The Anti-gout Effect And Mechanism Of Flavonoids In Selaginella Moellendorfii Hieron.via NLRP3 Signaling

Selaginella moellendorffii Hieron.,which belongs to the family Selaginellaceae,is a traditional Chinese folk herb described in Atlas of Meteria Medica.It has the effects of clearing heat and detoxifying,diuretic and phlegm,activating blood and swelling,and stopping bleeding and fever,and it was used for a variety of acute and chronic inflammation.Previous research by this research group showed that S.moellendorffii has anti-gout effect,and flavonoids are its main active components.But the mechanism of its for gout treatment is still unclear.Gout is an autoinflammatory disease.The core mechanism of gout attack is the activation of NLRP3 inflammasome.Therefore,this article intends to use the overall animal model to study the anti-gout effect of flavonoids from S.moellendorffii.And to explore the anti-gout mechanism of flavonoids from S.moellendorffii in cellular levels and animal levels based on previous research.Objective: By analyzing the main components of S.moellendorffii,screening the anti-inflammatory activity of different polar parts of S.moellendorffii,confirming their active parts and further studying the anti-inflammatory activity and anti-gout effect of main flavonoids in their parts.The effect of NLRP3 on inflammatory body activation was examined,and the mechanism of flavonoids from S.moellendorffii against gout was explored.Methods:(1)UPLC-MS technology was used to analyze and identify the components of S.moellendorffii extract.(2)(1)Establish a model of RAW264.7 cell inflammation ind uced by lipopolysaccharide(LPS),and screen the anti-inflammatory activity of flavonoids from different polar parts of S.moellendorffii,including amentoflavone(AM),apigenin(AP),4’-O-methylrobustafl avone(RO),6,8-di-C-β-D-glucopyranosylapigenin(YI),6-C-β-D-gluc opyranosyl-8-C-β-D-xylopyranosyl-apigenin(YII),6-C-β-D-xylopyrano syl-8-C-β-D-glucopyranosyl-apigenin(YIII).The detection indicators include: MTT method to detect cytotoxicity,Griess method to detect the effect on NO secretion,and ELISA method to detect IL-1β se cretion.(2)Establish an animal model of acute gouty arthritis induc ed by sodium urate(MSU),and screen anti-gout activities of flavon oids from S.moellendorffii.The detection indicators include: measuri ng the swelling of the ankle joints of mice in different groups,the level of IL-1β in the footpad tissues measured by ELISA,and the pathological changes of the footpad tissues were observed by HE staining.(3)THP-1 cells were induced into macrophages by PMA,and TPH-1cells sensitized by LPS were induced by MSU to establish NLRP3 inflammasome activation gout models;CCK-8 method was used to determine toxicity of flavonids in S.moellendorffii on THP-1 cells.The effect of flavonoids on LDH levels was measured using the LDH kit.PI staining was used to detect cell membrane integrity.Scanning electron microscope was used to observe cell surface morphology.THP-1 cells secreted IL-1β and TNF-α;RT-PCR was used to detect the expression of inflammation-related genes;Western Blot method was used to detect the expression of NLRP3 signaling pathway-related proteins.Results:(1)UPLC-MS analysis revealed that adenosine,guanosine,turquoise B,quercetin,neoselaginic acid,luteolin,coulisin,apigenin-6,8-Di-C-β-D-glucopyranoside,6-C-β-D-glucopyranosyl-8-C-β-D-xyranosyl apigenin,5-carboxymethyl-4’-Hydroxyflavones-7-O-β-D-glucopyranoside,6-C-β-D-glucopyranosyl-8-C-β-D-glucopyranosylapigenin,Ginkgoflavin,Ginkgoflavin,apigenin and other compounds,mainly flavonoids and nucleosides,among which there are 9 flavonoids,common components of Selaginella plants.(2)LPS-induced RAW264.7 cell inflammation model results showed that:(1)The ethyl acetate and n-butanol parts of S.moellendorffii significantly inhibited NO secretion,while the water part had no significant effect.(2)In the ethyl acetate part,25%(TF-1),50%(TF),75%(TF-2)were separated and purified by D101 macroporous resin.The TF and TF-2 ethanol-eluting part could significantly inhibit NO secretion.The flavonoid content was 32.22% and 22.76%,respectively.(3)AM,AP and RO have obvious inhibitory effects on the secretion of inflammatory factors such as NO and IL-1β,but flavonoid glycosides have no obvious effect.The TF,AM,AP,and RO EC50 values for inhibiting NO secretion were 40.47 μg / m L,116.0 μM,16.71 μM,15.21 μM,and the EC50 values for inhibiting IL-1β secretion were 38.43 μg / m L,25.90 μM,and9.49 μM,respectively.28.24 μM.The results of the mouse model of acute gouty arthritis showed that the thickness of the footpads of the model group significantly increased 4hours after the model establishment compared with the normal group(P<0.01),and the thickness of the foot pads reached the maximum at 24 hours.The level of IL-1β in the pad tissue increased significantly(P<0.01),and a large amount of inflammatory cell infiltration was observed by HE staining.Compared with the model group,the TF,AM,and AP administration groups could significantly reduce the thickness of the footpads of mice(P <0.05),reduce the level of IL-1β in footpad tissues(P <0.05),and reduce the infiltration of inflammatory cells.(3)LPS-sensitized THP-1 cells induced by sodium urate(MSU)were used to construct an in vitro gout model,and the anti-inflammatory mechanism of flavonoids from S.moellendorffii was explored.It was found that S.moellendorffii could significantly reduce lactate dehydrogenase(LDH)levels compared with the model group(P <0.01)at a concentration of 100-50 μg / m L,improve cell surface morphology,and reduce the number of PI stained cells.At a concentration of 100-25μg/m L,S.moellendorffii significantly inhibited the secretion of IL-1β and TNF-α(P <0.01),and inhibited the expression of IL-1β,TNF-α,Caspase-1 and NLRP3 m RNA.At the same time,flavonoids from S.moellendorffii could inhibit ASC oligomerization,reduce ASC spots,reduce the fluorescence intensity of NLRP3 protein,and reduce the expression of IL-1β /pro-IL-1β,Caspase-1 / pro-caspase-1.Conclusion: The 50% ethanol elution part,apigenin,amentoflavone,and Robusta-4’ methyl ether from S.moellendorffii have good anti-inflammatory activity.Flavonoids in S.moellendorffii could protect cell membrane integrity,improve cell morphology,reduce the release of inflammatory factors,and inhibit the expression of related proteins and m RNA on the NLRP3 / ASC / Caspase-1 axis to play a role in anti-gouty arthritis.Research has laid the foundation for the pathogenesis and treatment of gout.