The Role And Mechanisms Of Vitamin B6 In Lipopolysaccharide-induced Acute Cardiopulmonary Injury

Background:Vitamin B6(VitB6)includes pyridoxine,pyridoxal,and pyridoxamine and their respective phosphate forms.Pyridoxal 5'-phosphate(PLP)is the biologically active form of VitB6 in the body and a key cofactor for more than 100 enzymes.It plays a crucial role in processes such as amino acid metabolism,gluconeogenesis,ornithine cycle and heme biosynthesis.Clinical studies have found that supplementation of VitB6 can significantly reduce the level of inflammation in critically ill patients,rheumatoid arthritis.Alzheimer's disease,Parkinson and other patients.When VitB6 is lacking or depleted,it leads to hypochromic small cell anemia and elevated ferrous ions,and heme synthesis is blocked.In recent years,more and more evidences show that VitB6 can prevent hydrogen peroxide-induced lipid peroxidation and the generation of oxygen free radicals.In addition,VitB6 plays an important role in preventing oxidative stress caused by homocysteineSepsis is a systemic inflammatory response mainly caused by infection,which can cause multiple organ failure in the body,especially cardiac dysfunction.When the body is infected with exogenous bacteria,viruses or fungi,the immune system produces a series of cascades of reactions that cause myocardial depression and dysfunction Systemic sepsis results in multiple organ dysfunction or failure.The main causes of death in the intensive care unit are severe sepsis and septic shock.Although various antibiotics are widely used in clinical practice,sepsis is still an insurmountable problem.Septicemia-induced overproduction of inflammatory cytokines can cause heart failure in patients.Damage to the inflammatory process includes structural and functional damage.In sepsis-induced cardiomyopathy,myocardial tissue structure was severely injured.Clinical studies have found that high levels of inflammatory factors in patients with sepsis can cause left ventricular dysfunction,which can lead to heart failure.Lipopolysaccharide(LPS)is the main bioactive component of the cell wall of Gram-negative bacteria and triggers the inflammatory cascade reaction.Numerous studies have shown that LPS-induced cardiomyopathy has a significant inhibitory effect on structure and function.For years,many researches have shown that multiple mechanisms are involved in regulating cell death,such as apoptosis which is familiar with us.However,ferroptosis is a ne w type of cell death,which is distinct from apoptosis,necrosis and autophagy in its genetics,biochemistry and morphology.This type of cell death is characterized by the accumulation of intracellular reactive oxygen species(ROS)and lipid peroxidation products which are iron-dependent.A large number of studies have shown that ferroptosis is related to various diseases,such as cardiomyopathy caused by ischemia-reperfusion(I/R),kidney degeneration and neurodegenerative diseases and various cancers.Therefore,inhibiting or enhancing the role of ferroptosis will become a new strategy for treating related human diseases.Apoptosis is a form of programmed cell death that can be caused by physiological and pathological stimuli.In the inherent apoptotic pathway,various stimuli lead to the imbalance of Bcl2 family pro-apoptotic and anti-apoptotic proteins.However,exogenous pathways are involved in the activation of caspase,Numerous studies have shown that abnormal apoptosis is associated with many diseases.In this study,we investigated the effects of VitB6 on LPS-induced myocardial injury,as well as the effects on ferroptosis and apoptosis in vivo and in vitro.Objectives:1.To explore the protective effect of VitB6 on LPS-induced myocardial injury by constructing a mouse cardiomyopathy model.2.To investigate the effect of VitB6 on ferroptosis by detecting the expression of ferroptosis related protein molecules and the levels of oxidative stress.3.To investigate the effect of VitB6 on apoptosis by detecting the expression of apoptosis-related protein molecules and cell mortality rate.Methods:1.Establishment of mouse myocardial injury model and groupingTwenty-seven C57BL/6J male mice(body weight 25±2g)which were 12-week-old were randomly divided into 3 groups(n=9):control group,lipopolysaccharide(LPS)group,vitamin B6+lipopolysaccharide group(VitB6+LPS).C57BL/6J mice in the LPS group and the VitB6+LPS group were intraperitoneally injected with