intestinal flora richness index Chao1, Observed species and diversity index Shannon were all decreased (P<0.05), the diversity index Simpson was increased (P<0.05). Compared with the model group, the GMDI of rats in the acupuncture group and medication group was reduced (P<0.01, P<0.05), the gastric mucosal damage degree was reduced, and the intestinal flora richness index Chao1, Observed species and diversity index Shannon were all increased (P<0.05) and the diversity index Simpson decreased (P<0.05). Compared with the medication group, the GMDI of rats in the acupuncture group was reduced (P<0.01), the recovery of gastric mucosal injury was better than that of the medication group. CONCLUSION: Acupuncture can effectively improve gastric mucosal injury of SGU, and the mechanism may be related to increasing the diversity of intestinal flora and promoting the correction of the disordered intestinal flora.