Study On The Dynamic Changes Of Pulmonary Surfactant With The Animal Models Differentiated By Weifen, Qifen, Yingfen, And Xuefen Syndrome

Objectives:This paper aims to establish the lung injury Animal Models differentiated by Weifen, Qifen, Yingfen, and Xuefen syndrome, and then Screening appropriate indicators and indicators of the lung injury model, study the dynamic changes of Pulmonary Surfactant in that model, Seeks to reveal the material foundation of the lung injury and reveal the molecular mechanism of the lung injury.Method:1. establishing the model and groupingNew Zealand rabbits, half Male and female, were randomly divided into normal control group and ALI groups(2、6、12、24h four subgroups, Corresponding to weifen, qifen, yingfen and xuefen group), LPS(1mg/kg)was given by rabbits’ auricular vein intravenous injection in ALI group Under aseptic. The Injection rate is10ml/h. Normal control group received no treatment, direct access to the blood, and lavage, lung tissue specimens tested.2. Observed indicators and method①Observing morphology activities changes in the rabbits after establishing the model,Observing Food intake, faeces, urine, ears, conjunctiva, respiratory, heartbeat, mind, body, limbs, tongue and other changes.②plasma and serum samples collection after injection of LPS, intraperitoneal anesthesia with2%sodium pentobarbital at2,6,12,24h respectively, arterial blooding, determining the heart, liver, kidney function in the Supernatant,counting and classificating the neutrophils and lymphocytes in the blood, evaluating the levels of TNF-α and protein content in the serum using Elisa and coomassie blue staining(Bradford),③lung permeability index (LPI):Lavaging the left alveolar with N. S. evaluating the levels of TNF-α and protein content in the BALF using Elisa and coomassie blue staining(Bradford). calculating lung permeability index (LPI, LPI=protein concentration in the BALF÷protein concentration in the serum).④lung histopathology observing changes in right lung pathology by eyes, take Part of lung of the same size and position in4%paraformaldehyde for24h. After dehydration, embedded in paraffin,5μm thick sections, HE conventional staining, observing changes in lung pathology under light microscope.⑤The detemination of (surfactant protein A)、SP-B (surfactant protein B)、 SP-C (surfactant protein C) mRNA in the lung:According to the SYBR Premix Ex TaqTM RT-PCR reaction reagent for reaction system.⑥The determination of TPL:determined it with Bartlett method.⑦The determination of DSPC (unsaturated phosphatidylcholine) determined it with Mason method. The activity of PS is represented by saturated phosphatidylcholine/total protein (SatPC/TP).⑧The content study of the components of phospholipids in BALF:The standard include-phosphatidylcholine(PC), lysophosphatidylcholine(LPC), phosphatidyl-glycerol (PG), phosphatidylinositol (PI), phosphatidylethanolami ne(PE), phosphatidyl serine (Ps) and sphingomyelin (SPH), Chromatographic conditions:high performance liquid chromatography, Evaporative light-scattering detector.3.statistical processingThe data of each group are represented by (x+SD), the differences between the groups were compared with SPSS12.0statistical software by t test, if P <0.05, the differences were statistically significant.Result:1Observing morphology activities changes:Weifen group Appear Chills, with hair, curling up, trembling, ear vasoconstriction, aversion to cold, do not drink do not eat, fever, conjunctival hyperemia or ear was mild congestion, sane. Qifen group curled up with gradually disappeared, but the fever is more apparent, breathing and heartbeat is accelerated apparently, a fidgety, drinking, eating, auricular vascular hyperemia conjunctiva hyperemia, mild, sane, the tongue is red. Yingfen group the rabbit conjunctiva marked hyperemia, look tired, lethargy, the reaction is slowed down, dyspnea, stridor and lazy, slow heartbeat, ear vasoconstriction, oliguria, loose stools, slightly purple tongue. Xuefen group the rabbit conjunctiva no longer hyperaemia, pale ears, touch of cool or even cold, coma and sleepiness, sprawled or semicircle shape, loose stool, anuria, lips cyanosis, arrhythmia, breathless, screaming, convulsions, and death. In the same observation period, normal control animals response sensitively, diet as usual, no abnormal performance of the above. Prompt model group of animals are in the blood rheology and microcirculation disorder.and the disorder are more and more obvious as time went on.2Lung pathological changes:Macroscopic observation:wei fen group double lung are mild congestion and edema, but has no obvious bleeding. Qifen group double lung are more congestion than of Weifen group, bilateral pulmonary diffuse hyperemia edema, bulk increases apparently, individual pair of lungs diplay a few bleeding points, most congestion but no bleeding spots. Yingfen group, double lung volume increased significantly, edema surface has focal flaky bleeding point, with two lower lobe lung edge more obvious extravasated blood. Xuefen group, thymic hemorrhage intensively, double lung displays much larger sheets and cords congestion or hemorrhage, almost pervaded the whole lung, lung