saline or VitB6(20mg/kg).After 6 hours,intraperitoneal injection of lipopolysaccharide or saline 4mg/kg was performed,and the mice were euthanized after 24 hours.And collected the serum,heart,liver and other tissue specimens2.Cardiac function AssessmentThe mice were subjected to cardiac ultrasound before euthanasia.The mice were inhaled with isoflurane for volatile anesthesia and the hair was removed with a depilatory cream.The mice were fixed on a heated imaging platform,and then the coupling agent was applied.The Vevo 2100 imaging system is equipped with a 40 MHz high-frequency sensor(Visual Sonics,Toronto,Canada)and is used for non-invasive inspection.The parasternal long-axis M-mode echocardiogram was performed to measure left ventricular ejection fraction(EF)and shortened fraction(FS).3.Survival assay of the miceMice were pretreated with saline or VitB6(20mg/kg/day)for 7 days,and then mice were injected with LPS(20mg/kg)for 7 days.Monitored the mice three times a day for 7days.DMSO,tween 80,NMP and PEG400 were mixed in proportion,and Fer-1(2mg/kg)and emricasan(2.5mg/kg)dissolved in the above solvent respectively.Fer-1 and emricasan were given by intraperitoneal injection for 24 hours and then followed by LPS(20mg/kg).Monitored 3 times daily for 7 days.4.Detection of troponin I(cTnl),lactate dehydrogenase(LDH),reactive oxygen species(ROS),malondialdehyde(MDA)and superoxide dismutase(SOD)Different kits were used to detect the levels of cTnl,LDH.MDA and SOD in serum and the concentrations of MDA and SOD in myocardial tissue.Flow cytometry was applied to detect total ROS and lipid ROS5.Analysis of myocardial histologyParaffin sections of myocardial tissue were dewaxed and hydrated.Histomorphological analysis was performed by hematoxylin and eosin staining(H&E)and pictures were taken under a microscope(Nikon,Tokyo,Japan)6.Culture and treatment of H9C2 cellsEmbryonic rat heart-derived H9C2 cell line was obtained from American Type Culture Collection(ATCC).H9C2 cells were differentiated with all-trans retinoic acid(RA,10nM)before use and cultured in 1%fetal bovine serum for 5 days.The differentiated HPC2 cells were stored in DMEM which contained 4.5g/L of d-glucose and 10%fetal bovine serum and penicillin-streptomycin.VitB6(500?M)pretreated H9C2 cells for 2 hours,and then LPS(100ng/ml)was given for 24 hours.Or Fer-1 pretreated the differentiated H9C2 cells for 30 minutes,and then given LPS(100n/ml)to stimulate for 24 hours.All cells were cultured in a humid environment at 37?,95%air and 5%C02.7.Western BlotWe extracted the protein lysates of H9C2 cells and myocardial tissue and detected the expression levels of Bcl2,Bax,C-caspase3,GPX4,NQO1,Nrf2,HOI,Transferrin receptor,FPN1 and ferritin proteins by Western Blot.GADPH and ?-actin served as internal reference proteins.8.Real-time quantitative PCRThe total RNA of myocardial tissue was extracted and we measured the concentration of the total RNA,And then reverse transcription was performed into cDNA strictly in accordance with the instructions of the reverse transcription kit.9.Observation of mitochondrial structure by electron microscopeVitB6 pretreated differentiated H9C2 cells for 2 hours,and then followed by LPS(100ng/ml)for 24 hours.Discarded the medium without washing.Added electron microscope fixing solution to fix the cells and scraped the cells.Collected the cells by centrifugation and discarded the fixing solution.New fixing solution was added to the EP tubes and fixed at room temperature for 2 hours and then transferred to 4? for storage.Fixed cells were transported to an electron microscope photographing company for subsequent smearing,staining,and photographing10.Flow cytometry assayDiluted DCFH-DA with serum-free medium at a ratio of 1:1000 to a final concentration of 10?M.We added 1 ml of diluted probe to each well and incubated at 37? for 30 minutes.We then used serum-free medium to rinse the cells 3 times,scraped the cells and collected them by centrifugation into a flow tube.Discarded serum-free medium and 500?l of PBS was added to each tube.Tested on the machine by selecting the FITC channel for detectionResults:1.VitB6 improved LPS-induced myocardial damagePrevious studies have shown that VitB6 improved myocardial ischemia.To study the protective effect of VitB6 on LPS myocardial injury,mice were pretreated with normal saline or VitB6(20mg/kg)for 6 hours,and then intraperitoneally injected with LPS(4mg/kg)for 24 hours.Compared with the control group,ejection fraction(EF)and shortened fraction(FS)of the mice in LPS group had significantly lowered;while in the VitB6+LPS group,cardiac dysfunction was significantly improved.There was no difference in left ventricular end-diastolic volume among the three groups of mice,while in the VitB6+LPS group,the left ventricular end-systolic volume decreased significantly compared with the mice in LPS group.The serum cardiac troponin ?