| objectiveObservation of polydatin(Polydatin,PD)on paraquat(Paraquat,PQ)poisoning rat lung wet dry ratio(Wet-to-dry weight ratio,W/D),the concentration of PQ in plasma and tissues,lung tissue and serum lipid peroxidation(Lipid peroxidation,LPO)and the effects of inflammatory cytokines,to explore the acute lung injury(Acute lung,injury,ALI)of the protective effect,providing a theoretical basis for the clinical application of PD treatment of PQ poisoning ALI.Methods(1)Experiment one: establish a rat model of acute paraquat poisoning suitable for this study.32 healthy female SD rats,weighing about(200 + 20)g,according to the normal living conditions after 2 days adaptive feeding fasting 12 h,while rats were observed no abnormality after weighing,and according to the number of randomly divided into 4 groups,normal control group,each dose group(each group 8);normal control group normal saline gavage 1ml/kg groups respectively with 20% PQ solution according to the mass fraction of low(25mg/kg),(50mg/kg),high(75mg/kg)three doses were administered once.After exposure to normal normal feeding for 7 days,no treatment was done during the period.Every morning the fixed time to observe the general condition of the rats,including mental reaction malaise,four claw nose lip cyanosis,dyspnea and pathological respiratory sound,eating water excretion abnormalities,aggressive activity decreased,fluffy hair color dark,bleeding and death or survival of a comprehensive and detailed records,in 8 days all survival rats responded to all 10% chloral hydrate(0.3ml/100g)intraperitoneal injection of anesthesia were dissected chest lung tissue,PBS buffer(pH = 7.3,0.1mol/L)repeatedly wash,10% Formaldehyde Solution refrigerator to be fixed,4 DEG C for gross pathology and pathological HE staining was observed in the lungs,the survival rate at the same time calculation of all groups of rats,so as to obtain the optimal dose for this study by PQ model.(2)Experiment two: Determination of paraquat in serum or tissue by UV Spectrophotometry30 healthy female Sprague-Dawley(SD)rats were selected and the body weight was about(200 + 20)g.All the rats were randomly divided into normal control group(n=5)and dye group(n=25).Take a one-time intragastric method to establish animal model: 20% paraquat exposure group by 25mg/kg;the control group was given the same amount of 1mL/kg saline;after the administration of each sampling time(6h,12 h,24h,48 h,72h five time points,n = 5),with 10% chloral hydrate(0.3ml/100g)intraperitoneal injection the supernatant were killed,serum was collected from three anatomical chloroacetic acid 600 L and 20% 100 L mixed with 12000r/min 0.2ml centrifugal 10 min,distilled water and 1.8mL by UV spectrophotometry(ultravioletspectrophotom-etry,Uv)for determination of paraquat in.In this experiment,low temperature and high speed centrifugation,20% three chloroacetic acid direct precipitation,impurity protein were used to separate and extract PQ from plasma or tissue of poisoned rats,and Uv quantitative method was established.The plasma,lung,heart,liver and kidney of PQ poisoned rats were detected by the established Uv detection method,which laid the foundation for further exploration of the relationship between PQ concentration in plasma or tissue and the prognosis of poisoning.(3)Experiment three: The therapeutic effect of polydatin on lung injury in rats with acute paraquat poisoningIn order to evaluate the therapeutic effect of PD on acute poisoning caused by PQ in rats,90 healthy female SD rats,weighing about 200 + 20 g,were randomly divided into 3 groups: normal control group(Con group n=20),exposure group(PQ group n=35)and PD intervention group(n=35).According to the results of the pre experiment,the rats in the infected group and the PD intervention groups were fed with 20% grass and withered water in one time by 25mg/kg,and the rat paraquat poisoning model was established.The rats in the PD intervention group were given 60 min polydatin after intervention.The rats in the exposure group and the normal control group were given the same amount of 1ml/kg normal saline for gavage,then 1 times a day for a total of 3 days.The rats in the intervention group were given the same amount of 1ml/kg normal saline.The toxic reaction of each group was observed at 6h,12 h,24h,48 h and 72 h at the beginning of the experiment.The rats were anesthetized and killed(at each time point,the exposed group and the PD intervention group 6,the normal control group 4).The serum and tissue specimens were collected at the-80 C refrigerator for preservation.In strict accordance with the biochemical method kit detection of rat serum superoxide dismutase(SOD),peroxidase(CAT),glutathione peroxidase(GSH-Px)and malondialdehyde(MDA),myeloperoxidase(MPO)content;enzyme linked immunosorbent assay(ELISA)detection of tumor necrosis factor alpha in serum(TNF-alpha),transforming growth factor beta 1(TGFbeta 1)and interleukin-6(IL-6)levels and changes;at the same time take part in lung tissue were detected by nuclear factor kappa B(NF-K B),hydroxyproline(HYP)and the wet / dry weight ratio(W/D)to observe the degree of pulmonary fibrosis;ultraviolet spectrophotometry(Ultravioletspectrophotometry,Uv)to detect the level of PQ in serum and different tissues,scavenging effect of PD on evaluating organizations within PQ.Results(1)Experiment one:All the rats in normal control group had no obvious change in the observation period,eat normal water,action quick,smooth and clean hair,four limb muscle strength is normal,the body weight increased significantly;after after modeling,the rats were in the 2H gradually began to appear symptoms of poisoning,gradually show different degrees of mental retardation and eating the amount of water,reduce the action response is slow,fluffy,dry fur four limb muscle strength decreased