section has a lot of dark red bloody foam liquid formation. The control group double lung’ s appearance was pink, no edema, section without effusion. Prompt model group animal lung damage was more and more serious over time.Light microscopy:weifen group, alveolar septal was mild hypertelorism, capillary wall was mild congestion. Qifen group, alveolar septal thickening of capillary was moderate, with a small amount of congestion, neutrophil exudate. Yingfen group, alveolar had destructed structure, pulmonary capillary was congestion, severe thickening of alveolar septa, alveolar and interstitial lung were with massive neutrophil and erythrocyte. In addition, Xuefen group besides with main Yingfen group’s pathological features, and fibrin exudation, airway epithelial cell shedding and hyaline membrane formation. The control groups of alveolar septal and vascular wall were without edema, without, thickening of alveolar cavity, without obvious exudation, under a light microscope the structure of lung tissue was normal basicly. The result suggests that model group animal lung were more and more inflammatory infiltration, alveolar structure was severely damaged as time went on, Results consistent with the naked eye.The pathological score results: Compared with normal group, the pathological injury of all model group were significantly heavier than the control group and the difference is significant (P<0.05), pathological score differences among each subgroup are also significant (P<0.05), prompting the extension of time is more and more obvious pathological changes of lung. It suggests that pathological changes of lung are more and more obvious with the passage of time.3Measurement of body temperature:Compared with normal group, the temperature in Xuefen group, Qifen group, Yingfen group, Xuefen group rised significantly (P<0.01), there was a increased trend progressively, among the four groups, there was no significant difference between the groups in body temperature. It suggests that the temperature changes of rabbit are more and more obvious with the passage of time.4Changes of CK. LDH:Compared with normal group, CK level in weifei group, qifen group, yingfen group, xuefen group increased significantly (P<0.05), LDH levels has a continuly increased trend, LDH level in yingfen group is higher than xuefen group (P<0.05), the two groups are back down in the xuefen time, but still higher than normal group. It suggests that myocardial cells of the model animal may suffers serious injury or necrosis.5Changes of ALT、ASTM、AST/ALT:ALT is found primarily in the cytoplasm and AST is mainly present in the mitochondria in the cytoplasm. Elevated ALT reflects the liver cell membrane was damaged, elevated AST reflects the tips of liver cell injury reached to organelle level. The results of this study show:compared with normal group, the ALT and AST level in Xuefen group, Qifen group, Yingfen group, Xuefen group, rised progressively, there was a increased trend, ALT level in weifei group, qifen group, yingfen group, xuefen group increased significantly (P<0.05), AST level in yingfen group and xuefen group increased significantly (P<0.05), AST level in qifen group is higher than weifen group (P<0.05), the ratio of them in yingfen group, xuefen group increased significantly (P<0.05), the ratio of yingfen group is higher than qifen group (P<0.05), the ratio was back down in the xuefen time, but still higher than the normal group. Results suggest that the liver function abnormalities.6Changes of Urea、Cr: Compared with normal group, Urea level in weifei group, qifen group, yingfen group, xuefen group increased significantly (P<0.05), Urea level differences among weifei group and qifen group, qifen group and yingfen group,, yingfen group and xuefen group are significant (P<0.05), Cr level in xuefen group increased significantly (P<0.05), as Urea, the level in weifei group, qifen group, yingfen group display a increased trend, back down in the xuefen time, but still higher than the normal group. The results suggest that animals renal may dysfunction after LPS injection.7Peripheral white blood cell counting and classification:Compared with normal group, neutrophil levels in weifen group, qifen group, yingfen group, xuefen group increased significantly (P<0.05), lymphocyte levels in weifen group is higher than control group(P<0.05), neutrophil levels and lymphocyte levels in qifen group were higher than weifei group and yingfen group(P<0.05), microscopically neutrophil increase thickening, platelet distributed in single, easy to see. It reveal that there are plenty of inflammatory substances and cytokines, and they are released into blood.8Measurement of TNF-α in serum and BALF:Compared with normal group, TNF-α in BALF increased significantly (P<0.05), TNF-α in BALF in qifen group is higher than yingfen group (P<0.05), TNF-α in serum in yingfen group is higher than xuefen group (P<0.05), the two subjects between the other group showed no significant difference, It reveal that TNF-α in the serum of the model group has no obvious change, TNF-α in the BALF fluid rise in gas phased phased decline Yingfen blood, possibly lung injury, pulmonary inflammation