Baicalin Intervenes Mycoplasma Gallisepticum-induced Inflammation Via Regulating NLRP3-Autophagy Pathway And Energy Metabolism

Mycoplasma gallisepticum(MG)belongs to the class Mollicutes,the smallest microorganism which lack cell wall and capa ble of self-replication outside a host.MG is one of the primary etiologic agents of chronic respiratory diseases in chicken and caused major economic losses.MG-infection reduced weight gain,feed intake and egg production in chickens and caused mortality of young chickens.MG lack cell wall and therefore antibiotics that act on the cell wall are ineffective against MG infection.Hence,it is especially important to develop effective and safe drugs for the treatment of MG infection.Currently,traditional Chinese medicinal compounds are famous for the prevention and treatment of MG infection.For instance,the flavonoid,baicalin possess multi-fold therapeutic properties,and mainly found in the roots of Scutellaria baicalensis Georgi.Baicalin have been reported to possess antibacterial,antiviral,anti-cancer,anticonvulsant,antioxidant,hepatoprotective and neuroprotective effects.The pharmacological properties of baicalin are due to their abilities to scavenge reactive oxygen species(ROS)and modulati on of various signaling molecules associated with apoptosis,inflammation,autophagy,cell cycle,mitochondrial dynamics and cytoprotection.However,the protective effects of baicalin on MG infection in chicken and macrophages,and its underlying molecula r mechanisms are still unclear.(1)Baicalin alleviated MG-induced oxidative stress and inflammation via modulating NLRP3 inflammasome-autophagy pathway in HD11 cellsIn the present study,chicken like macrophages(HD11 cells)were cultured for in vitro experiments.HD11 cells were divided into six experimental groups including control group,MG-infection group(400 MOI),MG-infected plus baicalin-treated low(400 MOI,50?g/m L),medium(400 MOI,100?g/m L),high dose group(400 MOI,150?g/m L)and baical in-alone treated group(150?g/m L).The results of cell's viability showed that significant(p<0.05)cell death was observed at a dose greater than 400 MOI at 4 h and 6 h,and at a dose greater than 200 MOI at 8 h compared to the control group.Thus,MG 400 MOI for 6 h was used as the optimum infection dose for subsequent experiments.While,Baicalin concentrations less than 200?g/m L for 12 h has no potential cytotoxic effects on the cell's viability of HD11cells.Thus,baicalin concentrations of 50,10 0 and 150?g/m L for 6 h after infection were chosen for subsequent experiments.Oxidant status revealed that malondialdehyde(MDA)content and lactate dehydrogenase(LDH)enzyme activity was significantly(p<0.05)increased and catalase(CAT)and glutath ione peroxidase(GSH-Px)activities were significantly(p<0.05)reduced in the MG-infected HD11 cells.The level of intracellular ROS was also significantly(p<0.05)enhanced in the MG-infected HD11 cells.Meanwhile,baicalin alleviated the alterations in the MDA content,CAT,GSH-Px and LDH activities,and significantly(p<0.05)reduced the level of total intracellular ROS in a dose-dependent manner.Light microscopy revealed the detachment of HD11 cells and shrinkage of cell bodies in the MG infected group compared to the control group.MG-infection induced mitochondrial and cellular damage in HD11 cells along with the disappearance of the nuclear membrane.However,baicalin-treatment partially attenuated these abnormal ultrastructural changes compared to the MG-infection group,and prevented the loss in??_M(dose-dependently)in HD11 cells.Moreover,MG-infection caused an increase in toll-like receptor(TLR)-2A,Nuclear factor kappa B(NF-?B),myeloid differentiation primary response 88(MYD88),tumor necrosis factor(TNF-?),cyclooxygenase(Cox)-1,Cox-2,caspase-1,NLR family pyrin domain containing 3(NLRP3),interleukin(IL)-1?,and IL-18 m RNA expression.At the same time,baicalin treatment significantly(p<0.05)reduced the m RNA expression of these genes compared to the MG infection group.MG-infection significantly(p<0.05)increased TLR-2 protein expression along with the phosphorylation of P65 and IKB-?,NLRP3,mature caspase-1 and mature IL-1?in the MG-infected HD11 cells.The phosphorylation of P65 and IKB-?,and protein expressions of IL-1?were also reduced with baicalin in a dose-dependent manner.Besides,baicalin intervention decreased IL-1?,IL-18,TNF-?and IL-18 activities.MG-infection caused reduction in the mammalian target o f rapamycin(m TOR)m RNA expression in HD11 cells.While,the m RNA expression of Beclin-1,autophagy related gene(ATG)-7,microtubule associated protein 1 light chain 3(MAP1-LC3),ATG-5 and Unc-51 like autophagy activating kinase(ULK-1)were increased in the MG-infected HD11 cells.The protein expression of m TOR were significantly(p<0.05)reduced in HD11 cells.While,the protein level of ATG-5 and sequestosome-1(P62)were enhanced compared to the control group.Meanwhile,baicalin treatment restored m TOR m RNA and protein level,and enhanced Beclin-1,ATG-7,MAP1-LC3,ATG-5 and ULK-1m RNA expression and protein expressions of ATG-5 and P62 in a dose-dependent manner compared to the MG-infection group.