Experimental Study On The Therapeutic Effect Of High Dose Ambroxol Against Rats Pulmonary Surfactant By Paraquat Poisoning

0bjective:The paraquat (Paraquat, PQ) is in the present worldwide scale uses one of scope widespread weed killers, has to the human and animals compares toxic. Because its toxicant lethal dose is small, and at present still special effect antidote, therefore the mortality rate is high, the domestic individual report case fatality rate may reach above 80%. After the lung is the paraquat poison, the main harm's target organ, may create acute breath poverty-stricken syndrome (ARDS), is the PQ poison patient's main cause of deathAt present thought generally the paraquat poison creates the lung damage mainly with the oxygen free radical, the excessive lipin peroxidization has the lipin peroxidation thing as well as in vivo return to original state Gu Guanggan the peptide (GSH) content reduction concerns. Now on clinical main application sugar medicines and so on cortical hormone, immunosuppression, medicinal preparation and anti-free radical, decrease the PQ induction the peroxidation damage. Some research indicated that the paraquat poison may create the pulmonary alveolus surface-active substance (ps) exceptionally, possibly creates the reason which the paraquat poison acute lung . injury。The hydrochloric acid ammonia bromine rope is one kind has many kinds of biology effect mucilage lytic agent In recent years studied indicated that the large dose ammonia bromine rope could promote the lung surface-active substance synthesis, reduced the pulmonary alveolus surface tension; Suppresses the activation the neutral granular cell's response, erupts including the breath, dissolves the enzyme body's release to reduce the hyperoxide anion and the hydrogen peroxide and so on production, reduces it to lung's oxidized damage; Reduces the pulmonary alveolus inflammation extent of reaction. Therefore, this experiment passes the abdominal cavity injection paraquat to establish the big mouse acute lung damage model, the observation large dose hydrochloric acid ammonia bromine rope - returns to original state various targets influence to pulmonary alveolus surface activity protein c(sp-c) and the lung oxidation, the appraisal large dose hydrochloric acid ammonia bromine rope the therapeutic action which damages to the paraquat poison result acute lung.Methods:One hundred and thirty-six healthy male Sprague-Dawley rats,weighting 250-300g,were divided into three groupsand each of the three groups were separately dividedinto four subgroups randomly according to four different intervals(1,3,5 and 7 days).(1)NS group(n=24):the same volume of 0.9%saline solution was injected into peritoneal cavity every day.(2)PQ group(n=56):2%paraquat(25mg/kg) was injected into peritoneal cavity at the first day.(3)AT group(n=56):ambroxol(35mg/kg)was injected into peritoneum daily after praraquat intoxication.There were twenty-four rats in NS group and each of subgroups has six rats.Each of 1d,3d,5d subgroups in PQ group and AT group had twelve rats and there were twenty rats in 7d subgroups respectively.Fatality rate was observed in 7d subgroups.After anesthetized animal, takes the venous blood from the vena cava inferior place。The number of WBC,the percentage of PMN,the content of MDA and GSH-Px,SOD activity in blood was routinely measured. Right lung tissue was observed through light microscope(LM). The expression of SP-C was observed under multimedia color pathological image pattern after immunohistochemical SP staining.Analysis was performed for statistical analysis with aid of SPSS11.5 software.All data were expressed as mean+standard error of the mean.Differences among groups were tested by one-way analysis of variance(ANOVA)followed by a least significant difference(LSD)test when significant differences were detected.P values less than 0.05 were considered statistically significant.Results:1 The fatality rate of group 7days .There were no dead in NS group.Sixteen rats were dead in PQ group(fatality rate 80%).Ten rats were dead in AT group (fatality rate 50%).There is significant difference between PQ group and group (P<0.05)2 The results of blood2.1 The quantity of WBC in blood.There was no significant difference among NS group.The quantity of WBC was increased after the administration of praraquat.PQ group or ATgroup comparied with NS group at the same time had significant difference(P<0.05,P<0.01).It was lower in groupAT than that in group PQ at the same time.There was significant difference at 3d,5d