Crocus sativus L.produces anti-inflammatory effects and regulates the NLRP3–NF-κB pathway

Objective:This study aimed to evaluate the anti-inflammatory effects of petal and stamen extracts of saffron crocus(Crocus sativus)and explore the underlying mechanism.Methods:Local and systemic inflammation models were used to investigate the anti-inflammatory effects of C.sativus.A xyleneinduced inflammation model or lipopolysaccharide(LPS)-induced inflammation model was used in this study.C.sativus petal and stamen extracts were each administered to the mice in the xylene and LPS models by gavage for 14 d at 0.1 and 0.4 g/kg doses,respectively.Enzyme-linked immunosorbent assay(ELISA)was used to measure the concentrations of tumor necrosis factor(TNF)-αand interleukin(IL)-1βin mouse serum.Hematoxylin and eosin(H&E)staining was used to observe the pathological changes in the ear in the xylene-induced inflammation model and in the spleen in the LPS-induced inflammation model.NOD-like receptor thermal protein domain associated protein 3(NLRP3)protein levels within the nuclear factor-kappa B(NF-κB)pathway were assessed using western blotting.RAW264.7 cells were treated with LPS(5μg/mL)and LPS+C.sativus(0.05,0.1,and 0.2 mg/mL)for 24 h,and a Cell Counting Kit-8 was used to measure cell proliferation.Changes in NLRP3 and NF-κB levels were evaluated by western blotting.Results:Petal and stamen extracts of C.sativus attenuated the anti-inflammatory effects in local or systemic inflammatory models and repaired pathological changes in the ear in the xylene-induced inflammation model and spleen in the LPS-induced inflammation model.These extracts also decreased the concentrations of TNF-αand IL-1βin the mouse serum in the LPS-induced inflammation model.C.sativus downregulated NLRP3 protein level through the NF-κB pathway and downregulated LC-3 and BECLIN1 in vivo and in vitro.Carbonyl Cyanide3-ChloroPhenylhydrazone(CCCP)weakened the effects of C.sativus on the NLRP3–NF-κB pathway.Conclusion:C.sativus has anti-inflammatory effects and regulates the NLRP3-NF-κB pathway.

PANoptosis-like cell death in ischemia/reperfusion injury of retinal neurons

PANoptosis is a newly identified type of regulated cell death that consists of pyroptosis,apoptosis,and nec roptosis,which simultaneously occur during the pathophysiological process of infectious and inflammatory diseases.Although our previous lite rature mining study suggested that PANoptosis might occur in neuronal ischemia/repe rfusion injury,little experimental research has been reported on the existence of PANoptosis.In this study,we used in vivo and in vitro retinal neuronal models of ischemia/repe rfusion injury to investigate whether PAN optosis-like cell death(simultaneous occurrence of pyroptosis,apo ptosis,and necroptosis)exists in retinal neuronal ischemia/repe rfusion injury.Our results showed that ischemia/repe rfusion injury induced changes in morphological features and protein levels that indicate PANoptosis-like cell death in retinal neurons both in vitro and in vivo.Ischemia/repe rfusion inju ry also significantly upregulated caspase-1,caspase-8,and NLRP3 expression,which are important components of the PANoptosome.These results indicate the existence of PANoptosis-like cell death in ischemia/reperfusion injury of retinal neurons and provide preliminary experimental evidence for future study of this new type of regulated cell death.

Maraviroc promotes recovery from traumatic brain injury in mice by suppression of neuroinflammation and activation of neurotoxic reactive astrocytes