(2)Baicalin attenuated MG-induced inflammation and apoptosis by restoring energy metabolism in the chicken lungs120 one-day old White Leghorn chickens were randomly divided into control group,MG-infection group(1×10⁹ CCU/m L,0.2 m L),MG-infected group treated with baicalin at a dose of 450 mg/kg and baicalin-alone treated group(450 mg/kg).After 7 days of post-treatment,samples of blood serum,lungs,thymus and bursa of fabricius(BOF)were collected for experimental analyses.Serum TNF-?,IL-8,IL-6 and IL-1?(p<0.05)enzyme activities were increased in the MG-infection group.Histopathological assessment showed inflammatory cells infiltrate,increased neutrophils,alveolar wall thickening and hemorrhages in the lungs of MG-infected chickens.Ultrastructural analysis revealed mitochondrial swelling,chromatin condensation and membrane disruption in the MG-infection group.However,baicalin-treatment alleviated these abnormal pathological changes and reduced MG-induced increase in serum cytokine activities compared to the MG-infection group.The m RNA expression of TLR-2A,NF-?B,MYD88,IL-6,IL-1?,inducible nitric oxide synthase(i NOS),prostaglandin E2 receptor 2(PTGE),interferon gamma(IFN-?),Cox-1 and Cox-2 were enhanced in the MG-infection group.In addition,the protein expressions of NF-?B,IL-6,and IL-1?were enhanced in MG-infected chickens.The m RNA and protein expression levels of these inflammation-related genes were reduced with baicalin treatment compared to the MG-infection group.TUNEL assay revealed that the number of positive-stained nuclei were significantly(p<0.05)enhanced in the MG-infection group compared to the control group.However,baicalin intervention significantly(p<0.05)reduced positive-stained apoptotic nuclei compared to the MG-infection group.The ATPase activities including Mg²⁺-ATPase,Ca²⁺ATPase,Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase activities were significantly(p<0.05)decreased in the MG-infected chickens.Besides,MG infection caused a decrease in the m RNA expression of energy metabolism-related genes such as phosphofructokinase(PFK),pyruvate kinase(PK),aconitase 2(ACO ₂),hexokinase 1(HK)₁,HK₂,succinate dehydrogenase complex-B(SDHB),lactate dehydrogenase(LDH)A and LDHB gene.In addition,the protein expression level of PK,PFK,HK ₁ and HK₂ were significantly(p<0.05)reduced compared to the control group,except ACO ₂(p>0.05).However,baicalin treatment significantly(p<0.05)restored MG-induced decrease in Mg²⁺-ATPase,Ca²⁺ATPase and Ca²⁺-Mg²⁺-ATPase activities along with the m RNA expression of PFK,PK,ACO₂,HK₁,HK₂ SDHB,LDHB and LDHA compared to the MG-infection group.Besides,baicalin-treatment significantly(p<0.05)alleviated the decrease in protein expression level of PK,PFK,HK ₁ and HK₂ enzyme compared to the MG-infection group,except the ACO₂(p>0.05)protein level.(3)Baicalin mitigated MG-induced oxidative stress and apoptosis in chicken thymus through the Nrf2/HO-1 defence pathwayIn chicken thymus tissues,superoxide dismutase(SOD),CAT and GSH-Px activities were significantly(p<0.05)decreased in the MG-infection group compared to the control conditions.Meanwhile,MDA content and gamma-glutamyl transferase(?-GT)activity were significantly(p<0.05)enhanced in the MG-infected group.At the same time,baicalin treatment significantly(p<0.05)ameliorated the alteration in CAT,SOD,MDA and?-GT activities compared to the MG-infection group,except the GSH-Px activity(p>0.05).Histopathological results revealed increased inflammatory cells infiltrate,nuclear debris,and a few vacuoles in the thymocytes of chickens from the MG-infected group compared to the control group.Ultrastructural examination revealed many apoptotic fea tures,such as mitochondrial swelling,chromatin condensation and cell damage,in the MG-infected group.However,these abnormal morphological and ultrastructural changes were partially disappeared with baicalin treatment compared to the MG-infection group.Besides,baicalin alleviated MG-induced increase in TNF-?,IL-1?,IL-6 and IL-8 enzyme activities.The m RNA expressions of p53,Cytochrome-C,Bax,Caspase-3,Caspase-8 and Caspase-9 were significantly(p<0.05)increased in the MG-infected group,while B-cell lymphoma-2(Bcl-2)was