Protective Mechanism Of Pinus Massoniana Pollen Polysaccharides On LPS-induced Septic Myocardial Injury In Animals

Sepsis is a systemic inflammatory response syndrome caused by pathogenic microorganism infection.In the field of veterinary medicine,sepsis is one of the leading causes of death in poultry,and also a major"killer"of morbidity and mortality in domestic animals such as newborn foals and calves.Cardiomyopathy is a common complication of sepsis,with50%of septic patients exhibiting myocardial dysfunction,and sepsis-induced myocardial damage is ultimately the main cause of death in affected animals,so there is an urgent need to explore new therapeutic strategies.The use of natural polysaccharides for the treatment of related diseases has become an effective strategy in modern medicine.Pre-laboratory studies have confirmed the anti-inflammatory effect of pine pinus massoniana pollen polysaccharides（PPPS）,which can be speculated to have a potential protective effect in septic myocardial injury in livestock and poultry.In this study,LPS was injected into Hy-Line brown chicks and Wistar rats to simulate the sepsis model of poultry and domestic animals,and to explore the protective effect of PPPS on septic myocardial injury in vitro and in vivo.High-throughput sequencing of cardiomyocytes in vitro was performed to investigate the anti-injury mechanism of PPPS,screen out the target proteins and signaling pathways of PPPS,and further explore its role in septic myocardial injury.These findings provide a theoretical basis for understanding the mechanism of sepsis-induced myocardial injury and the development of related drugs.This research was divided into four parts:1.Protective effect of PPPS on sepsis-induced animal myocardial injury in vivo.In order to explore the effects of PPPS on myocardial injury in septic animals,the effects of PPPS on septic myocardial injury were comprehensively evaluated by detecting myocardial enzymes,myocardial tissue morphology,oxidative stress level,apoptosis and inflammation-related indicators.The results showed that PPPS could alleviate the abnormalities of myocardial enzymes induced by LPS,reduce the levels of AST and ALT in the serum of septic chicks,and reduce the levels of AST,LDH,and CK-MB in the serum of septic rats.Pathological injury and inflammatory cell infiltration in the myocardial tissue of LPS-induced chicks and rats were also significantly alleviated after PPPS treatment.In addition,apoptosis and oxidative stress damage were also found in the LPS-induced sepsis model group,and PPPS treatment was able to inhibit apoptosis but not alleviate oxidative stress damage.Excessive inflammatory response is the main reason of septic myocardial injury,so the changes of related inflammatory indicators were detected by blood cell analyzer,ELISA and q RT-PCR.The results of blood cell analyzer showed that the average number of white blood cells in the sepsis model group was significantly increased(chicks:7.07×10¹⁰/L,rats:6.26×10⁹/L),but had no effect on the number of red blood cells.PPPS treatment significantly reduced the number of white blood cells in the blood of septic rats to 5.34×10⁹/L,but did not have the same effect in the septic chick model.The expression levels of inflammatory-related mediators in serum and myocardial tissues were subsequently examined,and the results showed that PPPS treatment had a certain inhibitory effect on the increase of related inflammatory mediators and the expression of inflammatory genes in the myocardial tissue of septic chick and rat models,but the anti-inflammatory effect of PPPS on septic rats was better than that of chicks.This part of the study demonstrated that PPPS had some protective effects against septic myocardial injury induced by LPS in both chicks and rats.2.Protective effect of PPPS on sepsis-induced cardiomyocyte injury in vitro.In order to explore whether PPPS has protective effect on septic cardiomyocyte injury in vitro,H9c2 cardiomyocytes were treated with LPS/ATP to establish the sepsis model,and/or co-treated with PPPS.Then,the protective effect of PPPS in vitro was comprehensively evaluated by detecting the related indicators of cell viability,oxidative stress,apoptosis and inflammation.The results showed that PPPS could antagonized the LPS/ATP-induced reduction of cell number and LDH release,and inhibited the LPS/ATP-induced reduction of cell viability.Oxidative stress indicator showed that the levels of ROS and MDA were significantly increased and GSH was significantly decreased in the LPS/ATP model group,PPPS treatment could significantly reduce the levels of ROS and MDA,but did not improve the level of GSH.PPPS also had anti-apoptotic effect in vitro,which could reduce the apoptosis rate induced by LPS/ATP from 18.63%to 11.17%,and down-regulate the expression of apoptotic proteins cleaved caspase3 and cleaved caspase9.In addition,PPPS could inhibit LPS/ATP-induced inflammatory response,reduce the levels of IL-1βand NO,inhibit the phosphorylation of IKBαand NFκB and the expression of NLRP3,and