Acacetin protects against cerebral ischemia-reperfusion injury via the NLRP3 signaling pathway

Acacetin(5,7-dihydroxy-4′-methoxyflavone), a potential neuroprotective agent, has an inhibitory effect on lipopolysaccharide-induced neuroinflammatory reactions. However, whether acacetin has an effect on inflammatory corpuscle 3(NLRP3) after cerebral ischemia-reperfusion injury has not been fully determined. This study used an improved suture method to establish a cerebral ischemia-reperfusion injury model in C57BL/6 mice. After ischemia with middle cerebral artery occlusion for 1 hour, reperfusion with intraperitoneal injection of 25 mg/kg of acacetin(acacetin group) or an equal volume of saline(0.1 mL/10 g, middle cerebral artery occlusion group) was used to investigate the effect of acacetin on cerebral ischemia-reperfusion injury. Infarct volume and neurological function scores were determined by 2,3,5-triphenyltetrazolium chloride staining and the Zea-Longa scoring method. Compared with the middle cerebral artery occlusion group, neurological function scores and cerebral infarction volumes were significantly reduced in the acacetin group. To understand the effect of acacetin on microglia-mediated inflammatory response after cerebral ischemia-reperfusion injury, immunohistochemistry for the microglia marker calcium adapter protein ionized calcium-binding adaptor molecule 1(Iba1) was examined in the hippocampus of ischemic brain tissue. In addition, tumor necrosis factor-α, interleukin-1β, and interleukin-6 expression in ischemic brain tissue of mice was quantified by enzyme-linked immunosorbent assay. Expression of Iba1, tumor necrosis factor-α, interleukin-1β and interleukin-6 was significantly lower in the acacetin group compared with the middle cerebral artery occlusion group. Western blot assay results showed that expression of Toll-like receptor 4, nuclear factor kappa B, NLRP3, procaspase-1, caspase-1, pro-interleukin-1β, and interleukin-1β were significantly lower in the acacetin group compared with the middle cerebral artery occlusion group. Our findings indicate that acacetin has a protective effect on cerebral ischemia-reperfusion injury, and its mechanism of action is associated with inhibition of microglia-mediated inflammation and the NLRP3 signaling pathway.

Neuroprotection mediated by the Wnt/Frizzled signaling pathway in early brain injury induced by subarachnoid hemorrhage

The Wnt/Frizzled signaling pathway participates in many inflammation-linked diseases. However, the inflammatory response mediated by the Wnt/Frizzled signaling pathway in experimental subarachnoid hemorrhage has not been thoroughly investigated. Consequently, in this study, we examined the potential role of the Wnt/Frizzled signaling pathway in early brain injury in rat models of subarachnoid hemorrhage.Simultaneously, possible neuroprotective mechanisms were also investigated. Experimental subarachnoid hemorrhage rat models were induced by injecting autologous blood into the prechiasmatic cistern. Experiment 1 was designed to examine expression of the Wnt/Frizzled signaling pathway in early brain injury induced by subarachnoid hemorrhage. In total, 42 adult rats were divided into sham(injection of equivalent volume of saline), 6-, 12-, 24-, 48-, 72-hour, and 1-week subarachnoid hemorrhage groups. Experiment 2 was designed to examine neuroprotective mechanisms of the Wnt/Frizzled signaling pathway in early brain injury induced by subarachnoid hemorrhage. Rats were treated with recombinant human Wnt1(rhwnt1), small interfering Wnt1(siwnt1) RNA, and monoclonal antibody of Frizzled1(anti-Frizzled1) at 48 hours after subarachnoid hemorrhage. Expression levels of Wnt1, Frizzled1, β-catenin, peroxisome proliferator-activated receptor-γ, CD36, and active nuclear factor-κB were examined by western blot assay and immunofluorescence staining. Microglia type conversion and inflammatory cytokine levels in brain tissue were examined by immunofluorescence staining and enzyme-linked immunosorbent assay. Our results show that compared with the sham group, expression levels of Wnt1, Frizzled1, and β-catenin were low and reduced to a minimum at 48 hours, gradually returning to baseline at 1 week after subarachnoid hemorrhage. rhwnt1 treatment markedly increased Wnt1 expression and alleviated subarachnoid hemorrhage-induced early brain injury(within 72 hours), including cortical cell apoptosis, brain edema, and neurobehavioral deficits, accompanied by increasing protein levels of β-catenin, CD36, and peroxisome proliferator-activated receptor-γ and decreasing protein levels of nuclear factor-κB. Of note, rhwnt1 promoted M2-type microglia conversion and inhibited release of inflammatory cytokines(interleukin-1β, interleukin-6, and tumor necrosis factor-α). In contrast, siwnt1 RNA and anti-Frizzled1 treatment both resulted in an opposite effect. In conclusion, the Wnt/Frizzled1 signaling pathway may participate in subarachnoid hemorrhage-induced early brain injury via inhibiting the inflammatory response, including regulating microglia type conversion and decreasing inflammatory cytokine release. The study was approved by the Animal Ethics Committee of Anhui Medical University and First Affiliated Hospital of USTC,Division of Life Sciences and Medicine, University of Science and Technology of China(approval No. LLSC-20180202) in May 2017.

Electroacupuncture Delays Cartilage Degeneration by Modulating Nuclear Factor-κB Signaling Pathway

Objective: To illustrate the molecular mechanisms underlying the therapeutic effects of electroacupuncture(EA) on knee osteoarthritis(OA). Methods: Twenty-seven six-month-old New Zealand white rabbits were allocated into three groups in accordance with a random number table: normal group(no surgery-induced OA;without treatment), model group(surgery-induced OA;without treatment) and EA group [surgery-induced OA;received treatment with EA at acupoints Dubi(ST 35) and Neixiyan(EX-LE 5), 30 min twice a day]. After eight consecutive weeks of treatment, the histopathological alterations in cartilage were observed using optical microscopy and transmission electron microscopy, cartilage degeneration was evaluated by modified Mankin’s score principles, the synovial fluid concentration of interleukin-1β(IL-1β), interleukin-6(IL-6), tumor necrosis factor-α(TNF-α) and matrix metalloproteinase-3(MMP-3) were evaluated by enzyme-linked immunosorbent assay, and the protein expression levels of IL-1β, IL-6, TNF-α, MMP-3, IκB kinase-β(IKK-β), nuclear factor of α light polypeptide gene enhancer in B-cells inhibitor α(IκB-α) and nuclear factor-κB(NF-κB) p65 were quantified by Western blot analysis. Results: EA treatment significantly improved cartilage structure arrangement and reduced cellular degeneration. The IL-1β, IL-6, TNF-α and MMP-3 of synovial fluid in the EA-treated group were significantly decreased compared with the model group(all P<0.01). Compared with the model group, the IL-1β, IL-6, TNF-α, MMP-3, IKK-β and NF-κB p65 protein expressions in cartilage of EA-treated group were significantly decreased(all P<0.01), whereas IκB-α expression was significantly up-regulated(P<0.01). Conclusion: EA treatment may delay cartilage degeneration by down-regulating inflammatory factors through NF-κB signaling pathway, which may, in part, explain its clinical efficacy in the treatment of knee OA.