(cTnI)and lactate dehydrogenase(LDH)of mice in VitB6+LPS group were much lower than those in LPS group.H&E staining of myocardial tissue showed that the myofibril structure of the control group was normal,myocardial tissue in the LPS group appeared swollen and broken,and VitB6+LPS group showed mild swelling.However,there was no significant difference in collagen deposition in three groups2.VitB6 reduced LPS-induced oxidation stress in serum and myocardial tissueMDA is one of the most important products of membrane lipid peroxidation,and superoxide dismutase(SOD)is an important antioxidant enzyme that can scavenge free radicals.Glutathione(GSH)is a tripeptide composed of glycine,glutamic acid and cysteine.GSH is an important antioxidant that can protect hemoglobin from being oxidized.Levels of MDA in serum and myocardial tissue of mice induced by LPS were significantly increased,and those in the VitB6+LPS group were obviously reduced.In LPS group,SOD activity and GSH levels were significantly reduced,while this phenomenon was reversed in the VitB6+LPS group.Nrf2,an important transcriptional activator in the antioxidant response,could be activated by a variety of stimuli including LPS.To determine whether VitB6 is involved in LPS-stimulated Nrf2 expression in myocardial tissue,we first pretreated mice with VitB6 and then stimulated the mice with LPS.We found that VitB6 increased the expression of Nrf2.The relative mRNA levels of Nrf2 in the myocardium are consistent with the protein expression trend.These results indicate that VitB6 inhibits LPS-induced oxidative stress and peroxidation by activating Nrf2.3.In myocardial cells,VitB6 improves LPS-induced oxidative stress and lipid peroxidationTo further investigate the changes caused by LPS in cells,we examined SOD,ROS,and lipid peroxidation levels.VitB6 pre-treated the cells for 2 hours and then followed by LPS for 24 hours.As we predicted,in LPS-induced cells,MDA and ROS levels increased and SOD activity decreased.However,in VitB6+LPS group,the changes of MDA.ROS and SOD were reversed.These results were consistent with in vivo experiments.4.VitB6 promoted the expression of Nrf2 protein and related antioxidant enzymesNumerous studies have shown that inactivation of GPX4 triggered ferroptosis,and up-regulation of Nrf2 signal inhibited ferroptosis in many diseases.NQO1 and HO1 were important antioxidant-related enzymes and played crucial roles in the Nrf2 signaling pathway.To clarify the effect of VitB6 on these proteins,in this study,H9C2 cells were pretreated with VitB6 for 2 hours,then treated with LPS 24 hours and detected the levels of these proteins by Western Blot.LPS decreased the expression of Nrf2,GPX4,HO1 and NQOl.In contrast,VitB6 significantly increased the expression of the aforementioned proteins.These results indicated that VitB6 altered the synthesis of ferroptosis-related proteins5.VitB6 inhibited LPS-induced the increase of non-heme iron and hemeFerroptosis is characterized by an iron-dependent accumulation of intracellular reactive oxygen species(ROS)and lipid peroxidation products.To investigate the effects of LPS and VitB6 on non-heme iron and heme,we examined iron and heme in serum and myocardial tissue.It was found that compared with the control group,the levels of iron and heme in the serum and myocardial tissue of mice in the LPS group were significantly increased,while in VitB6+LPS group this trend was changed.It was suggested that VitB6 may mediate the metabolism of iron and heme6.In myocardial cells,VitB6 regulated the expression of iron regulatory proteins.The level of iron in the body is closely related to many diseases.Transferrin transports iron to the cell,while ferropotinl(FPN1)transports iron to the outside of the cell and regulates intracellular iron levels.Ferritin,an iron-binding protein,stores iron.This experiment explored changes in transferrin receptor,FPN1 and ferritin.Cells were pretreated with VitB6 for 2 hours