subgroup(P<0.05,P<0.01).2.2 The percentage of PMN in blood.There was no significant difference among NS group.The percentage of PMN was higher obviously in PQ group or AT group then that in NS group(P<0.05,P<0.01)at the same time.It was lower in AT group than that in PQ group and there was significant difference betweens AT group and NS group at3d,5d time point(P<0.05,P<0.01).3 The results of MDA,SOD and GSH in plasma3.1 The MDA content in plasma.There was no significant difference among NS groups.It was obviousty higher in group PQ than that in group NS(P<0.01).It was also higher in groupAT.There was significant difference between group AT and group NS at 1d,3d (P<0.05).The content of MDA was obviously lower than that in group PQ at 3d,5d,(P<0.01).3.2 The SOD activity in plasma.There was no significant difference among groups NS.It was obviously lower in PQ group than that in NS group at 1d ,3d(P<0.01,P<0.05).The SOD activity of AT group was obviously lower than that of NS group at 1d(P<0.05).There was significant difference between group AT and group PQ at 1d 3d,5d(P<0.01).3.3 The GSH content in plasma.There was no significant difference among groups NS.It was obviously lower in PQ group than that in NS group(P<0.01).AT group also decreased,there was significant difference between group AT and group NS at 1d,3d,5d(P<0.05,P<0.01).The activity of GSH was higher in AT group than that in PQ group at 1d,3d(P<0.01). 4 Pulmonary morphologic and pathologic findings4.1 Gross Appearance:lung samples in NS group had normal pink smooth appearance and outline.The lung of rats in PQ group were swell with extensive dark red patchy bleeding focus and moderate quality.There was more light pink or white foam-like liquid exudation could be seen in the cut face.While in AT group,the lung had less volume as well as less hemorrhage and exudation,pulmonary edema abated.The lung was soft and most pulmonary tissue approached the normal pigmentation.4.2 Light microscopic Findings:In NS group alveolar structure was clear and integrated with clean alveolar cavity. There was no alveolar septa widen and congestion.There was no inflammatory cell infiltration and hemorrhage.After toxicity,alveolar collapsed and leakage of blood and fibroprotein into the alveolar space could be seen.The capillary of alveolar wall dilated and congested and the alveolar septa widened. Surrounding the vessels and trachea,there was different degree of edema and inflammatory cell infiltration.There was free neutrophil and macrophage aggregated in the alveolar space and septa.Fibroblasts proliferation,inflammatory granulation tissue,capillaries hyperplasia and alveolar atrophy could be seen.After using Ambroxol,all the pathologic alterations palliated.5 The SP- Cimmunohistochemistry show that:The alveolar epithelial typeⅡcells were the primary target cell for SP-C.5.1 In NS group,SP-C were intensely stained and micro particles distributed scatterly on inter-wall of alveolar lumen.There was a layer of positive substance on the small bronchial lumen.5.2 In group PQ,SP-C were weakenly stained.Line or short line shape.There was positive substance deposition on the small bronchial lumen.5.3In group AT,SP-C were intensely stained and the positive substance alveolar lumen and small bronchial lumen was obviously increasing.5.4There was no significant difference among NS group.The Date of positive cells in group PQ was obviously lower than that in group NS(p<0.01)at the same time,and decreased to valley at 7days.The percentage of positive cells in AT group was lower also.There was significant difference between AT group and NS group at 1d,3d,5d(p<0.01).It was obviously higher in AT group than that in PQ group at 3d,5d.(p<0.05,p<0.01).Conclusions:1 Peritoneally injection of PQ caused acute pulmonary impairment with MDA increasing andGSH, SOD decreasing in plasma.The imbalance of oxidation and anti-oxidation resulting from hyper production of free radical is the causation of acute lung injury induced by paraquat poisoning.2 After PQ poisoning,SP-C expression in alveolar epithelial typeⅡdecreased which indicated decreasing of pulmonary surfactant may play an important role in ALI/ARDS caused by paraquat.3 After high dose of ambroxol therapy,the pulmonary exudation and PMN cells decreased,MDA content declined,SOD,GSH levels elevated and SP-C expression enhanced.Our study shows that ambroxol has effect of anti-inflammation,anti-oxidation and facilitating synthesis and secretion of pulmonary surfactant protein A.Ambroxol has obviously therapeutic effect in paraquat poisoning.