The Expression Of NF-κB,iNOS,MMP-9 In The Pulmonary Tissue Of Paraquat-induced Rats And The Effect Of Salvia Miltiorrhiza Monomer IH764-3

Objective: Paraquat (PQ) , has been widely used as a general herbicide throughout the world. As is widely used on agricultural land within 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 antidote 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 orgarn,which also determines the major reason 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 monomer IH764-3 on it, structure, ultrastructure and the expression changes of nuclear factor kappaB(NF-κB),inducible nitric oxide synthase(iNOS) and matrixmet alloproteinase 9 (MMP-9) were studied in lung of PQ-poisoned rat.Methods: Ninety adult healthy Sprague-Dawley (SD) rats (45 female, 45 male) were divided into three groups at random- the control group (group A) , 6 rats, the poisoned group (group B) , 42 rats and 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 immediately after the administration of the PQ. The rats in group A and 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 B and C were anesthetized with ketamine at a period of 1, 3, 7, 14, 21, 28 and 35 day after paraquat treatment. Rats were dissected , Part of the left lung was stained with hematoylineosin to observe its pathology by HE staining and Masson staining .The expression of NF-κB,iNOS,MMP-9 in lung tissues were detected by immunohistochemistry staining The other part of left lung was stained to observe the ultrastructure by using electron microscope. 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 day 1~day 3, including the respiratory system, psychological system , neural system and diges-tive system, etc. However , in group C all the rats demonstrated slighter clinical changes than those in group B (especially in the respiratory system).2 HE stain: Group A: Alveolar structure was clear and integrated with clean alveolar cavity. There was no alveolar sep-ta widen and congestion. There was no inflammatory cell infiltration. Group B: There were obvious changes of acute alveolitis on day 1 and 3, such as pulmonary edema, hyaline membrane, diffuse pulmonary hemorrhage and inflammatory cell infiltration. It was the same with that of day 7. On day 14, 28 and 35 there were widened alveolar septa and fibroblastial hyperplasia to 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 fibes 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 percentage of positive area than that of group A (P<0.01). In group C, interfered in Salvia miltiorrhiza monomer IH764-3,1d～14d collagen fibes accre- tion were not found obviously. After 21d, collagen fibes increases gradually,but also lower than group B(p<0.05).4 Electron microscope: There was no obvious change in ultrastructure of lung in group A. While in group B on day 3, there were a large number of efflusion in the alveolar space with some smudge cellular organ in it. Inflammatory cell infiltrated in interstitial. Alveolar epithelia type I was injured, and its cilia were lost. Basal membrane broke. On day 14, alveolar septum was thickened with lots of hyperplasia and hypertrophic alveolar epithelia type II in it. Lamellar body was vacuolized. On day 35, 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 NF-κB in group A, while it kept in a sustained significantly higher level in group B compared with group A from 1d to 14d (p<0.01). In contrast ,the expression of NF-κB in group C was significantly lower than that in group B from 1d to 14d (p<0.01), though it was also obviously higher than that in group A from 1d to 7d (p<0.01).②There was only a very weak expression of iNOS in group A. while in group B, there was already a significant higher expression of iNOS from 1d to 14d after PQ poisioning (p<0.05), and returned to the similar level with group A on the 21th day (p>0.05). The expression of iNOS in group C was significant lower than that in group B from 1d to 14d (p<0.01) ,though still statistically higher than that in group A from 1d to 7d (p<0.01).③The expression of MMP-9 was very weak in group A , while a significant higher expression of MMP-9 was already found in group B compared with group A from 1d to 14d (p<0.05). In constrast , the expression of MMP-9 in group C was significantly lower than that in group B from 1d to 14d (p<0.01),though it was markedly higher than that in group A from 1d to 7d (p<0.01).Conclusions: 1) PQ intragastrically poisoning can induce acute lung injury and fibrosis. 2) PQ intragastrically poisoning is a reliable method of making a model of lung fibrosis. It can imitate the clinic of PQ poisoning well and is worth of more widespread use. 3) After PQ intragastrically poisoning, the changed gene expression of NF-κB,iNOS and MMP-9 can probably unbalance the synthesis and degradation of ECM,which may probably be a cause of lung fibrosis. 4) Salviamiltiorrhiza monomer IH764-3 can affect the gene expression of NF-κB,iNOS and MMP-9 to a certain extent, and lighten the acute lung injury and fibrosis.