and followed by LPS for 24 hours.We detected the changes in iron regulation-related protein expression by Western Blot.After LPS treatment,transferrin receptor and ferritin levels increased significantly and FPN1 expression decreased.The synthesis of transferrin receptor,FPN1 and ferritin in the VitB6+LPS group was reversed.7.VitB6 alleviated apoptosis of myocardial tissue induced by LPSStudies have reported that VitB6 can protect intestinal epithelial cells from ionizing radiation-induced apoptosis.To investigate whether VitB6 could inhibit LPS-induced cardiomyocyte apoptosis,we used TUNEL method to study the effect of VitB6 on LPS-induced apoptosis.After LPS stimulation,the number of TUNEL-positive cells increased significantly,while in VitB6+LPS group,the number of positive cells decreased distinctly.Western blot was used to detect apoptosis-related proteins.LPS increased the levels of Bax protein in myocardial tissue and decreased the expression of Bcl2,while VitB6 reversed the expression trend of Bax/Bcl2.These data indicated that VitB6 regulates the intrinsic apoptotic pathway activated by LPS.8.In myocardial cells,VitB6 suppressed LPS-induced apoptosisCells were seeded in 96-well plates,pretreated with VitB6(500?M)or PBS for 2 hours,and then stimulated with LPS at different concentrations for 24 hours.Cell viability was measured using Cell Counting Kit 8(CCK8).VitB6 significantly reduced cell death induced by LPS(100ng/mg and 1000ng/ml).To further demonstrated that VitB6 inhibited apoptosis,H92C2 cells were pretreated with VitB6(500?M)for 2 h,and then stimulated with LPS(100ng/ml)for 24 h.The effect of VitB6 on LPS-induced cell death was detected using a TUNEL kit.TUNEL-positive cells in the LPS group increased significantly,while the number of TUNEL-positive cells in the VitB6+LPS group decreased.Apoptosis-related proteins were detected by western blot.LPS increased the expression levels of cleaved caspase3 and Bax,while the expression levels of Bcl2 was decreased.9.In myocardial cells,VitB6 improved LPS-induced mitochondria damageThe mitochondrial membrane of H9C2 cells in the control group was basically completed and the mitochondrial ridges were clear.However,mitochondria swelled after LPS stimulation,and the internal condyles rupture or even disappeared.Although mitochondria were still a little swollen,VitB6 significantly improved the morphology of mitochondria induced by LPS.10.In vivo,Fer-1 and emricasan mimicked pharmacological effects of VitB6 decreasing the mortality of LPS-induced miceIn order to further study the protective effect of VitB6 on LPS-induced mice,mice were pretreated with VitB6(20mg/kg/day)for 7 days,and then intraperitoneally injected with LPS(20mg/kg).Mice were monitored 3 times a day for 7 day.The survival rate of mice in the VitB6+LPS group was higher than that in the LPS group.We suspected that VitB6 potentially suppressed ferroptosis and apoptosis caused by LPS.To confirm this hypothesis,we pretreated mice with Fer-1(2mg/kg),ferroptosis inhibitor and emricasan(2.5mg/kg),apoptosis Inhibitors for 24 hours and then administered with LPS(20mg/kg).Monitored 3 times daily for 7 days.The survival rate of mice pretreated with Fer-1 and emricasan was significantly higher than that of LPS group.Conclusion:1.VitB6 ameliorated LPS-induced myocardial injury.2.The myocardial protective effects of VitB6 were related to oxidative stress,ferroptosis,apoptosis and Nrf2Background:Inflammatory diseases are mostly the result of uncontrolled inflammation,resulting in damage and destruction of normal tissue structure.Pneumonia is an inflammatory disease of the lower respiratory tract,such as the terminal airway,alveoli and pulmonary interstitium,which is mainly caused by pathogens,immune damage and inhalation physicochemical factors.Clinical pneumonia is mainly caused by pathogenic microorganisms,such as endotoxin produced by gram-negative bacilli.Lipopolysaccharide(LPS),as an endotoxin,is the main active component of the cell wall of gram-negative bacilli and the key substance causing inflammatory response.Therefore,elucidating the underlying mechanism of inflammation is of great significance for the development of effective new strategies for the treatment of pneumonia.A variety of immune cells,including macrophages,lymphocytes and neutrophils,play an important role in the development of pneumonia.For example,pathogenic factors activate macrophages and