| Asthma is a chronic inflammatory disorder of the airways in which many cells and cellular elements play a role, characterized by inflammation, hyperresponsiveness, and remodeling of the airway. The airway remodeling include marked goblet cell, airway smooth muscle, and fibroblast hyperplasia. Increased deposition of collagen and extracellular matrix proteins occur in the reticular basement membrane and throughout the bronchial mucosa, together with prominent airway angiogenesis. Airway remodeling has been recently one of the main goals in asthma research. Shema Zhichuan Decoction is been used to treat asthma for many years, the effect is significant. This project intended to further explored the mechanism in the treatment of asthma on the basis of the previous study and the level of molecular biology.Objective:To observe the expression of MMP-9, TGF-β1, Smad3, Smad7,OPN, and PAI-1in asthmatic rats, evaluating the effect of Jiawei Shema Zhichuan Decoction on the airway remodeling of asthmaMethods:72SD rats were randomly divided into6groups:control group(A);asthma group(B); high(C), middle(D) and low(E) dosage Chinese medicine group, and dexamethasone group(F), Each group contained12rats. Each rats were sensitized with lOmg OVA in200mg aluminum hydroxide by intraperitoneally injection on days1,8except Control group. This was followed by additional intranasal challenges for20min with OVA Three times a week for8wk(1,2weeks with1%OVA;3and4weeks with1.5%OVA;5, and6weeks with2%OVA; and7,8weeks with2.5%OVA). The rats in group A was administered PBS instead of OVA for sensitization and challenges.Each rat in group C, D, and E were treated with Chinese medicine at the doses of40g/kg,20g/kg,10g/kg by intragastric administration, and the rats in group F were treated with dexamethasone(0.5mg/kg) by intragastric administration half an hour before each ultrasonic atomizing inhalation. Rats in group A were replaced by sodium chloride. Lung tissue were sliced and hematoxylin and eosin stained. Rats were sacrificed by drawing blood24hours after the last allergen challenge, left lungs were snap-frozen, and right lungs were perfused with4%paraformaldehyde to preserve pulmonary structure, fixed in4%paraformadehyde, and paraffin-embedded. The levels of PAI-1and OPN in serum were measured by ELISA (enzyme linked immunosorbent assay). And pulmonary histopathological analysis was performed to observe the infiltration of inflammatory cells, the hyperplasia of airway global cells and the deposition of collagen. The pulmonary expression of MMP-9, TGF-p1, Smad3and Smad7proteins were evaluated by immunohistochemistry. The mRNA expressions of MMP-9, TGF-β1,Smad3and Smad7were measured by Quantitative real-time PCR (polymerase chain reaction).Result:1. HE staining showed that there were obvious inflammatory cells infiltration in the lung tissues, loss of cilia in airway epithelium, airway smooth muscle hypertrophy and sub-epithelial fibrosis in group B, which were significantly decreased in group C, D, E and F.2. WAm/Pbm and WAi/Pbm were significantly decreased in group C,D and E when compared with group B(P<0.01), but still higher than those in group A (P<0.01), and there was no significant difference when compared with group F(P>0.05), except group C (P<0.05)3. Immunohistochemical showed that the MMP-9protein mainly existed in the lung tissue around the airway, which was weak positive. The MMP-9protein expression was significantly enhanced in group B when compared with group A (P<0.05). The protein level of MMP-9in group C and F was significantly decreased when compared with group B(P<0.01,P<0.05). And there was still no significant change in group D and E(P>0.05). QRT-PCR showed that the relative expression of MMP-9mRNA was significantly decreased in group C,D when compared with group B (P<0.05), and there was no significant change in group E and F(P>0.05).4. ELISA showed that that:(1) the level of OPN was signi ficantly decreased in group B when compared with group A (P<0.01). There was significantly decreased in group C when compared with group B(P<0.01), and no significant change in group D, E and F(P>0.05).(2) The level of PAI-1was significantly decreased in group B when compared with group A (P<0.01). There were significantly decreased in group C, D, and E when compared with group B(P <0.01, P<0.05, P<0.05), and no significant change in group F(P>0.05).5. Immunohistochemical showed that:(1)The TGF-β1protein mainly existed in the lung tissue around the airway, which was weak positive. The TGF-β1protein expression was significantly enhanced in group B when compared with group A (P<0.01). The protein level of TGF-p1in group C, D and F was significantly decreased when compared with group B(P<0.01). And there was still no significant change in group E(P>0.05).(2) The Smad3protein mainly existed in the lung tissue around the airway, which were weak positive. The Smad3protein expression was significantly enhanced in group B when compared with group A (P<0.01). The protein level of Smad3in group C, D and F was significantly decreased when compared with group B(P<0.05). And there was still no significant change in group E(P>0.05).(3) The Smad7protein mainly existed in the lung tissue around the airway, which was strong positive. The Smad7protein expression was significantly weaken in group B when compared with group A (P<0.01). The protein level of Smad7in group C, D, E and F was significantly increased when compared with group B(P<0.05).QRT-PCR showed that:(1)The relative expression of TGF-β1mRNA was significantly decreased in group C, D when compared with roup B (P<0.05), and there was no significantly difference when compared with group F(P>0.05). And there were still no significantly change in group E(P>0.05).(2) The relative expression of Smad3mRNA was significantly decreased in group C, D,E when compared with group B (P<0.01), and there was no significant difference when compared with group F(P>0.05).(3) The relative expression of Smad7mRNA was significantly increased in group C, D when compared with roup B (P<0.01). And there was still no significant change in group E and F(P>0.05).Conclusions:1. Intervention with Jiawei Shema Zhichuan Decoction could inhibit proliferation of smooth muscle, alleviate the asthmatic airway remodel ing and partly restore the appropriate structure of airway wall.2. Intervention with Jiawei Shema Zhichuan Decoction could decrease the expression of MMP-9in the lung tissue on asthmatic rats, adjust the MMP-9 /TIMP-1balance, reduce the extracellular matrix, thus delay the process of airway remodeling.3. Intervention with Jiawei Shema Zhichuan Decoction could decrease the expression of OPN and PAI-1in the lung tissue on asthmatic rats, adjust the dynamic balance of extracellular matrix, reduce the extracellular matrix, restrain the migration and proliferation of smooth muscle cell and fibroblasts, etc, thus delay the process of airway remodeling.4. Intervention with Jiawei Shema Zhichuan Decoction could adjust the expression of TGF-β1, smad3, and smad7, restrain TGF-p1signal to the cytoplasm and cell nuclear transcription, reduce the collagen fiber composure and airway sub-epithelial fibrosis, thus delay the process of airway remodeling.
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