Mechanisms Of PKM2 Tetramer Regulating Endotoxin Tolerance In Macrophages

BackgroundSepsis is a common complication with dangerous onset and high mortality.Endotoxin tolerance is an important endogenous protective way for the body to cope with sepsis.Pyruvate kinase M2,(PKM2)is the rate-limiting enzyme in glycolytic pathway,and PKM2 has three configurations: monomolecular,dimer and tetramer.It was found that macrophages,stimulated by lipopolysaccharide(LPS),promoted the transformation of PKM2 tetramer(cytoplasm)to mono/dimeric(nucleus),and mediated inflammatory by inducing nuclear translocation of mono/dimeric PKM2.TEPP-46 is a small molecule agonist of PKM2,which can induce the formation of PKM2 tetramer.Studies have shown that macrophages treated with TEPP-46 can inhibit LPS-mediated inflammatory response by inhibiting nuclear translocation of mono/dimeric PKM2.TEPP-46 can not only inhibit the nuclear translocation of mono/dimeric PKM2,but also promote the formation of PKM2 tetramer.It is not clear how PKM2 of tetramer can regulate endotoxin tolerance of macrophages.To study the mechanism of PKM2 tetramer in macrophage endotoxin tolerance is of great significance to elucidate the endogenous protective effect in sepsis.In this study,we took C57BL/6 mice,peritoneal macrophage(PMs),Kupffer cells(KCs)and RAW264.7 macrophage strain as the research objects,took PKM2 as the research target,and constructed endotoxin tolerance and non-tolerance models of mice or macrophages by stimulating with LPS to explore the role of PKM2 in the formation of endotoxin tolerance.This provides a new theoretical basis for further studying the molecular mechanism of endotoxin tolerance and the endogenous protective pathway in sepsis.Part ?: The effect of PKM2 tetramer on endotoxin tolerance of macrophagesObjective: To study the regulatory effects of PKM2 tetramer on endotoxin tolerance of PMs,KCs and RAW264.7 macrophages in vivo and in vitro.Methods:(1)In mice,endotoxin tolerance and non-tolreance models were established by intraperitoneal injection of LPS with different doses,and then PMs and KCs of mice in each group were extracted.Western blot and cellular immunofluorescence was used to detect the protein expression of PKM2 in PMs and KCs of endotoxin tolerant and non-tolreant mice.ELISA was used to detect the levels of tumor necrosis factor-?(TNF-?)and imerleukin-6(IL-6)in serum of mice.(2)PMs and KCs were extracted from the mice,and LPS was used to stimulate the cells to construct endotoxin tolerance and non-tolreance models of PMs,KCs and RAW264.7 cells.Western blot was used to detect the protein expression of PKM2 in PMs and KCs.ELISA was used to detect the levels of TNF-? and IL-6 in supernatant of cells.(3)TEPP-46 is a small molecule agonist of PKM2,which can promote the expression of PKM2 tetramer.Western blot experiment was used to explore the best stimulation concentration and treatment time of TEPP-46.The effect of TEPP-46 on the activity of RAW264.7 cells was detected by CCK8 experiment.(4)TEPP-46 was used to induce the formation of PKM2 tetramer in RAW264.7 cells,and then high-dose LPS was used to treat the cells.ELISA was used to detect the the level of inflammatory factors in supernatant.We discussed the effect of TEPP-46-induced PKM2 tetramer formation on endotoxin tolerance of macrophages.Results:(1)In vivo,compared with endotoxin non-tolreant mice(sepsis group),the expression of PKM2 tetramer in PMs and KCs in endotoxin toleraned mice was increased,while the expression of mono/dimers were decreased,which inhibited LPS-mediated nuclear translocation of PKM2.The levels of TNF-? and IL-6 in serum of mice in endotoxin tolerance group were lower than that in endotoxin non-tolerance group(p<0.05).(2)In vitro,compared with the endotoxin non-tolreance group,the expression of PKM2 tetramer in PMs,KCs and RAW264.7 cells in endotoxin tolerance group was increased,while the expression of mono/dimer were decreased,which also inhibited LPS-mediated nuclear translocation of PKM2.The levels of inflammatory factors TNF-? and IL-6 in cell supernatant of endotoxin tolerance group were also lower than that in endotoxin non-tolerance group(p<0.05).(3)TEPP-46 promoted the formation of PKM2 tetramer in macrophages,and the protein expression of PKM2 tetramer in macrophages approached the peak when stimulated by TEPP-46 at 50nmol/ml for 2 h;CCK8 test confirmed that TEPP-46 at 50 nmol/ml had no obvious toxic effect on macrophages after stimulation for 2 h(p>0.05).(4)The RAW264.7 cells were pre-stimulated with 50 nmol/ml TEPP-46 for 2 h,and then treated with a large dose of LPS.It was found that TEPP-46 pretreatment could significantly reduce the secretion of inflammatory factors TNF-? and IL6 mediated by LPS,and induce endotoxin tolerance of macrophages(p<0.05).Conclusions: Macrophage endotoxin tolerance promotes the high expression of PKM2 tetramer,and the formation of PKM2 tetramer is also the key step of macrophage endotoxin tolerance.Part ?: The molecular mechanism of TEPP-46-mediated PKM2 tetramer formation regulating macrophage endotoxin toleranceObjective: To study the molecular mechanism of TEPP-46-mediated PKM2 tetramer formation regulating macrophage endotoxin tolerance.Methods: Firstly,we discussed the effect of TEPP-46 on mitochondrial function.