significantly(p<0.05)decreased in the MG-infected group compared to the control group.Meanwhile,a significant(p<0.05)reduction was noted in the m RNA expression of p53,Bax,Caspase-8,Caspase-3,Cytochrome-C and Caspase-9 with baicalin intervention.In contrast,the m RNA expression of Bcl2 gene was significantly(p<0.05)increased in the baicalin-alone group and MG-treated baicalin group compared to the MG-infected group.Consistently,baicalin alleviated the changes in the protein expression levels of apoptosis-related genes in the thymus tissues,and effectively reduced the number of positive-stained brown nuclei in the MG-treated baicalin group.More importantly,the transcription factor Nrf2,NAD(P)H quinone dehydrogenase 1(NQO1),heme oxygenase-1(HO-1)and Glutathione S-Transferase A2(GSTA2)m RNA and protein expressions of Nrf2 and HO-1were also significantly enhanced with baicalin treatment compared to the control group and the MG-infection group.(4)Baicalin attenuated immune impairment during MG infection in chicken BOF through modulation of autophagy and inhibited inflammation and apoptosisHistopathological examination showed abnormal morphological signs and cellular damage including increased lymphocyte i nfiltration and interstitial cells in the MG-infection group.Besides,electron microscopy showed obvious tissue damage,swollen mitochondria and broken mitochondrial cristae in the MG-infection group compared to the control group.However,the above abnormal pathological and ultrastructural changes in the chicken BOF were partially ameliorated with baicalin-treatment compared to the MG-infection group.The m RNA expressions of NF-?B,IL-6,IL-1?and TNF-?were increased in the MG-infection group.Besides,the protein expression level of NF-?B,IL-6,IL-1?and TNF-?were enhanced in the MG-infection group.Meanwhile,baicalin significantly(p<0.05)suppressed the m RNA and protein expressions of NF-?B pathway-related genes,and alleviated the increased activities of TNF-?,IL-6,IL-8 and IL-1?activities compared to the MG-infection group.Bax,p53 and cytochrome C m RNA were significantly(p<0.05)enhanced in the MG-infection group compared to the control conditions.In contrast,Bcl2 m RNA was significantly decreased(p<0.05)in the MG-infection group.Consistently,the protein expression of Bax,p53 and cytochrome C levels were significantly(p<0.05)enhanced in the MG-infection group with the exception of Bcl-2.In addition,the number of positive-stained nuclei were significantly(p<0.05)increased in the MG-infection group compared to the control group.Notably,baicalin significantly alleviated the alterations in the m RNA and protein expression level of apoptosis-related genes and reduced the numbe r of positive-stained apoptotic nuclei in BOF tissues compared to the MG-infection group.Besides,MG-infection significantly(p<0.05)reduced the m RNA expression of MAP1-LC3,ATG-5,F-actin,Dynein,?-tubulin and Beclin-1 in BOF tissues.While,the m TOR m RNA expression was significantly(p<0.05)increased in the MG-infection group.The protein levels of m TOR were significantly(p<0.05)enhanced,and Beclin-1 and ATG-5 were reduced in the MG-infection group compared to the control group.Moreover,MG infection significantly increased the expression of dynamin-related protein 1(DRP1)and mitochondrial fission factor(Mff)proteins in chicken BOF tissues.While,the protein expression of optic atrophy 1(Opa1)has been reduced in the MG-infection group.Meanwhile,baicalin-treatment significantly restored the m RNA and protein expressions of autophagy and mitochondrial dynamics-related genes.Furthermore,MG-infection caused a significant decrease in the number of CD8⁺cells in the chicken BOF.While,baicalin significantly restored the number of CD8⁺cells compared to the MG-infection group.The data suggested that baicalin could alleviate the dysfunction in mitochondrial dynamics and immune impairment by restoring the number of CD8⁺cells in the BOF tissues.In conclusion,the results of this study indicated that baicalin could protect the structural integrity and inhibited MG-induced nuclear and cellular damage in the macrophages,lungs,thymus and BOF tissues.In addition,baicalin effectively alleviated oxidative stress,inhibited inflammation and apoptosis and protected mitochondrial function and energy metabolism dysfunction during MG infection.Baicalin provided cytoprotection by the activation of Nrf2 signaling pathway and activated autophagy vi a the suppression of NLRP3inflammasomes and TLR-2-NF-?B pathway.The present research revealed new therapeutic drug targets against MG infection,and provide a theoretical basis for the application of baicalin in veterinary clinics.