Effects Of Ag On The Imbalance Of MMP-9, MMP-13 And TIMPs In The Experimental Pulmonary Fibrosis Of Rats

Objective: Pulmonary fibrosis (PF) is a kind of progressive interstitial disease in lung, characterised by excessive extracellular matrix deposition (mainly collagens). It is well known that the imbalance between matrix metalloproteinase (MMPs) and tissuse inhibitor of metaloproteinases (TIMPs) is one of the most machanisms of pulmonary fibrosis. It is found that a variety of causes may damage alveolar epithelial cells, which could expose the basement membrane. Matrix metalloproteinase-9 (MMP-9),known as the typeⅣcollagenase, can degrade the basement membrane, which results in fibroblasts migrating into alveolargap and proliferating. This promotes the further thickened of alveolar septum. Matrix metalloproteinase-13 (MMP-13) is a rodent mainly interstitial collagenase, whose functions include degradating typeⅠ,ⅡandⅢcollagen. TIMPs can specifically inhibit the activity of MMPs. Therefore, the imbalance between MMPs/TIMPs takes part in the formation of pulmonary inflammation, basement mambrance damage and ECM remodeling.Bleomycin (BLM) is a common anti-cancer chemotherapy drug. The main side effect of it was to result in sub-acute alveolitis and fibrosis in lung. So it is recognized that intratracheal instillation of BLM became a useful method of pulmonary fibrosis. Our previous studies have found that aminoguanidine (AG), an inducible nitric oxide synthase (iNOS) inhibitor, effectively prevent and treat PF induced by BLM. However, the mechanism of its actions is not known. There is no report showing whether the preventive action of AG on fibrosis in lung is associated with its ameliorating the imblance of MMP-9, MMP-13 and TIMPs.In order to illuminate the mechanism of AG on pulmonary fibrosis, our present study was undertaken to observe effect of AG on the content and activity of MMP-9,MMP-13, as well as the activity of TIMPs in the early phase (1-14d) and late phase (14-28d) of BLM-induced fibrosis in lung.Methods: Gelatin zymography was adopted to detecte the content and activity of MMP-9, MMP-13. Reserve zymography was adopted to detecte the activity of TIMPs. Sirius Red staining and hydroxyproline (HYP) of lung tissue were used for determining the degree of pulmonary fibrosis. HE staining was utended to judg the typical morphological changes and assist the positions of collagen in pulmonary interstitial for Sirius Red staining.58 SD rats(♂) (170-180g) were randomly divided into three groups: 1. Dynamic observation group (n=12): Rats were killed on day 8, day 15, and day 29 after intratracheal instillation of a single dose of BLM (5 mg/kg) or a single dose of normal saline (NS, 0.5 ml/kg), respectively. The lungs were used for detecting the activity of MMP-9, MMP-13 and TIMPs. 2. The early treatment group (1-14d, AG, i.p) (n=16): 14dNS+14dNS rats (n=3) were given NS (1 ml/kg, i.p) for 14 days after intratracheal instillation of a single dose of normal saline (NS, 0.5 ml/kg). The 14dM+14dNS rats: (n=8) were given NS (1ml/kg, i.p) for 14 days after intratracheal instillation of a single dose of BLM (5 mg/kg). The 14dM+14dAG rats (n=5) were given AG (20mg/kg/d, i.p) for 14 days after intratracheal instillation of a single dose of BLM (5 mg/kg). The above rats were killed on day 15 after intratracheal administration. The lungs were used for detecting the content and activity of MMP-9, MMP-13, and the activity of TIMPs, as well as for observing morphological change in lungs (Sirius Red and HE staining). 