Neuroinflammation and the NACHT,LRR,and PYD domains-containing protein 3 inflammasome play crucial roles in secondary tissue damage following an initial insult in patients with traumatic brain injury(TBI).Maraviroc,a C-C chemokine receptor type 5 antagonist,has been viewed as a new therapeutic strategy for many neuroinflammatory diseases.We studied the effect of maraviroc on TBI-induced neuroinflammation.A moderate-TBI mouse model was subjected to a controlled cortical impact device.Maraviroc or vehicle was injected intraperitoneally 1 hour after TBI and then once per day for 3 consecutive days.Western blot,immunohistochemistry,and TUNEL(terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling)analyses were performed to evaluate the molecular mechanisms of maraviroc at 3 days post-TBI.Our results suggest that maraviroc administration reduced NACHT,LRR,and PYD domains-containing protein 3 inflammasome activation,modulated microglial polarization from M1 to M2,decreased neutrophil and macrophage infiltration,and inhibited the release of inflammatory factors after TBI.Moreover,maraviroc treatment decreased the activation of neurotoxic reactive astrocytes,which,in turn,exacerbated neuronal cell death.Additionally,we confirmed the neuroprotective effect of maraviroc using the modified neurological severity score,rotarod test,Morris water maze test,and lesion volume measurements.In summary,our findings indicate that maraviroc might be a desirable pharmacotherapeutic strategy for TBI,and C-C chemokine receptor type 5 might be a promising pharmacotherapeutic target to improve recovery after TBI.

当归补血汤及其活性成分通过抑制NLRP3炎症小体活化改善糖尿病肾病的实验研究

目的:探究不同剂量的当归补血汤及其活性成分黄芪甲苷改善糖尿病肾病的作用机制及与NOD样受体蛋白3(nod-like receptor protein 3,NLRP3)炎症小体的相关性。方法:5 w~8 w的雄性GK大鼠分为模型对照组、阳性药格列喹酮10 mg/kg、盐酸贝那普利片10 mg/kg、当归补血汤2、4、8 g/kg组、黄芪甲苷40 mg/kg组,每组10只;10只雄性Wistar大鼠作为正常对照组。各组灌胃相应药物,正常对照组和模型对照组灌胃生理盐水,连续8 w。检测随机血糖、空腹血糖;计算肾脏系数;Elisa法检测大鼠尿液中白蛋白含量;HE染色观察肾脏组织病理改变;Western Blot法检测大鼠肾脏NLRP3、ASC、Caspase-1蛋白的表达。结果:与正常对照组相比,模型对照组大鼠的血糖、肾脏系数、尿白蛋白含量、炎症蛋白表达量均显著升高(P<0.01);与模型对照组比较,当归补血汤4、8 g/kg及其活性成分黄芪甲苷0.04 g/kg均可改善GK大鼠的体质量、血糖、肾脏肥大的情况,显著降低尿白蛋白含量,能有效抑制NLRP3小体的活化,其中8 g/kg组对调节血糖、降低尿白蛋白含量效果最为显著(P<0.01)。结论:不同剂量当归补血汤及其活性成分黄芪甲苷对GK大鼠肾脏均具有一定的保护作用,其机制与抑制NLRP3炎症小体密切相关。

Upper limb arthromyodynia can be treated with Notopterygium by down-regulating P2X3 to mediate PKC-induced inflammatory response

Rheumatoid arthritis(RA)is one of the most common refractory diseases in the world,and traditional Chinese medicine Notopterygium(NE)has been used in the treatment of upper limb pain for a long time.NE can significantly reduce the expression of inflammatory pain target P2X3 receptor in rats with upper-limb arthritis.To verify the relationship between the mechanism of NE for“upper limb paralysis”and the P2X3 receptor-mediated PKC inflammatory response pathway,UPLC was taken to measure the exact medicinal substance of ethyl acetate from NE.Sprague Dawley rats were randomly divided into a blank group,a model group,a live-action group,and a positive group.The joint cavity was removed after 21 d.Moreover,a model group,a live group,and a positive group were also set up with RA-FLS cells in our in vitro study.The expressions of P2X3 and PKC inflammation pathway indicators were detected by Western blotting analysis.A P2X3 inhibitor(A-317491)acted on RA-FLS cells,and a model group and a positive group were set.Then the protein expression of PKC was detected.NE reduced the expressions of P2X3,Rab7,PKC,and NF-κB at the protein level in both systems.NE and P2X3 receptor antagonists reduced the expressions of key proteins in the PKC pathway in RA-FLS cells to similar extents,and their effects were not additive.NE could effectively improve the“forelimb pain”of RA rats,with a mechanism closely related to the P2X3/Rab7/PKC/NF-κB pathway.