| Objective: Paraquat (PQ) , has been widely used as a general herbicide throughout the world. As is widely used on agricultural land in China , PQ poisoning is also gradually increasing . Although PQ is very effective as a herbicide, it is highly toxic to humans and animals . PQ is readily absorbed through skin,pneogaster or enteron.But the poisoning mech- anism has not been fully elucidated, and there is lack of anti- dote heretofore.The mortality rate in clinical practice is very high. So PQ-induced changes of pathophysiology are becoming a hotspot in toxic therapeutics. The lung is the most susceptible targart organ. The lung injury induced by PQ is the major rea- son of death .The exact mechanism of pulmonary toxicity of PQ has not been fully educidated.To study the mechanism of PQ- induced lung fibrosis and the effect of Salvia miltiorrhiza mono- mer IH764-3 on it, structure, ultrastructure and the expression changes of matrix metalloproteinase-2 (MMP-2),tissue inhibi- tor of metalloproteinase-1(TIMP-1)and Transforming growth factor-β1(TGF-β1)were studied in lung of PQ-poisoned rat.Methods: Ninety adult healthy Sprague-Dawley (SD) rats (45 female, 45male) were divided into three groups at random. The control group (group A): 42 rats, the poisoned group (group B) : 42 rats ,the Salvia miltiorrhiza monomer IH764-3 treated group (group C):42 rats. The rats in group B and C were treated intragastrically with 1 ml of PQ (50mg/kg) diluted with normal saline. The rats in group A were treated intragastrically with the same dose of normal saline as that of group B and C. The rats in group C were given 1 ml of Salvia miltiorrhiza monomer IH764-3 intraperitoneally at a dose of 40mg/kg diluted with normal saline once a day after the administration of the PQ. The rats in group B were treated intraperitoneally with the same dose of normal saline once a day as those in group C. Six rats respectively from each of group were anesthetized with ketamine at a period of the 1st, 3rd, 7th, 14th, 21st, 28th and 35th day after paraquat treatment. Rats were dissected , the same part of the left lung was stained with hematoylineosin to observe its pathology by HE staining and Masson staining .The expression of MMP-2,TIMP-1,TGF-β1 in lung tissues were detected by immunohistochemistry staining .The other part of left lung was stained to observe the ultrastructure by using electron micro- scope. The six rats in group A were treated the same.Results: 1,Clinical signs and appearances after PQ poisoning: In group B and C,all of the rats began to demonstrate the changes to different extent in their clinical signs in 30mins~2hrs, and were especially severe on the 1st~7thday, including the respiratory system, psychological system , neural system,uropoiesis system and digestive system, etc. However , in group C all the rats demonstrated slighter clinical changes than those in group B (especially in the respiratory and uropoie- sis system).2,HE stain: Group A: Alveolar structure was clear and integrated with clean alveolar cavity. There was no alveolar septa widen and congestion. There was no inflammatory cell infiltration. Group B: There were obvious changes of acute alveolitis on the 1st and 3rd day, such as pulmonary edema, hyaline membrane, diffuse pulmonary hemorrhage and inflam- matory cell infiltration. It was the same with that of the 7th day. On the 14th , 21th ,28th and 35th day there were widened alveolar septa and fibroblastial hyperplasia in different degrees. Hyaline change could also be seen . The aforementioned changes were also shown up in group C, but were more palliative than group B. A little bit of collagen fibre was hyperplastic in the late phase.3,Masson staining: In group A,only a bit of stained green collagen fibres were found in the areas around bronchia and the insterspace between alveoluses, whereas in group B,after the 14th day,fibrous thickening occurred in bronchia walls and the alveolus interspacea,in some areas with an irregular colloca- tion of collagen fibres,with a higher level of MOD than that of group A (P<0.01). In group C, interfered with Salvia miltiorrhiza monomer IH764-3, collagen fibes accretion were not found obviously on the 1st～14th day. After the 21st day, collagen fibres increases gradually,but also lower than that of group B(p<0.05).4,Electron microscope: There was no obvious change in ultrastructure of lung tissues in group A. While in group B, there were a large number of efflusion in the alveolar space with some smudge cellular organ in it on the 3rd day. Inflammatory cell infiltrated in interstitial. Alveolar epithelia type I was injured, and the cilia of it were lost. Basal membrane broke. On the 14th day, alveolar septum was thickened with lots of hyperplasia and hypertrophic alveolar epithelia type II in it. Lamellar body was vacuolized. On the 35th day, collagen fibrils spread to alveolar epithelia and capillary basement membrane.Pathologic changes in group C were mild.5,Immunohistochemistry(IH)staining:①There was only a very weak expression of TGF-β1 in group A, while it kept in a sustained significantly higher level in group B compared with group A from the 1st day to 35th day (p<0.01). In contrast ,the expression of TGF-β1 in group C was significantly lower than that in group B along with the whole time points (p<0.01), though it was also obviously higher than that in group A all over the period (p<0.01).②There was only a very weak expression of MMP-2 in group A. while in group B, there was already a significant higher expression of MMP-2 from the 1st day to the 28th day after PQ poisioning (p<0.05), and it returned to the similar level with group A on the 35th day (p>0.05). The expression of MMP-2 in group C was significant lower than that in group B from the 1st day to 28th day (p<0.05) ,but still statistically higher than that in group A from the 1st day to the 21st day (p<0.01).③The expression of TIMP-1 was very weak in group A , while a significant higher expression of TIMP-1 was already found in group B compared with group A from the 1st day to 35th day(p<0.01). In constrast , the expression of TIMP-1 in group C was significantly lower than that in group B along with the whole time points (p<0.01),but it was markedly higher than that in group A all over the period (p<0.01).Conclusions: 1,PQ intragastrically poisoning can induce acute lung injury and fibrosis. It manifests that PQ intragastri- cally poisoning is a reliable method of making a model of lung fibrosis. 2,After PQ intragastrically poisoning, the expression of TGF-β1,MMP-2 and TIMP-1 is changed ,Which cause the the expression of MMP-2 and TIMP-1 derangement and proba- bly unbalance the synthesis and degradation of ECM , which may probably be a cause of lung fibrosis. 3,Salvia miltiorrhiza monomer IH764-3 can affect the expression of TGF-β1,MMP -2 and TIMP-1 in the lung tissues of PQ poisoned rats in a certa- in extent, regulate the rate of MMPs and TIMPs and lighten the acute lung injury and fibrosis.
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