Therapeutic Effects Of Magnesium Isoglycyrrhizinate On Pulmonary Injury Induced By Paraquat In Rats

Background and objectives Paraquat is the most widely used herbicide, and causes the highest mortality rate (65%) upon acute poisoning. It raises the worldwide problem of curing acute paraquat poisoning by efficient physio-medical therapeutics for the fact that most traditional or exploratory treatment scheme, via either the administration of high doses of glucocorticoid or immunodepressant, or the employment of blood purification, could hardly increase the survival rate of patients. Paraquat depletes NAPDH and generates a large amount of free radicals through causing a series of redox reactions in vivo, thus resulting in a cascade of cytokines involved in the pathology of pulmonary fibrosis which include the decrease of superoxide dismutase (SOD) and Glutathione (GSH) and the increase of Malondiadehyde (MDA), Intercellular adhesion molecule-1(ICAM-1), Matrix metalloproteinase-9 (MMP-9) and Hydroxyproline (HYP). These changes severely impair the body, particularly the lung. Affected patients ultimately dead of respiratory failure or severe infection resulted from pulmonary fibrosis, whereas some severely poisoned patients are directly killed by acute hepatic and renal failure. Animal model experiments and clinical studies found that Magnesium Isoglycyrrhizinate (MgIG) exerts its powerful anti-poisoning effects of anti-inflammation, anti-oxidation, anti-apoptosis, anti-fibrosis, stabilization of cell membrane, immuno-modulation and increase of endogenous steroid, both intra- and extrahepatically. Foreign investigators have revealed that MgIG could evidently reverse the progression of carrageenin-induced lung damage in rats by blocking the up-regulation of ICAM-1, TNF-α, IL-1α, NF-κB and HYP. Based on a comprehensive review of literature and a large number of theory demonstrations, we found that, intriguingly, the pharmacological mechanism of MgIG could be nicely applicable to target the clinical and pathological characteristics upon PQ poisoning, suggesting a practical use of MgIG for the treatment of acute PQ poisoning. In this study, we had established a rat model of PQ poisoning, and based on this animal model, we evaluated the practical use of MgIG injection during the intervention of PQ poisoning by lines of experimental evidences, hoping to explore a new therapeutic scheme for human PQ poisoning.Methods 1, Establishment of rat model of PQ poisoning. 15 clean SD rats (male, 180-200g) were raised under normal condition for 1 week, weighed and randomly assigned into 3 groups (5 animals each). 3 different dosages of 20% PQ solution, low (15mg/kg), medium (20mg/kg) and high (25mg/kg), were administered to animals in each group by intraperitoneal injection, respectively. Animals were then raised for 15 days, life signs including the movement, breathing, food-taking, hair color, excreta, hemorrhage, death and survival were recorded every day. Afterwards, animals were sacrificed. Lung tissues of each animal were sampled, embedded, sectioned and stained by HE staining. The optimum PQ dose was calculated by the histological observations.2, The study of protective effect of MgIG on acute lung injury in rat model of acute PQ poisoning. 30 clean SD rats (male, 180-200g) were raised under normal condition for 1 week, weighed and randomly assigned into 5 groups (6 animals each) which were labeled as the blank group (0mg/kg PQ, 0mg/kg MgIG), the control group (15mg/kg PQ, 0mg/kg MgIG), the low dose group (15mg/kg PQ, 15mg/kg MgIG), the medium dose group (15mg/kg PQ, 30mg/kg MgIG) and the high dose group (15mg/kg PQ, 45mg/kg MgIG), respectively. One-shot injection of 20% PQ solution was followed 24 hours later by 1 dose of intraperitoneal MgIG injection per day. Life signs including the movement, breathing, food-taking, hair color, excreta, hemorrhage, death and survival were recorded every day. 14 days after the MgIG treatment, animals were anaesthetize by 50mg/kg pentobarbital and sacrificed for further studies. Inferior vena cava blood samples were tested for SOD, MDA, GSH, ICAM-1 and MMP-9 concentration; lung tissue samples were briefly examined, ~100mg left lung tissue samples were cut into ~10mg pieces for the measurement of HYP content by the method of basic hydrolysis, the right lung tissue samples were formalin-fixed, embedded and sectioned for the histopathological observations including pulmonary alveoli inflammation by HE staining, lung fibrosis by Masson's trichrome staining. The lung tissue was detected by immunohistochemistry for the ICAM-1 and MMP-9 protein expression .Results 1, After PQ injection, all the animals were tardy, dispirited and suffered from hypoalimentation in various degrees. Medium and high dose of PQ usually caused oral/nasal hemorrhage and hematuria. Based on HE histology, hyperplasia of interstitial tissue, increased effusion and destroyed type I and type II alveolar cells within all the poisoned animals indicated a successful establishment of lung fibrosis model. As for the lethality rate, 1 rat in the low dose group died on 3rd day since the PQ injection, 3 in the medium dose group died within 5 days, 4 in the high dose group died within 3 days. Since the survival of experimental animals was required within an experimental period of 2 weeks, low dose PQ (15mg/kg) was chosen for the further studies.2, As compared with the blank group, all poisoned group showed a significant decrease of serumal SOD and GSH concentration (P<0.05), indicating a successful model establishment. As compared with the control group, the medium MgIG dose group showed a significant decrease of serumal concentration of MDA, ICAM-1 and MMP-9, and lung tissue HYP (P<0.05 or P<0.01). However, the increase of serumal SOD was not evident (P>0.05). Either the low dose group or the high dose group showed an obvious reduction in serumal MMP-9 and lung tissue HYP concentration, but significant changes of serumal ICAM-1, SOD and GSH were not observed. The histopathological observations of lung tissue revealed that all poisoned animals, particularly those in the control group, showed lung edema and congestion, and partial lung consolidation. Interestingly, all the MgIG-treated groups, particularly the medium dose group, benefited from MgIG treatment as indicated by reduced lung edema and congestion, improved pulmonary alveolitis and fibrosis, and down-regulated expression of ICAM-1 and MMP-9.Conclusion MgIG injection following PQ poisoning, particularly of medium dose, could efficiently improve the lung injury via delaying the progression of lung fibrosis by mechanisms including down-regulation of ICAM-1 and MMP-9, inhibition of lipid peroxidation and reduction of release of inflammatory mediators whereby the anabolic-catabolic homeostasis of ECM components was maintained and thus the accumulation of collagen was suppressed in lung tissue.