| Part one:Study the protective role of oridonin against cerebral ischemia/reperfusion injury in miceObjective:Oridonin is a natural bioactive product which is firstly extracted from the herbal plant,Rabdosia rubenscens.Existing evidence indicates various pharmacological activeities of oridonin,including anti-inflammation,anti-oxidation,neuroprotection and anti-tumor.Moreover,a recent study reported that oridonin could effectively alleviate myocardial ischemia/reperfusion injury through modulating multiple metabolic pathways.However,the effect of oridonin on cerebral ischemia/reperfusion(I/R)injury and its underlying mechanism has not been explored so far.Hence,the purpose of this part was to determine whether oridonin could relieve cerebral I/R injury in mice and to uncover its molecular mechanism,as well as to provide the theoretical basics for the use of oridonin as a potential agent for the prevention and treatment of cerebral I/R injury in clinical setting.Methods:1.Experimental treatment groups:Group one:Sham group(n=5),I/R group(n=5)Group two:I/R group(n=5),I/R+oridonin low dose(20 mg/kg)group,I/R+oridonin high dose(40 mg/kg)group.2.Cerebral I/R injury model and oridonin administration:After the deep anaesthesia with pentobarbital sodium by intraperitoneal injection,the I/R and Ori-treated group of mice received a manipulation of middle cerebral artery occlusion(MCAO)for 1h,and get reperfusion for another 24 h.Specially,animals in the Ori-treated group were respectively administrated with 20 mg/kg or 40mg/kg Ori by intraperitoneal injection 1h prior to MCAO.Moreover,equal volume of DMSO was injected into mice of I/R group 1h before the MCAO.The mice of sham group underwent surgical procedures but without ischaemic insult.3.Neurological deficit score:The Neurological deficit score were conducted according to the 18-point scoring system.4.TCC staining was used to calculate the brain infarction volume of mice.5.Detection of brain water content:The ischemic brain tissue was collected in a clean 5-ml EP tube with known weight.The weight of EP tube with fresh tissue was recorded before being put in a 75℃drying oven for 72 hours.The tube weight was re-measured after drying.The weights of fresh and dried tissue were calculated,and the wet to dry ratio was deduced.6.Nissl staining and Fluoro-Jade B staining determined the neuronal injury and death.7.Immunohistochemical staining examined the expression level of Iba-1 in cerebral ischemic tissues.8.Double immunofluorescence staining confirmed the co-localization of Iba-1 and NLRP3 in cerebral ischemic tissues.9.Western blotting detected the protein levels of NLRP3,cleaved caspase-1,IκBα,pIκBα,cytoplasmic NF-κB p65,and nuclear NF-κB p65 in cerebral ischemic tissues.Results:1.TTC staining shows the marked infarct volume in I/R mice,while low-and high-dose oridonin could effectivelyreduced the infarct volume.The infarct volumes in sham group,I/R group,I/R+L-oridonin dose group,and I/R+H-oridonin dose group were 0%,(27.78±2.62%),(18.83±2.09%),and(10.76±1.07%),respectively.The difference among each group is of statistical significance(P<0.0001).2.The neurological injury was assessed by 18-point scoring system and results indicated that no abnormal behavior and neurological injury was found in sham group mice after ischemia-reperfusion for 24h.In contrast,mice in I/R group presented significant neurological injury with notably deceased neurological score.The neurological behavior scores in sham group and I/R group were 17.83±0.41 and 9.17±1.17(P<0.0001),respectively.Both low-and highdose oridonin could effectively reverse I/R-induced neurological injury.Neurological scores were 14.67±1.37 and 12.83±2.23(P<0.01,P<0.0001),respectively.3.The analysis on the wet/dry ratio of brain tissue indicated significant brain edema in I/R group after ischemia/reperfusion for 24h,when compared with sham group(P<0.0001).High-dose oridonin treatment significantly reduced water content of brain tissue(P<0.0001),while no obvious difference on the water content of brain tissue in low dose oridonin-treated group and I/R group was observed(P>0.05).The wet/dry ratios of brain tissue in sham group,I/R group,low dose oridonin andhigh dose oridonin-treated group were 76.22±1.77%,84.41±1.75%,82.11±1.18%,and 78.47±1.30%,respectively.4.Nissl and Fluoro-Jade B staining results showed significant lost of Nissl bodies and decreased survival,as well as increased degenerative and necrotic morphological changes in I/R group in comparison with sham group(P<0.0001).However,oridonin treatment led to the increased survival and decreased degenerative and necrotic alterations,when compared with I/R group(P<0.001,P<0.0001).5.Immunohistochemical staining results revealed the number of Iba-1-positive cells of I/R group was dramatically elevated after ischemia/reperfusion for 24h,when compared with sham group(P<0.0001).While both low and high dose oridonin treatment significantly decreased the number of Iba-1-positive cells,indicating the inhibitory effect of oridonin on microglia activation(P<0.01,P<0.0001).6.Double immunofluorescence staining results demonstrated both amount of NLRP3-and Iba-1-positive cells in brain tissues were notably