Shikonin Controls Inflammatory Disease Through Inhibiting NLRP3 Inflammasome Activation

Background:NLRP3 inflammasome is a cytosolic polymer protein complex consisting of NLRP3,ASC,and pro-caspase-1.When receiving external stimuli or perceiving endogenous danger signals,the NLRP3 inflammasome activates macrophages through a series of reactions to ward off fungal,bacterial,and viral infections and eliminate internal danger signals.However,inflammation is a double-edged sword.Overactivated inflammasome promote the release of a large amount of IL-1β and IL-18,leading to cell death and inflammatory tissue damage,and then participate in the emergence and development of a variety of diseases such as ulcerative colitis,acute respiratory distress syndrome,gout,diabetes,etc.Therefore,targeted inhibition of the activation of the NLRP3 inflammasome signaling pathway not only helps to deepen the understanding of the occurrence and development of inflammatory diseases but also provides new ideas to find potential therapeutic targets for such diseases.The acute inflammatory disease models used in our research are ulcerative colitis and acute respiratory distress syndrome,which have been reported to be related to the abnormal activation of NLRP3 inflammatory bodies.Chinese herbal medicine has gained worldwide attention as a complementary treatment for diseases and a source for the discovery of new medicines.Due to their advantages of availability,and lower toxic side effects.SHK is a naphthoquinone compound extracted from “Radix Lithospermi”.As an immunosuppressant,SHK plays an immunomodulatory role in immune-associated diseases,in the treatment of diseases,or the relief of clinical symptoms.Studies have shown that SHK inhibits inflammasome activation by inhibiting caspase-1 activation,however,the exact mechanism by which SHK inhibits the inflammatory response remains unclear.Therefore,we speculate that SHK exerts anti-inflammatory effects by inhibiting NLRP3 inflammasome activation.Purpose:To explore whether SHK improves inflammatory diseases by inhibiting NLRP3 inflammasome activation and elucidating its mechanism of action using vitro and vivo experiments.Methods:1.In vitro experiment: BMDMs and PMs were separated for the following research:(1)The effect of SHK on caspase-1 and IL-1β maturation and secretion.LPS-primed PMs and BMDMs were treated with different concentrations of SHK(0.5 μM,1 μM,and 2 μM)for different times(0 h,0.5 h,1 h,and 2 h),followed by incubated with various NLRP3 agonists Nigericin(20 μM for 45 min),ATP(2.5 m M for 45 min),and MSU(150 μg/m L for 6 h).The supernatants(Sup)and lysates(Lys)were collected.The expression of NLRP3 inflammasomes and cleaved-caspase-1(P20)and cleaved-IL-1β(P17)was detected by WB,and ELISA detected the level of IL-1β,IL-18,TNF-α in Sup.(2)The effect of SHK on other inflammasome activation and the first signal of NLRP3 inflammasomes.LPS-primed PMs were treated with different concentrations of SHK for 30 min,followed by incubation with Nigericin for 45 min.Cells were collected and the expression of IL-1β and NLRP3 m RNA was detected by q PCR and the expression of related proteins was detected by WB.(3)The effect of SHK on NLRP3 inflammasome assembly.LPS-primed PMs were treated with different concentrations of SHK for 30 min,followed by incubation with Nigericin for 45 min.Cells were collected to detect oligomerization of ASCs with cross-linking agents,ASC speck with IF,and the interaction between NLRP3 and ASC with co-IP.(4)The effect of SHK on ROS production,mt DNA release,and ox-mt DNA production.LPS-primed PMs were treated with different concentrations of SHK for 30 min,followed by incubation with Nigericin for 45 min.Cells were collected,ROS changes were detected by FCM,mt DNA expression was detected by q PCR,mt ROS expression was detected by multifunctional microplate reader fluorescence analysis,and JC-1,UCP2,ox-mt DNA expression was detected by IF.2.In vivo experiment:(1)Dextran sulfate sodium(DSS)-induced UC model.A 3% DSS-induced acute UC model of mice was established,and the C57/6N was randomly divided into 4 groups,including the Normal group,the DSS model