Study On A Drug Combination For The Treatment Of Diabetes Mellitus Complicated With Staphylococcus Aureus Pneumonia

Staphylococcus aureus（S.aureus）is a common opportunistic pathogen.Its infection is characterized by high incidence rate and high mortality rate,and patients with diabetes are more susceptible to infection.Among the virulence factors secreted by S.aureusα-Hemolysin has stronger virulence,and it is not easy to produce drug resistance when it is used as a target to intervene bacteria.Diabetes is a common clinical chronic metabolic disease,easy to concurrent lower respiratory tract infection.In the pathological state of high glucose environment,hyperglycemia itself is also beneficial to the growth and reproduction of pathogenic bacteria in vivo under the condition of high body immunity and impaired lung function.The risk of infection of S.aureus is higher in diabetics than in those with normal blood glucose.At present,diabetes is becoming more and more common in domestic animal.Because of the low immunity and special structure of respiratory animal,the risk of infection is significantly increased.So the cases of diabetes complicated with S.aureus pneumonia are also increasing.When S.aureus invades the body,alveolar macrophages fight S.aureus and other pathogens in the lung.TLR2,MAPKs and NLRP3 inflammasome family are the main participants.In this study,in order to explore the high glucose,S.aureus and other factorsα-Hemolysin,we will also explore the TLR2,MAPKs and NLRP3 pathways related to the pathogenesis of the diseaseα-Hemolysin（8325-4）and S.aureus withα-hemolysin deficiency（DU1090）was used to study the hypoglycemic,antibacterial and inhibitory effects of S.aureus on diabetic pneumonia.The discovery of drug combination ofα-hemolysin activity laid the foundation.Anti-virulence factor therapy is through the inhibition of S.aureus Anti-inflammatory treatment pathway forα-hemolysin activity has been reported.It has been reported that the use of antibiotics combined with hypoglycemic agents is better in the treatment of diabetic patients with pulmonary bacterial infections.Insulin（INS）is a protein hormone in the body.It is the only protein hormone that can reduce blood glucose in the body.Exogenous insulin is mainly used in the treatment of diabetes and can act on the lungs.Therefore,insulin is chosen as the hypoglycemic agent in this study.Linezolid（LZD）is a synthetic oxazolidinone against Gram-positive bacteria.It also has strong antibacterial activity against multidrug-resistant S.aureus.It is not easy to produce bacterial resistance when induced in vitro.It has high tissue penetration and can play a role in alveolar epithelial lining fluid and pulmonary interstitial fluid.Now it is widely used in the clinical treatment of Gram-positive bacterial pneumonia,so linezolid was selected as the antibacterial drug in this study.In this study,the effects of insulin and linezolid on hyperglycemia,S.aureus and diabetes were investigated by in vitro and in vivo tests and preliminary clinical case analysisα-Hemolysin.In vitro and in vivo models ofα-hemolysin associated diabetes mellitus and S.aureus pneumonia,as well as the inhibitory effect and therapeutic effect of inflammatory response in clinical cases.First of all,in order to examine the drug combination（insulin+linezolid）hypoglycemic,anti-S.aureus and inhibitionα-hemolysin.In order to study the pharmacodynamics ofα-Hemolysin in vitro,we studied the minimal inhibitory concentration（MIC）,growth curve and its mechanismα-Hemolysin activity,drug safety in vivo and in vitro were studied.The results showed that the MIC value of the drug combination against S.aureus was 0.5μg/mL by broth dilution method and chessboard method;Linezolid could effectively inhibit the growth of S.aureus at sub-inhibitory concentration（1/2 MIC）;1/2 MIC linezolid could completely inhibit the apoptosisα-Hemolysin activity and drug combination can completely inhibit bacteria;CCK-8 showed that the drug combination could effectively reduce the cell damage caused by high glucose.Therefore,1/2 MIC linezolid（0.25μg/mL）.And insulin（50 nmol/L）.The above results showed that the combination of drugs could reduce blood glucose,resist S.aureus and inhibit the growth of S.aureusα-Hemolysin has high activity and high safety in vivo and in vitro.Through the construction of high glucose,S.aureus and S.aureus in vitroα-Hemolysin.In order to further investigate the ability of drug combination to inhibit the related inflammatory pathway,the model of mice alveolar macrophage（MH-S）inflammation induced byα-hemolysin was established.Western blot was used to detect and analyze the inhibitory effect of drug combination on TLR2,MAPKs and NLRP3 inflammasome related protein expression in MH-S cells induced by 8325-4 strain.The results showed that high glucose,S.aureus andα-Hemolysin can activate TLR2,MAPKs,NLRP3inflammatory body pathway and significantly up regulate the protein expression level.Linezolid and drug combination can significantly