reduce the number of p-NFκB positive cells.This part of the study demonstrated that PPPS had a protective effect on LPS/ATP-induced septic cardiomyocytes in vitro by increasing cell viability,inhibiting ROS and MDA production,alleviating cell apoptosis and antagonizing inflammation-related responses.3.Transcriptome analysis and verification of PPPS against septic cardiomyocyte injuryIn order to clarify the mechanism of PPPS anti-cardiomyocyte injury,the gene expression changes in LPS/ATP group and LPS/ATP+PPPS group were detected by high-throughput sequencing.The molecular mechanism of PPPS was revealed by cluster analysis,KEGG pathway enrichment analysis and protein interaction analysis of the differential genes.The results showed that 183 genes were up-regulated and 150 genes were down-regulated in LPS/ATP+PPPS group.KEGG enrichment analysis showed that the differential genes in PI3K/AKT signaling pathway accounted for the largest proportion and had significant differences.In addition,the Top3 genes in protein interaction network analysis also belonged to PI3K/AKT signaling pathway,indicating that PPPS may achieve protective effects through PI3K/AKT signaling pathway.Therefore,the expression of PI3K/AKT signaling pathway was examined in vitro and in vivo.The results showed that the phosphorylation levels of PI3K（p85α）and AKT proteins were significantly increased,and the protein expression of PTEN was significantly decreased in myocardial tissues and cells of the sepsis model group,which could be reversed by PPPS treatment.These results indicated that the PI3K/PTEN/AKT signaling pathway was activated in the sepsis model group,and PPPS could significantly inhibit the activation of this pathway in vitro and in vivo.In addition,the intervention of PI3K/PTEN/AKT pathway activator antagonized the inhibitory effect of PPPS on inflammatory mediators（IL-1βand NO）.The decreased phosphorylation of IKBαand NFκB and the decreased expression of NLRP3 induced by PPPS were also significantly restored after the intervention of the activator.This part of the study confirmed that PPPS could inhibit sepsis-induced activation of PI3K/PTEN/AKT signaling pathway in vitro and in vivo,and the intervention of PI3K/PTEN/AKT signaling pathway activator could block the anti-inflammatory effect of PPPS,indicating that PI3K/PTEN/AKT signaling pathway is involved in and mediates the anti-inflammatory activity of PPPS.4.PPPS inhibits the activation of PI3K/PTEN/AKT signaling pathway in septic cardiomyocytes via p110β.p110β,a subunit of PI3K family protein,was found to be the key protein against sepsis-induced myocardial injury.In the present study,it was found that the expression of p110βwas significantly decreased after treatment with LPS/ATP,PPPS could increase the level of p110β.In addition,the expression of p110βwas more significantly increased after treatment with only PPPS compared with the control group.In order to explore the mechanism of p110β,the protein interaction prediction of p110βshowed that PTEN might interact with p110β.The results of cell immunofluorescence co-localization and immunoprecipitation also confirmed the interaction between PTEN and p110β.In addition,the interaction between the two was significantly weakened after LPS/ATP treatment,while PPPS treatment enhanced the binding between them.To explore whether p110βis the key protein of PPPS against inflammatory injury in cardiomyocytes,pik3cb（p110β）-p EGFP-C3 recombinant plasmid was constructed and transfected into cells.The effect of p110βon LPS/ATP-induced activation of PI3K/PTEN/AKT signaling pathway and inflammatory protein activation was observed.The results showed that p110βoverexpression significantly inhibited the activation of PI3K/PTEN/AKT signaling pathway induced by LPS/ATP.In addition,the LPS/ATP-induced increase in phosphorylation levels of IKBαand NFκB and the up-regulation of NLRP3 protein were also significantly reversed by overexpression of p110β.Thus,this part of the study suggests that p110βis a key protein of PPPS against inflammatory injury in cardiomyocytes,that PPPS can target and increase the expression of p110βprotein and promote its interaction with PTEN,and overexpression of p110βdirectly affects the activation of PI3K/PTEN/AKT signaling pathway and mediates its downstream inflammatory response.In conclusion,PPPS have protective effects on septic myocardial injury in both chick and rat models,and PPPS also showed good protective effects in an in vitro model of myocardial cell injury.PI3K/PTEN/AKT signaling pathway plays an important role in PPPS anti-septic myocardial injury,and p110βhas been confirmed to be the key protein of PPPS anti-septic myocardial injury.In a word,this study suggests that PPPS may be a promising candidate for the treatment of sepsis-induced myocardial injury,and the inhibition of PI3K/PTEN/AKT pathway and the targeted activation of p110βmay be a potential strategy for the treatment of septic myocardial injury,providing theoretical basis for the research and development of related drugs.