significantly,such as shortness of breath;exposure to 1~3d after the symptoms worsened significantly,rat death peak;in low dose group in response to the light,and appeared in several hours after exposure,while the middle and high dose groups are different the degree of the nose lips and limbs cyanosis,bloody discharge phenomenon,and the gradual emergence of dyspnea and hypoxia,severe symptoms of death.From HE staining,all exposed rats had lung pulmonary interstitial thickening,inflammatory cell infiltration and fibrin exudation increased,a large number of changes in alveolar structure destruction and pathological collapse,pathological changes in low dose group and high dose group than in similar,but to a lesser degree,can be regarded as the preparation of rat model of acute PQ the requirements of poisoning.The experimental period of pretest design in this study is 1 weeks.From the mortality rate of rats,the high dose group died 7 days in 7 days,while the middle dose group died 6 days in 7 days,while the small dose group died 1 days in 7 days.To sum up,the time requirements for our pre test should be exposed to the dose of 25mg/kg,so we should use the dose of 25mg/kg to prepare the acute PQ poisoning rat model.(2)Experiment two:quantitative method to detect serum Uv of rats exposed to paraquat concentration in the linear range for 0.08~10 in g/mL,the regression equation was y=0.0935x+0.2964,the correlation coefficient(R)was 0.9955,the recovery rate was 90.63%~103.0%,RSD,3.6%~8.12%,RSD and intraday fluctuations in the 1.1%~ 5.19% and 2.2%~ 6.04%,detection limit 0.05 g/mL.According to the standard curve regression equation,the serum PQ concentration at different time points was calculated.It can be seen that the concentration of PQ in serum decreases gradually from early to 6h,and 72 h can still detect PQ in serum,indicating that its concentration in serum can help to predict the prognosis of poisoning.During the sample processing,Uv used three chloroacetic acid to precipitate protein to treat rat serum samples.It has high reliability and less interference.It has the advantages of simple operation,quick analysis and accurate results.(3)Experiment three:in addition to the control group,the symptoms of the rats in each group were similar.Effect of 1.PD on experimental animal general observation results: normal control group,all rats without any abnormal changes in 72 h during the experimental period of good performance,the weight increased steadily,the rats were different symptoms of poisoning,the intervention group PD symptoms appeared relatively late,compared with the same time point in group was significantly reduced the symptoms were improved in different degrees.Impact of weight change of 2.PD of the experimental animal: the rats were exposed to body weight as the exposure time decreased,significantly lower than the normal control group,PD intervention group was given drug intervention,delaying the decline in body weight of rats poisoning the trend,there is a statistically significant difference with body weight in rats.The effect of 3.PD on the lung wet dry weight ratio(W/D)level of the experimental animals: at the corresponding time point,the lung W/D in the exposure group was significantly higher than that in the control group with the prolongation of the poisoning time.The W/D level in the PD intervention group was significantly lower than that in the exposure group at all time points.4.effects of PD on the concentration of PQ in serum and tissues in experimental animal: at different time after exposure in blood,lung,heart,distribution and contents of PQ in liver and kidney were significantly different,were significantly higher than the normal control group,PD intervention at different time points were significantly lower than control group,and then presented with time gradually decreased,but still significantly higher than the normal control group.5 Effects of.PD on lipid peroxidation markers in the serum level of experimental animal: exposure group and PD intervention group at each time point of serum MDA,MPO levels were higher than the normal control group,with significant difference(P<0.05 or P<0.01),CAT,SOD,GSH-PX levels were lower than the normal control group(P<0.05 or P<0.01)PD MDA,MPO;intervention levels of serum and tissue group at each time point than the exposure group decreased antioxidant enzymes CAT,SOD,GSH-Px,redox system level,and increased with time gradually increased,compared with exposure group(P<0.05 or P<0.01).6 Effects of PD on inflammatory cytokines in serum and lung tissue in the experimental animal: compared with normal control group,exposure group lung tissue at different time points HYP,NF-kappa B content increased gradually(P <0.05 or P<0.01),PD intervention group and exposed group,lung tissue HYP,NF-K B content decreased obviously,the difference was statistically significant(P,<0.05 or P<0.01);compared with the normal control group,exposure group at each time point in serum TGF-beta 1,IL-6 and TNF-alpha were significantly increased,the difference was statistically significant(P,<0.05 or P<0.01);compared with the control group,PD group of serum beta 1,TGF-IL-6,TNF-a level at each time point increased significantly reduced,the differences were statistically significant(P,<0.05 or P<0.01).ConclusionPD can significantly reduce the lung wet / dry weight ratio,PQ concentration in plasma and tissues of lung tissue and serum,reduce lipid peroxide and inflammatory factors of poisoning rats in tissues and plasma levels in patients with acute PQ,to reduce the response to acute lung injury induced by PQ,and had a strong anti oxidative stress,inhibiting the release of inflammatory factors is closely the relationship between the function,has good clinical application prospect. |
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