3. The late treatmen group (14-28d, AG, i.p) (n=16): 28dNS+14dNS rats (n=3) were given NS (1 ml/kg, i.p) from day 14 to day 28 after intratracheal instillation of a single dose of normal saline (NS, 0.5 ml/kg). The rats 28dM+14dNS rats (n=4) were given NS (1ml/kg, i.p) from day 14 to day 28 day after intratracheal instillation of a single dose of BLM (5 mg/kg). The 28dM+14dAG rats (n=5) were given AG (20mg/kg/d i.p) from day 14 day to day 28 after intratracheal instillation of a single dose of BLM (5 mg/kg). The above rats were killed on day 29 after intratracheal administration. The lungs were used for detecting the content of MMP-9, MMP-13, the activity of MMP-9, MMP-13, TIMPs, the content of HYP in lungs, as well as for observing the morphological changes in lung (Sirius Red and HE staining).The areas of intersititial collagen in lung were analysed by JEDA-801D. The content and activity of MMP-9, MMP-13 and the activity of TIMPs were analysed by Alpha FC analysis system. The data of MMP-9, MMP-13 and TIMPs were statistised by using SPSS-13.0 statistical software. P values less than 0.05 was viewed as statistically significant. The data of HYP and collagen were expressed as means±SD. After homogeneitic analysis, homogeneous data were analyzed with one-way anova analysis of variance and a post hoc test of least significant difference (LSD). P values less than 0.05 was viewed as statistically significant.Results: 1. General healthy state of rats: Reactions of the NS+NS rats were fine. The BLM+NS rats reacted slowly and the hair tarnished with poor appetite and hypoplasia. The above changes in lungs of BLM+NS rats were ameliorated by intraperitoneal injection of AG at the early and late phases of pulmonary fibrosis.2. Changes of activity of MMP-9, MMP-13 and TIMPs in lungs of rats: Compared with NS rats, the activity of MMP-9 and MMP-13 in BLM rats were enhanced on day 7, with a peak on day 14 and decreased on day 28. But there were difference between them . The activity of MMP-9 was still higher than NS rats, but the activity of MMP-13 was lower than NS rats. The activity of TIMP-1was higher than NS rats on day 7 , but the activity of TIMP-1 was lower both on day 14 and 28. Compared with NS rats, the activity of TIMP-2/3 in BLM rats was lower on day 7, higher on day 14, and lower on day 28.3 Effects of AG on the content and activity of MMP-9,MMP-13 and activity of TIMPs at the early and late phases of pulmonary fibrosis.3.1 Effects of AG on the content,activity of MMP-9,MMP-13 and activity of TIMPs at the early phase (1-14d) of pulmonary fibrosis: Compared with 14dNS+14dNS rats, the contents of MMP-9 and MMP-13 were increased (both P<0.05), the activity of MMP-9,MMP-13 in 14dM +14dNS rats were enhanced significantly (both P<0.01). But the activty of TIMPs have not changes (P>0.05). Compared with 14dM+14d NS rats, the content of MMP-9 in 14dM+14dAG rats was decreased (P<0.05) and the activity of MMP-9 was attenuated (P<0.01). Meanwhile, the content of MMP-13 was decreased (P<0.05). There were no difference on the activity of MMP-13 and TIMPs between 14dM+14dNS rats and 14dM+14dAG rats (P>0.05). There were no difference between 14dNS+14dNS rats and 14d M+14dAG rats on the content and activity of MMP-9, MMP-13 (P>0.05) ; at the same time there were no difference between the activity of TIMP-1 and TIMP-2/3 ( all P>0.05).3.2 Effects of AG on the content,activity of MMP-9,MMP-13 and activity of TIMPs at the late phase (14-28d) of pulmonary fibrosis: Compared with 28dNS+later 14dNS rats,the content of MMP-9 in 28dM+later 14 dNS rats was increased and the activity of MMP-9 was enhanced too (P<0.001), whereas the content of MMP-13 was markedly decreased (P<0.05). There were no difference on the activity of MMP-13 and TIMPs between them (P>0.05). Compared with 28dM+later 14dNS rats,the content of MMP-9 in 28dM + later 14dAG rats was decreased (P<0.001) and the activity of MMP-9 was attenuated too (P<0.05). At the same time, the content of MMP-13 was increased and the