down regulate the expression level of these proteins,and the drug combination has more significant inhibitory effect than linezolid alone,while insulin alone has no obvious inhibitory effect on these proteins.In conclusion,the combination of drugs through hypoglycemic,anti-S.aureus and inhibitionα-Hemolysin activity can significantly down regulate and inhibit the activation of TLR2,MAPKs and NLRP3 inflammatory pathways,thus exerting anti-inflammatory effect.Secondly,to verify the hypoglycemic,anti-S.aureus and inhibitory effects of the drug combination in vivoα-Hemolysin.In vivo pharmacodynamics of drug combinations（insulin plus linezolid）in diabetic mice with S.aureus pneumonia was investigated.First,When the concentration of linezolid was 25 mg/kg,80 mg/kg,160 mg/kg and insulin was 1 U/kg,the mice did not die,that is,the LD₅₀ of linezolid was not detected,and there was no death when the maximum dose(C_max=160 mg/kg,V_max=0.4 ml)was used for intragastric administration.Secondly,a diabetic model of S.aureus（8325-4 strain）was established,which was administered by 7 d[insulin（1 U/kg/12 h）+linezolid（25 mg/kg/12 h）,insulin（1 U/kg/12 h）+linezolid（80 mg/kg/12 h）.Insulin（1 U/kg/12 h）+linezolid（160 mg/kg/12 h）was combined with modeling and the weight and blood glucose level of diabetic mice before and after administration,and the CFUs and lung H.E in lung were determined.The optimal concentration of the drug combination was 1 U/kg/12 h,linezolid 80mg/kg/12 h and the related tests were carried out.In order to further investigate the drug in vivo against high glucose S.aureus and S.aureusα-Hemolysin Protection of S.aureus（8325-4 strain and DU1090 strain）infected pneumonia caused byα-Hemolysin infection in diabetic mice.Through 7 d administration,combined with modeling and before and after treatment,the body weight and blood glucose level,CFUs,W/D,H.E and ELISA of the diabetic mice were analyzed,and the drug combinations were hypoglycemic and anti-S.aureusα-Hemolysin in vivo.The effect ofα-Hemolysin on the treatment of diabetic mice with S.aureus pneumonia.The results showed that drug combination has high safety in vivo,8325-4 strain caused stronger inflammatory reaction than DU1090 strain,sustained hyperglycemia and S.aureus infection gradually reduced body weight;Insulin alone can significantly reduce the level of hyperglycemia,but it can slow down the weight loss of diabetic mice,the S.aureus infection of lung CFUs,the pathological changes and the levels of inflammatory cytokines(TNF-α、IL-1β、IL-10 and IL-18 had no significant effect;Linezolid alone does not reduce blood glucose levels,but it has a significant effect on the CFUs and pathological changes and inflammatory cytokines levels in the lungs of diabetic mice infected with S.aureus had obvious anti-S.aureus and anti-inflammatory effects;The drug combination has good effects on the blood glucose and body weight of diabetic mice in vivo.It can significantly reduce the number of bacteria infected by S.aureus in the model group of diabetic mice,and also reduce the inflammatory pathological changes and the levels of inflammatory cytokines and its therapeutic effect is better than linezolid alone.In conclusion,the results showed that the combination of drugs had a good effect on the treatment of pneumonia caused by S.aureus infection in diabetic mice.Finally,5 dogs with diabetes mellitus and S.aureus pneumonia from animal hospitals were analyzed and clinical treatment of combination of insulin and linezolid was carried out.First of all,routine examination,hematology examination,bacteriological examination and imaging examination were performed on the sick dog.Secondly,we used the combination of short acting insulin（0.3 U/kg～0.5 U/kg,twice a day）+long acting insulin（0.4 U/kg,twice a day）+linezolid（50 mg/kg）]to increase the resistance of the sick dogs.After 14 days of treatment,we compared with the routine examination,hematology examination,urine examination,urine examination before and after treatment Imaging examination showed that the number of white blood cells decreased to normal level,the content of glucose and ketone body in urine returned to recessive level,and all indexes were at normal level.Polydipsia,polyuria,polydipsia and weight loss basically disappeared.X-ray examination showed that pulmonary inflammation disappeared and the condition improved.The symptoms of diabetes in dogs were markedly improved and pulmonary function recovered.The results showed that the drug combination（insulin+linezolid）was effective in treating canine diabetic S.aureus pneumonia.In conclusion,this study not only developed a good combination of insulin and linezolid for diabetes and S.aureus pneumonia both in vivo and in vitro,but also clarified its mechanism through hypoglycemic,anti-S.aureusα-Hemolysin and inhibition.The expression ofα-Hemolysin inhibits the activation of TLR2,MAPKs and NLRP3 inflammatory pathways,which lays a solid foundation for the effective treatment of diabetes and S.aureus pneumonia.