release a variety of inflammatory factors such as reactive oxygen species,proteolytic enzymes and cytokines including tumor necrosis factor cytokine(TNF-?),interleukin-1 beta cytokines(IL-1?)and interleukin-6(IL-6),which further lead to severe inflammatory damage.Therefore,inhibition of macrophage activation is considered to be an important therapeutic strategy for lipopolysaccharide(LPS)induced pulmonary inflammation diseasesVitamin B6(VitB6)is a water-soluble vitamin,which mainly includes pyridoxol,pyridoxal and pyridoxine and their phosphate esters in vivo.As a coenzyme factor,VitB6 participates in a variety of metabolism-related activities in vivo,including amino acid,fat and glucose metabolism.As an active form of VitB6,5'-piridoxal phosphate(PLP)can reflect the long-term storage of VitB6 in vivo.Current studies have shown that decreased plasma PLP concentration is associated with increased risk of cardiovascular disease.The clinical study of Huang SC et al.found that exogenous supplementation of VitB6 in patients with rheumatoid arthritis could reduce the levels of inflammatory cytokines IL-6 and TNF-? levels in the plasma of patients.In addition,studies have shown that VitB6 has also been demonstrated to inhibit the expression of IL-1? induced by LPS.A large number of clinical studies have found that VitB6 can alleviate symptoms of Alzheimer's disease and Parkinson's disease.However,it remains unclear whether VitB6 has an effective effect on pneumonia.Adenosine 5 '-adenosine phosphate activated protein kinase(AMPK),a heterotrimeric protein composed of three subunits,?,?,and ? subunit,is an intracellular energy receptor.The a subunit serves as the catalytic subunit while the ? subunit contains a glycogen-binding domain that also regulates the activity.The y subunit forms the broad base of the protein and is required for AMP binding.AMPK is activated when the ratio of adenosine monophosphate(AMP)to adenosine triphosphate(ATP)increases,such as hypoxia and exercise.AMPK is a highly conserved serine/threonine kinase that plays a key role in the regulation of bioenergy metabolism.Many studies have shown that AMPK plays an important regulatory role in obesity,metabolic syndrome,type 2 diabetes mellitus and many other diseases through phosphorylating downstream targets Our previous findings suggest that AMPK activation plays a number of protective roles in cardiovascular disease by inhibiting oxidative stress.In addition,vascular endothelial knockout of AMPK in mice promoted the formation of hypoxic-induced pulmonary hypertension.The ligand protein family of downstream kinase 1(DOK1)and DOK3 protein mainly exist in immune cells.Lung cancer and histiocytic sarcoma were found in mice lacking DOK1 to DOK3 proteins,suggesting that the DOK protein family plays an important functional role in regulating cellular responses in vivo.DOK1 and DOK2 proteins play a negative role in LPS-induced the expression of TNF-? in macrophages.Humphrey MB et al.believe that in macrophages,DOK3 protein is associated with toll-like receptor(TLR)signaling pathways activated by LPS stimulation,and negatively regulates LPS response and endotoxin tolerance.Lam KP et al.found that DOK3 protein regulates the TLR signaling pathway by degradation of DOK3 protein or phosphorylation of tyrosine.The above studies proved that VitB6 may inhibit the activation of LPS-induced macrophages by activating AMPK and prevent acute pneumonia by inhibiting the phosphorylation of DOK3 induced by LPS.At present,the relationship between AMPK protein,macrophage activation and DOK3 protein has not been illuminated.The intention of this research was to investigate the role of VitB6 in acute pneumonia and its molecular mechanism.Our data suggest that VitB6 activates the AMPK signaling pathway and inhibits macrophage activation,thus preventing pulmonary inflammation.Objective:1.In vitro experiments,peritoneal macrophages were extracted from mice to explore the effect of VitB6 on AMPK level 2 protein2.In vitro experiments,peritoneal macrophages were extracted from mice to explore the effect of VitB6 on lps-induced expression of inflammatory cytokine proteins and genes3.In vitro experiments,constructing the acute lung injury model of mice was to detect the levels of lung tissue and systemic inflammation in mice4.In vitro experiments,peritoneal macrophages were extracted from mice,and interference of AMPK 2 gene was constructed