Study On The Mechanism Of Matrine Inhibiting Pams Secrete IL-1β And Its Combined Effect With Antibiotics

Porcine reproductive and respiratory syndrome virus(PRRSV)infection mainly results in reproductive failure and interstitial pneumonia,and leads to immunosuppression.It is often co-infected with other viruses and bacterial swine diseases,which causes great economic losses to pig production.Previous studies of our group have demonstrated that Matrine possess anti-PRRSV effect,has significant therapeutic effect against interstitial pneumonia induced by PRRSV/PCV2 co-infection and inhibits LPS-induced acute lung injury in mice.Inflammation is a common pathological process in clinic in which IL-1β is one of the most important multipotent inflammatory cytokines.Therefore,an inflammatory model was designed,in which primary porcine alveolar macrophages(PAMs)was co-stimulated with 4μg PRRSV 5’UTR RNA and 1 μg/m L LPS to investigate the anti-inflammatory mechanism of Matrine against the pro-IL-1β and mature IL-1β production process.Antibiotics with anti-inflammatory effects were screened and the combination of Matrine was used to detect the expression of IL-1β,the data obtained support the study of Matrine as an alternative antibiotics research.Results:1.Matrine can directly inhibit the PRRSV Nsp9 activity to influence the replication of PRRSV,so PRRSV cannot be used as an inflammatory factor to evaluate the anti-inflammatory effect of Matrine.2.After PAMs transfected with different doses of PRRSV 5’UTR RNA(1,2,4 μg)for 12 h,the results of q RT-PCR showed that 4 μg PRRSV 5’UTR RNA significantly increased the expression of IL-1β when compared with control groups(p < 0.05).When transfected with different doses of PRRSV 5’UTR RNA(1,2,4 μg)along with co-stimulation of 1 μg/m L LPS at the same time,q RT-PCR and Western blot analysis showed that IL-1β expression level was much higher in 5’UTR RNA and LPS co-stimulation group compared with 5’UTR RNA alone and LPS alone group(p < 0.05).The expression of IL-1β in 4 μg co-stimulation group was significantly higher compared with 2 and 1 μg co-stimulation group(p < 0.05).In addition,when PAMs were co-stimulated with 4 μg RNA and LPS for 12 h,the expression of IL-6,IL-8 and TNF-α were also increased significantly when compared with the control groups(p< 0.05).These results suggested that 4 μg PRRSV 5’UTR RNA and 1 μg/m L LPS co-stimulated PAMs induced the secretion of inflammatory cytokines,which can be used toevaluate the anti-inflammatory effect and mechanism of Matrine.3.q RT-PCR and Western blot results showed that Matrine inhibited the expression of IL-1β,IL-6,IL-8 and TNF-α.When compared with control groups,5’UTR RNA and LPS co-stimulation activated NF-κB signal pathway and NLRP3 inflammasome,significantly increased the m RNA expression of DHX36 and NOD2,and the m RNA and protein level of My D88 and RIPK2 also signifeicantly increeased(p < 0.05),but have no influence on the expression of TLR4 and NOD2(p > 0.05).Post Matrine treatment,the expression of NOD2,My D88,NLRP3,Caspase-1 and p-IκBα was significantly decreased(p < 0.05),the expression of p65 in cytoplasmic protein was significant increased(p < 0.05),but the expression of TLR4 、 DHX36 and RIPK2 have no significant difference(p > 0.05).Immunofluorescence showed that Matrine inhibited ASC speck formation.In conclusion,Matrine interferes with the secretion of IL-1β by inhibiting My D88/NF-κB signaling pathway and NLRP3 inflammasome activation.4.PRRSV 5’UTR RNA and LPS co-stimulation inflammatory model of PAMs was used to screen the effect of commonly used antibiotics on IL-1β.The results showed that Amoxicillin,Chlortetracycline,Doxycycline hydrochloride and Florfenicol could significantly inhibited the m RNA expression of IL-1β when compared with co-stimulated group(p < 0.05),Tilmicosina had no significant effect on the expression of IL-1β(p > 0.05).The inhibitory effect of Amoxicillin and Aureomycin treatment group was consistent with that of Matrine on IL-1β gene expression level.When Matrine was used in combination with Amoxicillin or Aureomycin,an interaction was noted between Matrine and Amoxicillin(p =2.89-8),while Matrine and Aureomycin lack such type of interation(p = 0.121).Western blot results showed that when compared with 1 mg/m L Amoxicillin alone treatment group,the combination group of Matrine and Amoxicillin(0.4 mg/m L + 1 mg/m L,0.2 mg/m L + 1mg/m L,0.4 mg/m L + 0.5 mg/m L)significant decreased the expression of IL-1β(p < 0.05).These results showed that Matrine and Amoxicillin had synergistic anti-inflammatory effect and could minimize the dosage of Amoxicillin,which indicates that Matrine could be used as a substitute for antibiotics.In conclusion,Matrine could inhibit the replication of PRRSV by inhibiting the activity of Nsp9.In order to investigate the efficacy of Matrine,an inflammation model was established by using PRRSV 5’UTR RNA and LPS co-stimulated PAMs,which described that Matrine could inhibit the production of IL-1β by preventing My D88/NF-κB signalingpathway and NLRP3 inflammasome activation,and thus plays the anti-inflammatory role.In addition,Matrine combined with Amoxicillin has synergistic effect and can minimize the dosage of some Amoxicillin.These results provide a new data for the research and development of Matrine as an anti-PRRSV drug,and laied a foundation for Matrine to be used as a substitute for antibiotics.