group,the olive oil solvent control group(Oil group),and SHK(12 mg/kg)treatment group,the Normal group was fed with normal drinking water.On day 1,day 3,day 5,and day 7 of modeling,refreshing drinking water with 3% DSS,and intraperitoneal injection of SHK on day 2,mice were recorded daily,and disease activity scores(DAIs)were performed.Mice were sacrificed on day 8 and peripheral blood,spleen,m LN,and colon was collected.Lymphocytes and macrophages in blood were detected with a hemocytometer;Macrophages number and polarization and CD3+T number and subtype in peripheral blood,spleen,and m LN of mice were detected with FAM.The expression of gene and protein levels of NLRP3 inflammasomes in colon tissues were detected with q PCR and WB.The histopathological changes in the colon were detected by hematoxylin-eosin staining(HE);Macrophage infiltration in colonic tissue was detected with IF.(2)LPS-induced ARDS model.Intraperitoneal 20 mg/kg LPS and 6mg/kg SHK for 8 h induced ARDS in C57/6N,mice were randomly divided into 3 groups,including the Normal group,the LPS model group,and the SHK(6 mg/kg)treatment group.The peripheral blood,BALF,and lung tissue of mice were collected.ELISA detected the levels of IL-1β,IL-18,IL-6,and TNF-α in plasma and BALF,and lung tissue pathological changes were detected by HE staining.Result:1.In vitro experiments,we explored the inhibition of NLRP3 inflammasome activation by SHK and its preliminary mechanism.(1)SHK inhibits the maturation and secretion of caspase-1 and IL-1β.The NLRP3 inflammasome was activated when PMs and BMDMs were treated with LPS plus Nigericin,ATP,MSU,and the secretion of P17 and P20 were increased,SHK treated with different concentrations and different time can inhibit the expression of P17 and P20 with dose-dependent.However,SHK did not affect the expression of TNF-α.(2)SHK does not affect the first signaling of NLRP3 inflammasome and other inflammasome activation.WB results showed that SHK had no effect on NLRP3 and pro-IL-1β gene and protein expressions,and had no effect on other inflammasome proteins(AIM2,NLRC4).(3)SHK inhibits the assembly of NLRP3 inflammasome.The results showed that SHK treatment inhibited ASC oligomerization,the formation of ASC spots,and blocked the interaction between NLRP3-ASC(4)SHK reduces the generation of ox-mt DNA via suppression of ROS production and mt-DNA release.SHK inhibits the production of ROS,increases mitochondrial membrane potential,and maintains mitochondrial homeostasis by upregulating the expression of mitochondria-associated proteins UCP2 and NCF1.In addition,SHK inhibits the production of mt DNA,mt ROS,and ox-mt DNA.After DMNQ was applied to cells,ROS,mt ROS,and ox-mt DNA expression levels recovered.2.In vivo experiment: we examined the therapeutic effect of SHK on NLRP3 inflammasomerelated diseases.(1)SHK alleviates DSS-induced UC in mice.In UC mice,we found that SHK treatment mice increased in body weight and colon length decreased in DAI score,a significant improvement in pathological damage in colon tissue,and a decrease in macrophage infiltration.WB and q PCR results showed that SHK reduced the expression of colonic inflammatory factor gene and protein levels.FCM results showed that SHK reduced the number of macrophages in peripheral blood,spleen,and m LN,and decreased the number of M1 macrophages and M2 macrophages in the SHK treatment group in the spleen and m LN,while CD3+ T cells and CD4+ T cells in the peripheral blood,spleen and m LN in the SHK treatment group were significantly reduced.(2)SHK alleviates LPS-induced ARDS in mice.In ARDS mice,we found that SHK treatment reduced the expression levels of IL-1β,IL-18,IL-6,and TNF-α in mouse plasma and BALF,and reduced inflammatory cell infiltration in lung tissue.Conclusion:1.SHK can improve related inflammatory diseases such as DSS-induced UC and LPS-induced ARDS by inhibiting the activation of NLRP3 inflammasomes.2.SHK inhibits the interaction between NLRP3 and ASC,preventing ASC oligomerization and complex assembly.3.SHK promotes mitochondrial homeostasis by regulating mitochondria-associated protein molecules to inhibit ox-mt DNA and mt ROS production.