and transfected to investigate the role of AMPK 2 gene in the inflammatory process induced by LPS5.In vivo and in vitro experiments,the role of DOK3 gene and VitB6 in the LPS-induced lung injury was investigatedMethod:1.Establishment and grouping of acute lung injury mouse models:Forty-five mice aged 8-12 weeks of B129 and DOK3-/-male mice(body weight 25±2g)were randomly divided into 3 groups(n=5):control group,lipopolysaccharide group(LPS),Vitamin B6+ lipopolysaccharide group(VitB6+ LPS).B129 and DOK3-/-mice in the LPS group and the VitB6+ LPS group were intraperitoneally injected with normal saline or VitB6(20mg/kg).After 6 hours,the mice were intraperitoneally injected with 500?g/kg of LPS or normal saline.Euthanasia was performed,and serum,lung,heart,liver and other tissue samples were retained.2.Immunohistochemical staining of lung tissueThe mouse lung tissue was fixed in 4%paraformaldehyde overnight.The lung tissue was washed with running water for 12-16 hours.The tissue was dehydrated to wax.Experiment of paraffin sections were dewaxed to water,and hematoxylin-eosin(H&E)staining was performed to detect the lung tissue morphology,alveolar cell size and arrangement.The expression levels of IL-1?,TNF-? and IL-6 in the lung tissue of mice were detected by immunohistochemistry.3.Extraction of mouse peritoneal macrophagesAt 8-12 weeks,male mice were intraperitoneally injected with 1 ml of 6%soluble starch after autoclaving.After 72 hours,the treated mice were sacrificed by dislocation and disinfected in 75%ethanol for 5 minutes.The mice were fixed and sterilized on a foam board and the abdominal skin of the mice was cut using sterilized scissors and tweezers.At the same time,the peritoneum was cut about 1 cm.The PBS was injected into the abdominal cavity and washed 3 times repeatedly to recover the washing solution in a centrifuge tube.And it was repeated several times until the washing solution became clear.Centrifuged the washing solution at 1000 rpm/min and discarded the supernatant.The cells were resuspended in a low-glucose DMEM medium containing 10%fetal bovine serum,streptomycin(100?g/ml)and penicillin(100U/ml)and the cells were directly seeded into 12-or 6-well plates.4.Cell culture and processingThe extracted primary peritoneal macrophages were cultured in a low-glucose DMEM medium containing penicillin(100U/ml),streptomycin(100?g/ml)and 10%fetal bovine serum and evenly seeded in 12 or 6-well plates.All cells were incubated in an atmosphere of 95%air and 5%CO2 which was humidified at 37?.In order to detect the effect of VitB6 on AMPK,VitB6 was treated with different concentrations(0,1,10,100,1000?M)and different times(0,5,15,30,60,120 min).In order to detect the effect of VitB6 on LPS-induced inflammation,macrophages were pretreated with different concentrations(0,1,10,100,1000?M)for 2 hours,followed by LPS(100ng/ml)stimulation for 24 hours and cells and supernatant were collected5.Western Blot analysisThe mouse lung tissue and peritoneal macrophage protein lysate were extracted and the protein expressions of pAMPK,AMPK,DOK3,and ?-actin were assayed by Western Blot.6.Enzyme Linked Immunosorbent Assay(ELISA)The cultured cell supernatant and mouse serum were collected and stored in a refrigerator at-80?.The concentrations of IL-1?,TNF-? and IL-6 in the supernatant and serum were measured strictly according to the instructions of the ELISA kit.7.Real-time quantitative PCRThe total RNA of peritoneal macrophages was extra2cted and the total RNA concentration was measured.Reverse transcription was performed into cDNA strictly according to the instructions of the reverse transcription kit.8.Macrophages transfection with AMPKa2 small interferenceMacrophages were seeded in plate and cell density was about 80%small interference is transfected with opti-MEM and RNAimax reagents and incubate at 37? and 5%C02 for 24 hours9.Transfection DOK3 overexpression adenovirus DOK3-/-mouse peritoneal macrophages were extracted and seeded in plates and cell density was about 80%.DOK3 overexpression adenovirus was transfected with normal medium and the medium was changed after incubation at 37? and 5%C02 for 24 hoursResults:1.In macrophages,VitB6 phosphorylates AMPKThe peritoneal macrophages were extracted,seeded in plates and given different concentrations of VitB6 for 2 hours and VitB6(1mM)in different times.VitB6 significantly increased the expression of phosphorylated AMPK at 30 