re-turn from light by weight of light process, which may be lung injury healing outcome requirement kind of inherent to reflect.9Pulmonary permeability index detection:Compared with normal group, LP1in weifen group, Qifen group, Yingfen group, Xuefen group, rised progressively, there was a increased trend, LP1in Xuefen group increased significantly(P<0.05), increased LPI showed increased pulmonary capillary permeability, May be the main reason to induce pulmonary edema/injury.10Determination of total phospholipids (TPL)、DSPC、PS activity:Compared with normal group, the TPL level in the weifen group, Qifen group, and Yingfen group persistent decrease, decline to a certain extent, increased slightly in Xuefen over time, the difference between Yingfen group and Xuefen group is significant (P<0.05), Rebound phenomenon may be associated with lung type II epithelial cells’compensatory secretion, because of the reduction of the TPL in alveolar.11Determination of protein in the serum and BALF:Compared with normal group, TP in BALF in weifen group, Qifen group, Yingfen group, Xuefen group and the subgroups showed no significant difference (P>0.05), there was no significant changes of TP in the BALF. Compared with normal group, TP in serum in Xuefen group, showed significant difference (P<0.05), the difference between Yingfen group and Xuefen group is significant (P<0.05), the difference between Yingfen group and Qifen group is significant (P<0.05), it suggest that the protein in serum change significantly until xuefen.12SP-A, SP-B, SP-C mRNA expression changes in pulmonary tissue:At different time points, SP-A mRNA expression of the model group in lung tissue decreased progressively, and the difference between each sub group and control group are significant(P<0.05), every two groups among xuefen,weifen, qifen,yingfen subgroups showed significant difference (P<0.05). SP-B mRNA expression has the same results. The trend of SP-C mRNA expression is same to the SP-A and SP-B mRNA expression. It reveals that diminished lung local immune function because of the reduced expression of SP-A, SP-B, SP-C mRNA may be one of the main mechanisms of the lung injury.13The variations of phospholipid in BALF:Compared with normal group, the PC and PE level elevated slightly in Weifen, in Qifen, Yingfen, Xuefen decreased progressively, there was a reduced trend, but compared with the normal group and the subgroups showed no significant difference. Lysolecithin (LPC), phosphatidyl glycerol (PG), phosphatidylinositol(PI), phosphatidyl serine (PS) and sphingomyelin (SPH) can not be determined in the normal group and model group samples. The reducment of PC relative content may be one of the main reason of the inhibition of PS activity.Conclusion:1. The model for lung injury differentiated by weifen, qifen, yingfen and xuefen syndrome established by rabbits’auricular vein infusion of lipopolysaccharide, in the database of its biological parameters, the Urea differences among weifen, qifen, yingfen and xuefen syndrome were significant, it can be used as the indicators to distinguish weifen, qifen, yingfen and xuefen, Temperature, ALT has a positive correlation with lung injury score, LPI has a very positive correlation with lung injury score, temperature、ALT、LPI can be used as the reference indicators to distinguish weifen, qifen, yingfen and xuefen. they are Objective material basis of the lung damage evolution in weifen, qifen, yingfen and xuefen.2. TPL, DSPC, PS activity decreased in bronchoalveolar lavage fluid in weifen, qifen, yingfen and xuefen syndrome compared with normal group, PS quality and quantity abnormality is an important mechanism of the occurrence and development of lung injury.3. SP-A、SP-B、SP-C mRNA expression decrease time-dependent in rabbit in weifen, qifen, yingfen and xuefen syndrome, that is one of the most important material basis of lung injury.4. The changes of phospholipid composition in BALF can be used as a reference indicator for early alveolar injury, continuous observation helps determine prognosis, the reduction of the relative content of PC maybe is one of the main reasons for inhibition of PS activity and lung damage.5. In conclusion, AST/ALT, Urea and LDH, CK and Cr, serum TNF-alpha index points increase in the weifen, qifen, yingfen syndrome, declined in the xuefen stages. In contrast, the activity indicator trends of the TPL, DSPC, PS, declined in the weifen, qifen, yingfen syndrome, increase in the xuefen stages. This may an inherent self-healing outcome requirements reflect lung injury; Body temperature、LDH、AST、ALT、AST/ALT、Urea、Cr、neutrophils、lymphocytes TNF-alpha in BALF、LPI、protein in serum, SP-A mRNA, SP-B mRNA and SP-C mRNA and DSPC indicators in the weifen, qifen, yingfen, xuefen syndrome one or several stages, there were significant differences, CK、levels of TNF-α in serum、protein in BALF、the PC and PE at any stage with the normal group compared to no significant difference. This shows that CK、levels of TNF-α in serum、 protein in BALF、the PC and PE relative to other indicators of lung injury, Less affected by lung injury during lung injury, is not suitable as Wei Qi Ying Xue dialectical reference index of lung injury.