Effect Of Baicalin On The Release Of HMGB1 From Piglets Induced By Haemophilus Parasuis And Lipopolysaccharide And Related Signal Pathways

Haemophilus parasuis(HPS)is a common bacteria that colonizes the upper respiratory tract of pigs and can cause Haemophilus parasuis disease in certain conditions.It is also called Glasser’s disease.The disease is characterized by meningitis,multiple serotonitis,arthritis as the main respiratory infectious diseases of pigs.It seriously harms pigs of all ages and has a high mortality rate.HMGBI protein is involved in the pathogenesis of a variety of infectious pathogens.In this study,we explored whether Haemophilus parasuis can induce HMGB1 secretion in porcine peripheral blood mononuclear cells.We also studied the effect of baicalin on HMGB1 secretion after LPS infection in piglets and analysis of changes in related signaling pathways after infection of peripheral blood monocytes by Haemophilus Parasuisthrough RNA-Seq.1.Establishment of HMGB1 release model in piglets induced by Haemophilus parasuis and lipopolysaccharide.In vitro test set blank control group,LPS group,HPS group,induced peripheral blood mononuclear cells of piglets 12-48hours;in vivo test LPS induced piglets o,3,6,9,12,24,36,48,72 hours after blood collection serum was separated to detect the release of HMGB1.Western blot was used to determine the expression of HMGB1 in monocyte culture supernatant.Q-RT PCR was used to determine the mRNA expression of HMGB1 in monocytes.ELISA was used to detect the content of HMGB1 in serum.The results showed that the protein expression of HMGB1 was significantly increased after 36 hours of infection with H.parasuis(P<0.01).and the mRNA expression of HMGB1 was highest at 24 hours.In addition,LPS can also induce monocyte releaseHMGB1,and the mRNA signiflcantly increased at 12,36and 48hours(P<0.01);in vivo serum ELISA results showed that compared with the control,the level of HMGB1 secretion was significantly increased from 3 to 48hours,and the release of HMGB1 peaked at 36hours and then decreased at 72hours.2.Effect of baicalin on the release of HMGB1 from porcine monocytes infected with Haemophilus parasuis in vitro.Tests were performed in blank control group,Haemophilus parasuis model group,acetylcysteine(NAC)treated group,and baicalin treated group(12.5,25,50.100 μg/mL).The drug treatment group was first pretreated with the corresponding concentrations of baicalin and 1 mM NAC for 1 hours,and then added to the mononuclear cells infected with Haemophilus parasuis.After 36 hours and 48 hours of culture,the cell culture supernatant was collected to test the content of HMGB1 in the supernatantby ELISA;the expression of HMGB1 mRNA in cells was determined by Q-RT PCR;the nuclear shift of HMGB1 was measured by indirect immunofluorescence.The results showed that after 36 hours of HPS-infected mononuclear cells,the release of HMGB1 in the cell culture supernatant was significantly higher than that in the control group(p<0.01),the effect of 1 mM NAC and 12.5 μg/mL baicalin on the down-regulation of HMGB1 in mononuclear cell culture medium induced by HPS infection was not significant.Baicalin at 25,50,and 100 μg/mL could significantly down-regulate the content of HMGB1(p<0.01).HPS infection after 48h of monocytes,the release of HMGB1 in cell culture supernatants was significantly increased(p<0.01)in the HPS model group compared with the control group,and mononuclear cell culture solution caused by 1 mM of NAC against HPS infection was observed that the down-regulation of HMGB1 was not significant,and 12.5 μg/mL baicalin significantly down-regulated the content of HMGB1(p<0.05).Baicalin at 25,50,and 100 μg/mL significantly down-regulated the content of HMGB1(p<0.01);HPS infected monocytes 24hours,HPS model group compared with the control group,monocyte HMGB1 mRNA expression levels increased significantly(p<0.01),The effect of 1mM NAC down-regulation of HMGB1 mRNA was not significant.Baicalin at 25,50,and 100 μg/mL can significantly down-regulatethe mRNA expression of HMGB1(p<0.01);The results of immunofluorescence showed that compared with the blank control group,the number of HMGB1 translocated to the nucleus increased significantly,and 1 mM NAC had a certain inhibitory effect on the nuclear transfer of HMGB1,The baicalin of 12.5,25,50,and 100 μg/mL have a certain inhibitory effect on the nuclear transfer of HMGB1,and the effect was most significant when the baicalin concentration was 100μg/mL.3.Effect of baicalin on the release of HMGB1 from serum induced by LPS in vivo in piglets.The test was divided into a blank control group and a baicalin treatment group.In the baicalin treatment group,baicalin was injected intramuscularly at 25 mg/kg 2 hours before LPS injection to the piglets,and twice daily,and the control group received only LPS injection.After LPS injection,serum was collected at 0,3,12,24,36,48,72hours to collect serum.ELISA was used to detect the content of HMGB1 in serum at each time point.Brain and lung tissues were taken and fixed with formaldehyde 72 hours later to make paraffin sections.Observe the histopathological changes.The results showed that the serum HMGB1 level in the piglets stimulated by LPS was highest after 36 hours and decreased to normal level after 72 hours.The effect of baicalin on the release of HMGB1 in LPS-induced immune stress was very significant(P<0.01).The piglets in the LPS injection group showed severe acute inflammation,but the piglets in the drug group showed a slight reaction.Baicalin injection at 25 mg/kg significantly increased the survival rate of the piglets in the drug group.Histopathological observation showed that piglets of LPS group exhibited extensive edema,congestion,and severe hemorrhage,fibroblasts proliferated in large numbers,and bronchioles were filled with a large amount of cell exudate,and LPS group piglets had parenchymal hyperemia and severe edema.However,the baicalin-treated piglets observed only minor pathological lesions in the brain and lungs.4.Transcriptome sequencing analysis of monocytic cells infected with Haemophilus parasuis in piglets.Pig peripheral blood mononuclear cells were isolated and the cell concentration was adjusted to 1.0 × 106/mL.The test was divided into blank control group and HPS group(blank control group:K1,K2,K3;HPS group:H1,H2,H3).Make 3 repetitions for each treatment group.2 mL of each cell lysate was cultured in a cell culture plate.The blank control group was added with 0.5 mL of cell culture medium,and the HPS group was added with 0.5 mL of HPS suspension having a final concentration of 1.0 × 106 CFU/mL.After 24 hours of co-cultivation,cells were harvested,Trizol reagent was added,and cDNA was subjected to RNA-seq using a Hiseq 2500.Sequencing results showed that 982 genes were significantly changed after infection with H.parasuis for 24 hours(fold change≥2,p<0.05),of which 646 genes were up-regulated and 336 genes were down-regulated;TheHPSgroup HMGB1 gene was up-regulated 0.21 fold;cytokine-cytokine receptor interactions,chemokine signaling pathways,and TNF signaling pathways were most abundant in mononuclear cell infection.In summary,Haemophilus parasuis and lipopolysaccharide can induce the release of HMGB1 in piglets.Baicalin can inhibit the release of HMGB1 induced by Haemophilus parasuis and lipopolysaccharide,and it can improve the immune response of piglets induced by LPS.The survival rate exerts anti-inflammatory effects The results of this study provide a new method for baicalin to prevent and control the infection of H.parasuis infection.