(1)RAW264.7 cells were stimulated with 50nmol/ml of TEPP-46 for 2 h,and the uptake of nonyl acridine orange(NAO)by cells was detected by flow cytometry.The protein expression of p62,LC3 and mitochondrial transcription factor A(mtTFA)were detected by Western blot.The relative copy number of mitochondrial DNA(mtDNA)was detected by RT-PCR.The general morphological changes of cells were detected by transmission electron microscope(TEM).Mitochondrial pressure test was used to detect oxygen consumption rate(OCR),basic respiration,ATP production and maximal respiration.(2)Then,we used small interfering RNA(siRNA)to silence PKM2 gene of cells,and repeated the above experiments.(3)We silenced mtTFA gene by using short hairpin RNA(shRNA),and we repeated the above experiments to investigat the regulation of mitochondrial biogenesis on endotoxin tolerance of macrophages induced by TEPP-46.(4)We silenced the PGC-1? gene by shRNA to study the regulation of PGC-1? in mitochondrial biogenesis induced by TEPP-46.(5)Western blot was used to detect the protein expression of p-PI3 K,PI3K,p-Akt and Ak,and then we discussed the regulation of PI3K/Akt signaling pathway in the promotion of PGC-1? expression by TEPP-46.Results:(1)TEPP-46(50 nmol/ml)stimulated RAW264.7 cells for2 h,which significantly increased the uptake of NAO,indicating that TEPP-46 promoted the increase of mitochondrial mass of macrophages.In addition,TEPP-46 increased the relative copy number of mtDNA in macrophages and promoted the protein expression of mtTFA.The results of TEM showed that the number of mitochondria increased after TEPP-46 stimulated cells,which indicated that TEPP-46 promoted mitochondrial biogenesis in macrophages.TEPP-46 had no significant effect on the protein expression of p62 and LC3I/II in macrophages,and the results of TEM showed that the number of lysosomes in macrophages had no significant change,suggesting that TEPP-46 had no significant effect on macrophage autophagy and mitophagy(p>0.05).TEPP-46 stimulated the cells to improve the OCR of macrophages,as well as the basic respiration rate,ATP production and maximal respiration,which indicated that TEPP-46 promoted the respiratory function of macrophages(p<0.05).(2)After silencing PKM2 with small interfering RNA,the above experiment was repeated.It was found that the promotion of TEPP-46 on mitochondrial biogenesis and OCR weakened or even disappeared after silencing PKM2,suggesting that the above functions of TEPP-46 depend on PKM2.(3)After inhibiting mitochondrial biogenesis by silencing mtTFA,the endotoxin tolerance of macrophages induced by TEPP-46 was effectively inhibited.(4)TEPP-46 can promote mitochondrial biogenesis by promoting the expression of PGC-1?.After inhibiting the expression of PGC-1? by lentivirus,the promotion effect of TEPP-46 on mitochondrial biogenesis of macrophages is weakened.(5)TEPP-46 can promote the expression of PGC-1? by inhibiting the activation of PI3K/Akt signaling pathway.After stimulating cells with PI3 K agonist 740Y-P or Akt agonist SC79,the promoting effect of TEPP-46 on PGC-1? weakened(p<0.05).Conclusions: TEPP-46-mediated PKM2 tetramer formation promotes PGC-1? expression by inhibiting PI3K/Akt signaling pathway activation,and inhibits LPS-mediated release of inflammatory factors by inducing macrophage mitochondrial biogenesis,thus promoting endotoxin tolerance of macrophages.Part ?: TEPP-46-mediated PKM2 tetramer formation regulates endotoxin tolerance in septic miceObjective: To study the regulation of TEPP-46-mediated PKM2 tetramer formation on endotoxin tolerance in septic mice in vivo.Methods:(1)Firstly,50 mg/kg TEPP-46 was injected into wild-type C57BL/6 mice by intraperitoneal injection,then PMs and KCs were extracted,and the protein expression of PKM2 in cells was detected by Western blot.The uptake of NAO by cells was detected by flow cytometry.The relative copy number of mtDNA was detected by RT-PCR.(2)The mouse sepsis model was established by intraperitoneal injection of 5 mg/kg LPS or cecal ligation and perforation(CLP),combined with intraperitoneal injection of 50 mg/kg TEPP-46.The effects of TEPP-46 on endotoxin tolerance of sepsis mice in vivo were investigated by observing the survival rate of mice,detecting the level of inflammatory factors in mice serum by ELISA and detecting the pathological changes of liver tissue by HE staining.(3)The mouse model of sepsis was established by injecting clodronate liposomes(CL)into the mouse through tail vein,and then LPS was injected intraperitoneally.Combined with intraperitoneal injection of 50 mg/kg TEPP-46,the role of monocyte/macrophage in endotoxin tolerance induced by TEPP-46 was discussed by detecting the level of inflammatory factors in serum.Results:(1)In vivo,TEPP-46 promoted the formation of PKM2 tetramer in PMs and KCs of mice,and TEPP-46 also induced macrophage mitochondrial biogenesis by promoting the expression of PGC-1?,nuclear respiratory factor 1/2(NRF1/2)and mtTFA.(2)TEPP-46 inhibits the inflammatory response in mice with sepsis induced by LPS or CLP,and promoted endotoxin tolerance and prolong the survival time of mice by inhibiting the secretion of inflammatory factors(p<0.05).(3)Injection of CL through tail vein can effectively eliminate PMs and KCs in mice(p<0.05).Elimination of PMs and KCs inhibited endotoxin tolerance in septic mice induced by TEPP-46.Conclusions: In mice,TEPP-46-mediated the formation of PKM2 tetramer promotes endotoxin tolerance of mice by inducing mitochondrial biogenesis of macrophages.