The Expression Of AGEs, TGF-β₁, MMP-9, TIMP-1 And The Effect Of Cordyceps Sinensis Cultivated Byartificial Fermentation And Rosiglitazone In The Lungs Of Diabetic Rats

Objective: Diabetes mellitus (DM) is a metabolic disorder syndrome caused by many reasons, which has chronic hyperglycaemia as the main feature, resulting in multi-organizational system diseases. As one of target organs of diabetes, Impaired lung is mainly realized as a dysfunction of pulmonary ventilation, diffusion impairment, basement membrane thickening, metabolic imbalance of synthesis and / or degradation in extracellular matrix (ECM), increased thickness of pulmonary interstitial tissue, eventually leading to pulmonary fibrosis. The pathogenesis of pulmonary fibrosis in diabetes is unclear at present. Non-enzymatic glycation of proteins caused by sustained hyperglycaemia can form advanced glycation end products (AGEs). AGEs combine with their receptors (receptors of advanced glycation end products, RAGEs) to start intracellular signaling pathways, which activate a series of cell-mediated response and lead to production and release of cytokine and growth factor resulting to tissue repair and protein denaturation. Transforming growth factor-β₁ (TGF-β₁) is involved in growth, proliferation, differentiation, migration and apoptosis of the cells in tissue and regulation the metabolism of ECM. TGF-β₁ is recognized as one of the most relative factors in the development of pulmonary fibrosis, among the cytokines that lead to fibrosis. Matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) may play an important role in metabolism of the ECM. Gelatinase B (MMP-9) mainly degrade the components of ECM such as elastin,â…£collagen, LM-based film, resulting in the damage of lung-based membrane scaffold. TIMP-1 can inhibit the activity of MMP-9 and reduce degradation of ECM. The changes of expression and activity of MMP-9 and TIMP-1 may be involved in the incidence and development of pulmonary fibrosis in diabetes. Cordyceps sinensis (Berk.) Sacc is a parasitic fungus from ergot Section on the complex of Hepialidae child seat of the insect larvae and the body of the larvae, which is a precious traditional Chinese medicine. It has the effects on nourishing kidney and lung, eliminating phlegm, preventing cough, regulating the immune system function and so on. Rosiglitazone (RSG) is a thiazolidinediones that can increase the insulin sensitivity. RSG play a role in regulating the metabolism of blood glucose and lipids and restraining inflammatory reaction by activating peroxisome proliferator activated receptorγ(PPARγ). The signals mediated by PPARγhas negative feedback regulation for activation and proliferation of T cells, macrophages and relative immune cells as well as transcription of the inflammatory gene. RSG may play an important effect on restricting local inflammation and interstitial fibrosis of lung tissue.In this study, rat model of type 1 diabetes was established by an high-dose intraperitoneal injection of STZ (streptozotocin, STZ). By given Cordyceps sinensis cultivated by artificial fermentation and RSG, we can detect the changes of morphology and expression of AGEs, TGF-β₁, MMP-9 and TIMP-1 in lung tissue though methods of pathology molecular biology. The purpose of this experiment is to explore the effect of AGEs, TGF-β₁, MMP-9 and TIMP-1 in diabetic pulmonary fibrosis and the protective effect on lung tissue by the application of Cordyceps sinensis cultivated by artificial fermentation and RSG.Methods: There were 60 male SD rats aged 8 weeks, 15 of which were randomly selected as normal control group (A group), and the remaining 45 as the experimental group. Rats in the experimental group were injected intraperitoneal by streptozotocin(55mg/kg). 72 hours later, the rats whose venous blood glucose higher than 16.7mmol / L in the continuous 3 days were considered type 1 diabetes animal models. The model of diabetic rats were randomly divided into diabetic control group (B group), group with treatment of Cordyceps sinensis cultivated by artificial fermentation (2.4g/kg/d) (C group), group with treatment of rosiglitazone (4mg/kg/d) (D group). This experiment lasted 12 weeks, blood glucose and body weight were measured at the beginning of the experiment and the end of 0, 4, 8 and 12 weeks, and 24 hours urine volumes were measured at the end of 12 weeks; at the end of 12th weeks, animals were sacrificed by separating femoral artery, and we removed bilateral lungs of the diabetic rats. Lung morphology and collagen fiber distribution was observed by HE and Masson staining in right lung. The location and protein expression of AGEs, TGF-β₁ in lung tissues were detected by immunohistochemistry. RT-PCR was used to detect the expression of MMP-9 mRNA and TIMP-1 mRNA in left lung. The experimental data were dealt with SPSS13.0. Analysis of variance was adopt among the comparison of multiple groups, and LSD test was used between each two groups comparison, when the data were in line with normal distribution and homogeneity of variance.Results:1 At 72-hour after injecting STZ and the end of 4, 8 and 12 weeks, the blood glucose of B, C and D group was significantly higher than group A, the difference was statistically significant (all P <0.01). At the end of 4, 8 and 12 weeks, the body weight of B, C and D group was obviously decreased compared with A group, the difference was statistically significant (all P <0.01).2 The changes of pulmonary pathology in B group were thickening of alveolar wall, widen of alveolar septum, infiltration of inflammatory cell, increase of fibroblasts in interstitial lung, collagen deposition, alveolar structural damage including alveolar collapse, narrow, distorted or even occlusion in some alveolar capillaries, compared with A group, by HE and Masson staining. C and D group had improved compared with B group.3 Immunohistochemistry showed that, compared with A group, the expression of AGEs, TGF-β₁ was significantly increased in B, C and D group, the difference was statistically significant (all P <0.01); compared with B group, the expression of AGEs, TGF-β₁ was decreased in C and D group, the difference was statistically significant (all P <0.01).4 RT-PCR reaction showed that, the expression of MMP-9 mRNA in B, C and D group was lower than A group, and the expression of TIMP-1 mRNA was higher than A group (all P<0.01); compared with B group, the expression of MMP-9 mRNA was increased in C and D group, and the expression of TIMP-1 mRNA was decreased in C and D group, the difference was statistically significant (all P<0.01).Conclusion:1 High-dose intraperitoneal injection of STZ and destruction of isletβcells could establish rat model of type 1 diabetes characterized as hyperglycaemia, polyuria and weight loss. This model was similar to characteristics of type 1 diabetes in human being.2 Morphological changes in diabetic lung tissue were consistent with the pathological changes of pulmonary fibrosis, and the expression of AGEs, TGF-β₁, MMP-9 and TIMP-1 was abnormal in diabetic rat lung tissue, suggesting that interaction among them might be involved in the occurrence and development of diabetic pulmonary fibrosis.3 the pathological changes of lung tissue were improved significantly by the treatment of Cordyceps sinensis cultivated by artificial fermentation and rosiglitazone. The improvement may contribute to regulation of the expression of AGEs, TGF-β₁, MMP-9 and TIMP-1 by the two drugs. So the two kinds of intervention had a significant protective effect in the lungs of diabetic rats.