increased in I/R group mice after ischemia-reperfusion for 24h,when compared with sham group(P<0.0001).Moreover,NLRP3 co-localized with Iba-1 in brain of cerebral I/R mice.Low and high dose oridonin treatment markedly reduced the number of NLRP3and Iba-1-positive cells(P<0.01,P<0.0001).7.Western blotting analysis results revealed that the protein levels of NLRP3,cleaved caspase-1,nuclear NF-κB p65,and p-IκBα,as well as the release of IL-1β and IL-16 were significantly elevated in I/R group mice after ischemia-reperfusion for 24h,when compared with sham group(P<0.0001).On the other hand,the above-mentioned changes on the expression of NLRP3,cleaved caspase-1,nuclear NF-κB p65,p-IκBα,IL-1β and IL-16 levels were markedly reversedabolished by the administration of low and high dose oridonin(P<0.01,P<0.0001).Conclusions:1.Oridonin treatment could effectively alleviate cerebral ischemia/reperfusion-induced neurological injury by the reduction in brain edema,infarct volume,and the death of neuronal cells.2.Oridonin might alleviate I/R-induced brain tissues injury through inhibiting the activation of NF-κB and microglia cells,withsubsequent reduction in NLRP3 inflammasome activation and the inflammatory injury.Part two:Study the effectof Oridonin on NF-κB/NLRP3 activation induced by OGD/R in cultured micrglia cell lineObjective:Recent studies have shown that the activation of NLRP3 inflammasome in microglia plays an important role in the development of cerebral ischemia.Moreover,the inhibition of NF-κB signal pathway is helpful to block the activation of NLRP3 inflammasome in microglia.Several in vitro and in vivo studies demonstrated that oridonin could exert its anti-inflammatory effect through inhibiting the activation of NLRP3 inflammasome.Our previous results confirmed that oridonin could effectively protect the mice from I/R injury.The purpose of this part was to explore whether oridonin could suppress the activation of NLRP3 inflammasome by inhibiting the NF-κB signaling,and subsequently reduce I/R injury.Methods:1.Experimental groups:Group one:Control group,OGD/R group.Group two:Control,OGD/R,OGD/R+L-Ori,OGD/R+H-Ori and OGD/R+JSH-23 group.2.Treatment:Cells in control group were cultured with normal condition,while cells in the OGD/R group were deprived of oxygen and glucose for 3 h and then reoxygenated for 24 h.Cells in OGD/R+L-oridonin or H-oridonin group were pre-treated with 5 μM and 10μM oridonin for 1 h,then treated with oxygen-glucose deprivation for 3 h and reoxygenation for 24 h(with the presence of L-oridonin or H-oridonin during OGD/R),and cells in OGD/R+JSH-23 group were treated with JSH-23(30 μM)for 1 h,followed by oxygen-glucose deprivation and reoxygenation.3.qRT-PCR detected the relative mRNA levels of NLRP3 in microglia.4.Western blotting analysis examined the protein levels of NLRP3,cleaved caspase-1,cytoplasmic NF-κB p65 and nuclear NF-κB p65 in microglia.5.The releases of IL-1β and IL-18 in cell supernatant were determined using ELISA kits.6.The distribution of NF-κB p65 in cytoplasm and nucleus of microglia was detected by immunofluorescence assay.Result:1.The results of qRT-PCR and western blot showed that the levels of NLRP3,cleaved caspase-1 mRNA and protein in microglia treated with OGD/R were significantly increased(P<0.05,P<0.0001)compared with control,while pre-treatment with H-oridonin could significantly reverse the increase of NLRP3 and cleaved caspase-1 induced by OGD/R(P<0.05,P<0.0001).No obvious statistical significance between L-oridonin dose group and control was found,despite a trend of reduction in NLRP3 and cleaved caspase-1.In addition,JSH-23,an inhibitor of NF-κB,could significantly down-regulate the levels of NLRP3 and cleaved caspase-1(P<0.01,P<0.001).2.ELISA results showed that the levels of IL-1β and IL-18 in the supernatant of BV2 cells in OGD/R group(129.145±11.910 pg/ml)were significantly higher than those in normal control group(16.778±3.254 pg/ml)(P<0.0001).Compared with OGD/R group,low and high dose oridonin as well as JSH-23 could reduce the secretion of IL-1β and IL-18 induced by OGD/R(P<0.05,P<0.001,P<0.0001).3.Immunofluorescence results showed that NF-κB p65 nuclear translocation in OGD/R group was significantly higher than that in normal control group,while H-oridonin and JSH-23 intervention could significantly reduce NF-κB p65 nuclear translocation.Similarly,compared with the normal control group,western blotting results also confirmed that the nuclear p65 level of OGD/R group was significantly increased,while the cytoplasmic p65 level was decreased(P<0.001,P<0.0001).The pre-treatment of H-oridonin and JSH-23 could effectively reverse the re-distribution of p65 from cytoplasm to nucleus(P<0.05,P<0.001,P<0.0001).Conclusions:1.Compared with control,the expression of NLRP3 was significantly elevated in OGD/R-induced BV2 microglia cells,while Ori-treatment diminished these alterations.Additionally,the significant down-regulated expression of cleaved caspase-1 and the restricted release of IL-1β and IL-18 were also found in Ori-treated BV2 cells2.Oridonin might inhibit NLRP3 inflammasome activation by suppressing the NF-κB signaling. |
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