activity of MMP-13 was enhanced (both P<0.05). There were no diffenence on the content,activity of MMP-9, MMP-13 and activity of TIMP-1 between 28dM+later 14dAG rats and 28dNS+later 14dNS rats (All P>0.05), but the activity of TIMP-2/3 was enhanced (P<0.05) .4. Effects of AG on the typical morphology (HE staining) and areas of interstitial collagen of rats on the early phase (1-14d) in BLM-induced fibrosis in lung (Sirius Red staining).4.1 HE staining: The lung structure of the 14dNS+14dNS rats were normal. The alveolar septum in 14dM+14dNS rats were obviously enlarged with increased fibroblasts and collagens. The above morphological changes in 14dM+14dNS rats were obviously improved in the 14dM+14dAG rats.4.2 Sirius red staining: The pencentage of interstitial collagen area in 14d NS+14dNS, 14dM+14dNS and 14dM+14dAG rats were (0.47±0.06) , (1.04±0.06) and (0.53±0.05). Compared with 14dNS+14dNS rats, areas of interstitial collagen in 14dM+14dNS rats were obviously enlarged (P<0.001). Compared with 14dM+14dNS rats, areas of interstitial collagen in 14dM+14dAG rats were reduced significantly (P<0.001. There were no difference between 14dM+14dAG rats and 14dNS+14dNS rats (P>0.05).5. Effects of AG on the typical morphology (HE staining) and areas of interstitial collagen of rats at the late phase (14-28d) of BLM-induced fibrosis in lung:5.1 HE staining: The lung structure of the 28dNS+later 14dNS rats was nomal. The alveolar septum in 28dM+later 14dNS rats were obviously enlarged. In 28dM+later 14dNS rats, mesenchymal cells infiltration exists in lung. The above morphological changes were obviously improved in the 28dM+later14d AG rats.5.2 Sirius Red staining: The pencentage of interstitial collagen areas in 28 dNS+later 14dNS, 28dM+later 14dNS and 28dM+later 14dAG rats were (0.46±0.01), (1.8±0.11) and (0.49±0.06). Compared with 28dNS+later 14dNS rats, areas of interstitial collagen in 28dM+later 14dNS rats was enlarged (P<0.001). Compared with 28dM+later 14dNS rats, areas of interstitial collagen in 28dM+later 14dAG rats was reduced significantly (P<0.001), and there were no difference between 28dM+later 14dAG rats and 28dNS+later 14dNS rats (P>0.05).5.3 Content of pulmonary HYP of rats: The contents of pulmonary HYP in 28d NS+later 14dNS, 28dM+later 14dNS and 28dM+later 14dAG rats were[(3.65±1.05)μg/mg lung weight], [(7.61±1.27)μg/mg lung weight] and [(4.86±1.51)μg/mg lung weight]. Compared with 28dNS+later 14dNS rats, contents of pulmonary HYP in 28dM+later 14dNS rats was increased (P<0.001). Compare with 28dM+later 14dNS rats, content of HYP in 28dM+later 14dAG rats was reduced significantly (P<0.001), but still higher than 28dNS + later 14dNS rats (P<0.05).Conclusion: 1. At the early phase of BLM-induced fibrosis in lung, AG completely inhibited the increased contents of MMP-9, as well as the enhanced activity of MMP-9 in lung in BLM model. At the same time, AG inhibited the increased contents of MMP-13, whereas there were no effects on the activity of MMP-13 and TIMPs. In addition, AG inhibited the thickened alveolar septum in BLM model, which suggested that the mechanism underling the blockage of AG on the thickened alveolar septum was associated with its inhibition on the increased content of MMP-9 and the enhanced activity of MMP-9 in BLM model.2. At the late phase of BLM-induced fibrosis in lung, AG completely inhibited the increased content of MMP-9 and the enhanced activity of MMP-9 , as well as the dereased content and activity of MMP-13 in BLM model. But AG has no effect on the activity of TIMPs. Furthermore, AG reduced the collagen accumulation in lung in BLM model. The above results suggested that the mechanisms of therapy on fibrosis in lung by AG was associated with it's ameliorated the imbalance between MMPs and TIMPs.