Matrine Inhibits Pneumonia Induced By PCV2 And Its Mechanisms In Kunming Mice

Objective: PCV2 was used to induce mouse pneumonia model to explore the anti-inflammatory effect and mechanism of matrine.Methods: Thirty-two female SPF-level Kunming mice aged 4 weeks,weighing 20±2 g,were randomly divided into 4 groups according to their body weight,with 8 mice in each group,namely: blank control group,PCV2 group,matrine group(40 mg/kg),positive control group(ribavirin 40 mg/kg).Except for the blank control group,mice in the other groups were challenged by intraperitoneal injection of 0.5 m L of105.4 TCID50/m L PCV2,while the blank control group was injected with an equal volume of normal saline,starting on the 5th day after PCV2 inoculation.The corresponding drugs were injected intraperitoneally,once a day at a fixed point,for 5 consecutive days.The mice in the blank control group were injected intraperitoneally with the same dose of normal saline as the drug.On the 11 th day after PCV2 inoculation(1 day after the end of the administration,6 days after the drug administration),the mice were autopsied,lung samples were collected,and lung tissue pathological sections were prepared,and hematoxylin-eosin(HE)staining was used for microscopic examination.Observe the histopathological changes of mouse lungs and score lung injury to determine whether the pneumonia model is successfully established.Lung tissue RNA and total protein were extracted,and the pro-inflammatory cytokines(IL-1β,IL-6,IL-8,TNF-α)in the lung tissues of each group were detected by real-time fluorescence quantitative method(q PCR)and Western blot method.The influence of m RNA and protein expression levels to determine whether matrine can inhibit the inflammatory response caused by PCV2.Western blot was used to detect the key proteins related to the regulation of IL-1β production including toll-like receptor 4(TLR4),myeloid differentiation factor 88(My D88),nuclear factor κB p65(NF-κBp65),phosphorylated nuclear factor kappa B inhibitor protein alpha(p-IκBα),nuclear factor kappa B inhibitor protein alpha(IκBα),aspartate-specific caspase-1(Caspase-1),apoptosis-related dot-like protein(ASC)and NOD receptor 3(NLRP3)protein in the total protein of each group of lung tissue to determine the potential mechanism of matrine against PCV2 induced inflammation.Results:1.Using HE staining,observe the effect of matrine on the pathological changes of the lungs of PCV2-infected mice under a light microscope and score the lung injury.The results showed that compared with the blank control group mice,the alveoli septum of the PCV2 group mice was significantly thickened,and the alveolar cavity was significantly reduced.At the same time,a moderate amount of inflammatory cell and red blood cell infiltration were observed,and the lung injury score increased significantly(p<0.001).Compared with the PCV2 group,the widening of the alveoli septum in the matrine group was relieved,and the lung injury score was significantly reduced(p<0.001).It shows that PCV2 can cause interstitial pneumonia,and the intervention of matrine can improve its symptoms.2.q PCR and Western blot were used to detect the m RNA and protein levels of IL-1β,IL-6,TNF-α,IL-8 in the lung tissue of mice after PCV2 infection for 11 days.The results showed that compared with the blank control group,the relative expression of the inflammatory factors IL-1β,IL-6,IL-8 and TNF-αm RNA and protein of PCV2 infected mice 11 d increased significantly(p<0.001).Compared with the PCV2 group,the expression levels of m RNA and protein of IL-1β,IL-6,IL-8 and TNF-α in the matrine group and the positive control group were significantly decreased(p<0.001).The results show that matrine inhibited PCV2-induced pneumonia in mice.3.Western blot was used to detect the protein expression levels of TLR4 and MyD88 in the lung tissues of each group.The results showed that compared with the blank control group,the protein expression levels of TLR4 and My D88 in the lung tissues of the PCV2 group were significantly increased(p<0.001).Compared with the PCV2 group,the protein expression levels of TLR4 and My D88 in the lung tissues of the matrine group and the positive control group decreased significantly(p<0.001).The results indicate that matrine inhibited the activation of the TLR4/My D88 signaling pathway.4.Western blot was used to detect the activation of NF-κB signaling pathway in the lung tissue of each group of mice.The results showed that compared with the blank control group,the expression levels of p-IκBα and NF-κBp65 protein in the lung tissue of the PCV2 group significantly increased,and the expression level of IκBα protein decreased(p<0.001).Compared with the PCV2 group,the expression levels of p-IκBα and NF-κBp65 protein in the lung tissues of the matrine group and the positive control group decreased significantly(p<0.001),and the expression level of IκBα protein increased significantly (p<0.001).The results indicate that matrine inhibited the activation of NF-κB signaling pathway.5.Western blot was used to detect the activation of NLRP3 inflammasomes in the lung tissues of each group of mice.The results showed that compared with the blank control group,the protein expression levels of NLRP3,ASC and Caspase-1 in the lung tissues of the PCV2 group of mice were significant increased(p<0.001).It shows that PCV2 infection induced the activation of NLRP3 inflammasomes.Compared with the PCV2 group,the protein expression levels of NLRP3,ASC and Caspase-1 in the matrine group and the positive control group were significantly decreased(p<0.001).The results show that matrine inhibited the activation of NLRP3 inflammasome.Conclusion: 1.Matrine ameliorated the widening of the alveoli septum in mice induced by PCV2 infection,and significantly reduced the expression of IL-1β,IL-6,IL-8 and TNF-α in the lung tissues of mice,which confirmed that Matrine inhibited mouse pneumonia induced by PCV2 infection.2.Matrine inhibited the activation of TLR4/My D88/NF-κB signaling pathway and NLRP3 inflammasome to relieve pneumonia induced by PCV2 in mice.