min.VitB6 and compound C treat macrophages simultaneously.VitB6-induced AMPK phosphorylation can be reversed by compound C.2.VitB6 inhibits phosphorylation of DOK3 proteinVitB6 stimulated wild-type peritoneal macrophages for 2 hours and then treated with LPS for 24 hours.The phosphorylated DOK3 protein in the LPS group was significantly increased compared to the control group.The phosphorylated DOK3 protein in the VitB6+LPS group was decreased compared to the LPS group.3.VitB6 inhibits LPS-induced inflammationVitB6 pretreated wild-type primary peritoneal macrophages for 2 hours and then treated them with LPS for 24 hours.Total mRNA was extracted from the cells to detect the mRNA and protein levels of inflammatory factors IL-1?,TNF-? and IL-6.Compared with the control group,the levels of mRNA and protein levels of inflammatory factors IL-1?,TNF-? and IL-6 increased significantly after LPS stimulation,but VitB6 was given LPS stimulation after 2 hours of pretreatment,compared with LPS group.The levels of mRNA and protein of IL-1?,TNF-? and IL-6 were significantly reduced.4.AICAR inhibits LPS-induced inflammation through DOK3 proteinIn vitro,AICAR pretreated wild-type primary macrophages for 30 minutes and then administered LPS for 24 hours.Cells were collected to extract total mRNA to detect the mRNA expression levels of inflammatory factors IL-1?,TNF-?,and IL-6 Compared with the control group,the levels of inflammatory factors was significantly increased after LPS stimulation.Compared with the LPS group,the expression of inflammatory factors was significantly reduced after AICAR pretreatment.In DOK3-/-mouse primary peritoneal macrophages,AICAR and LPS were used to treat DOK3-/-mouse primary peritoneal macrophages.Total mRNA was extracted from cells to detect inflammatory factors IL-1?,TNF-?,and IL-6 mRNA expression level in DOK3-/-mouse primary peritoneal macrophages.5.VitB6 exerts anti-inflammatory effects through AMPKIn vitro,wild type macrophages were transfected with AMPK siRNA or negative control siRNA(NC siRNA).After transfection for 24 hours,the medium was changed and treated with VitB6 and LPS.Total mRNA was extracted to detect the expression of inflammatory factors.In the NC group,the expression of inflammatory factors increased significantly after LPS stimulation.Compared with LPS stimulation,the expression of inflammatory factors in VitB6+LPS decreased significantly.However,in the group of AMPK siRNA,the expression of inflammatory factors increased significantly after LPS stimulation.Compared with LPS group,the expression of inflammatory factors in VitB6+LPS group did not change significantly6.VitB6 inhibits LPS-induced inflammation via DOK3 proteinDOK3-/-mouse peritoneal macrophages were extracted and seeded in plates.After 12 hours,they were treated with VitB6 and LPS for 24 hours.Total mRNA was extracted to detect the expression of inflammatory factors.Compared with the control group,the expression of inflammatory factors in the LPS group was significantly increased and the expression of inflammatory factors in the VitB6+LPS group was not significantly reduced compared with the LPS group.Adenovirus was transfected into peritoneal macrophages of DOK3-/-mice and the DOK3 gene was overexpressed.The medium was changed after transfection and then treated with VitB6 and LPS.In vector group,there was no significant difference in the expression of inflammatory factors between LPS group and VitB6+LPS group.However,in the adenovirus overexpression group,the levels of inflammatory factors in the VitB6+LPS group were significantly lower than those in the LPS group.In vivo experiments,DOK3-/-mice were injected intraperitoneally with VitB6(20mg/kg)or saline and treated with LPS(0.5mg/kg)after 6 hours and then euthanized after 24 hours.Serum and lung tissues were collected.The expressions of inflammatory factors IL-1?,TNF-? and IL-6 in serum and lung tissue were detected and the results were consistent with the results of cell experimentsConclusion1.In macrophages,VitB6 significantly increased the expression level of pAMPK protein.2.In macrophages,VitB6 significantly inhibited the expression of LPS-induced pDOK3.3.VitB6 significantly reduced LPS-induced inflammation through AMPK protein.4.VitB6 inhibited LPS-induced inflammation through DOK3 protein.5.In vivo and in vitro experiments,VitB6 inhibits LPS-induced acute lung inflammation through the AMPK-DOK3 signaling pathway.