Study On Safe Transfusion Based On Oxygen Supply And Demand In Coronary Heart Disease Rat

Objective:（1）On the basis of the model of coronary heart disease,the model of blood transfusion related acute lung injury was prepared.（2）Preparation of prokaryotic expression mutant（99,8 LysCys）ecombinant hemoglobin and further purification.To observe the effects of mutant Hb and rHb1.1 and rHb2.0 on the oxygen metabolism of coronary heart disease rats.The properties of mutant Hb in oxygen metabolism were identified.（3）Clear N-acetylcysteine loaded poly-L-lactic acid nanoparticles in the blood transfusion-related acute lung injury treatment.（4）This study aims to assess the effects of intrathecal dexmedetomidine and clonidine under ropivacaine anesthesia.Methods:（1）Based on the model of coronary heart disease,intraperitoneal injection of LPS was used to establish the model of blood transfusion related acute lung injury.After the model was established,the model of electrocardiogram,cardiac function,lung mass,lung histology score,myocardial tissue and lung tissue morphological observation were established.（2）Preparation of mutant Hb after purification.Coronary heart disease rats were randomly divided into five groups.In the normal control group and the model control group,human serum albumin was input into the control group.The mutant Hb and rHb1.1 were input into the human hemoglobin rHb1.1 and rHb2.0 group.The recombinant human hemoglobin rHb2.0 was input.The arterial and mesenteric venous blood values of rats were measured at 0,30,60,90 and 120 min after HAS,mutant Hb,rHb1.1 and rHb2.0 were measured and metabolic analysis.To observe the morphological changes of myocardial tissue in experimental rats.（3）Preparation of blood transfusion related acute lung injury animal model.After the model was successful,the lung tissue was observed to observe the pathological changes.The N-acetylcysteine-loaded L-lactic acid nanoparticles prepared by single-nozzle electrospray were subjected to in vitro intervention.Detection of the preparation of particles biochemical,electrochemical impedance method,toxicity.（4）P7 and P21 Sprague-Dawley rat pups were exposed to spinal anesthesia of 0.5%ropivacaine.Separate groups of rats were given intrathecal clonidine（0.03or0.1mg/kg b.wt）or dexmedetomidine（3or10μg/kg b.wt）dose along with ropivacaine.Immunohistochemistry for activated-caspase-3 expression and TUNEL assay were performed to assess neuroapoptosis in the spinal sections.Glial reactivity was measured by detecting glial fibrillary acidic protein and ionized calcium binding adapter molecule 1.The animals were tested for sensory and motor blocks following anesthesia.Results:（1）The dry ratio was significantly increased（P<0.05）.Alveolar cell KP-1staining showed alveolar macrophage cytoplasm staining was brown,the nucleus was not colored.In the lung injury model group,the pulmonary and pulmonary tissues were observed by HE staining.The bronchial wall was thickened,the alveolar septum widened,the alveolar cavity was enlarged,some alveolar rupture and fusion were found.Inflammatory infiltration was found in the alveolar cavity.Pulmonary interstitial infiltration of a large number of inflammatory cells.The level of proinflammatory cytokine TNF-αin serum of CHD rats was significantly higher than that of the control group（P<0.05）,reached a peak at 3 h,then decreased slightly,but remained high at 12 h Higher than the control group（P<0.01）.The relative expression of TNF-αwas measured by the ratio of TNF-αand GAPDH bands in gray scale,P<0.05.（2）MAPs of mutant Hb and rHb decreased within 30-60min.The Q_SMA of the mutant Hb group was significantly higher than that of the normal group(Q_SMA at the end of the experiment),the mutant Hb group vs the normal group,6.9土0.7vs3.6土0.5mmHg,P<0.01).The Q_SMA at baseline after rHb1.1 was slightly lower than that of baseline,but there was no significant difference（P>0.05）.The Q_SMA was significantly lower than that of the mutant Hb group at the end of the experiment（the infusion was 120min,4.6土0.4vs6.9土0.7ml/min,P<0.05）,and significantly higher than the pre-treatment level（4.6土0.4vs2.9土0.4,P<0.05）.At the same time,the input of mutant Hb and rHb can quickly and efficiently restore the pH and BE values of blood（P<0.05）,correct the symptoms of blood metabolic acidosis in hypoxic condition.The morphological changes of cardiomyocytes in short-term enter coronary heart disease rats were not changed compared with the normal control group.（3）The diameter distribution of NAC-PLLA nanoparticles varied between 115-650nm.There was no significant difference in the cytotoxicity of NAC-PLLA nanoparticles at different concentrations of 50 to 100μg/ml（P>0.05）.The impedance of NAC-PLLA treated samples was higher than that of NAC and control samples.The level of cytokines in the NAC-PLLA group was significantly lower than that in the NAC group（170±13vs240±23pg/mL,P<0.05）,significantly lower than that in the control group（170±13vs560±45pg/mL,P<0.05）.The concentrations of nitrite/nitrate in the control group,NAC group and NAC-PLLA group were 18.3±1.05,14.7±1.23 and 10.4±1.34μmol/L,respectively.There was significant difference between the two groups（P<0.05）.The concentration of MDA in lung tissue of NAC-PLLA group was lower than that of NAC group（159±11.5vs219.8±10.6nmol/g,P<0.05）and control group（159±11.5vs330.5±15.4nmol/g,P<0.05）.（4）Intrathecal injections of ropivacaine in combination with clonidine or dexmedetomidine resulted in dense blocks and as well extended the duration of spinal blocks.The latency of fall was shorter in rats exposed to ropivacaine when compared with clonidine or dexmedetomidine administration with ropivacaine spinal anesthesia.The apoptotic cell counts and glial activity was markedly lower in rats that received combined doses of clonidine or dexmedetomidine compared to the single drug injection of ropivacaine.Conclusion:（1）It is feasible to establish a transfusion-related acute lung injury model by intraperitoneal injection of LPS on the basis of coronary heart disease.Mutant hemoglobin and commercial recombinant hemoglobin to improve the oxygen supply and demand of coronary artery disease similar ability,mutant hemoglobin production costs lower.（3）NAC loaded PLLA nanoparticles have protective effect on acute lung injury,and have the advantages of long drug release time and targeted administration.（4）Clonidine or dexmedetomidine administration with ropivacaine increased the intensity and duration of spinal blockade thereby increased anesthetic effects and lowered ropivacaine induced neurotoxicity.