The Protective Effect Of Tanshinone â…¡A On Pulmonary Fibrosis And Its Mechanism In Rats

Idiopathic pulmonary fibrosis is a chronic, progressive and lethal disease ofunclear pathogenesis and no effective treatment, with a median survival of less thanthree years from diagnosis, it became a serious threat to people’s health and lives, hasalso been the difficulty and research focus of diagnosis and treatment of respiratorydiseases.IPF has complex etiology, and its pathophysiological processes involve theactivation of multiple signaling pathways, immune disorders, inflammation, fibrosis,and so on. In recent years, the researchers at home and abroad tend to consider that itspathogenesis of lung injury primarily is caused by a variety of reasons, earlypathological process is characterized by acute lower respiratory tract inflammation,including alveolitis, interstitial pneumonia, alveolar epithelial damage, fibroblastsproliferation, macrophages, neutrophils and other inflammatory cell infiltration, thesecretion of cytokines, inflammatory mediators and other biological active substances,such as tumor necrosis factor-α (TNF-α), interleukin (IL-1β and IL-6), etc., resultingmetabolic disorders of the extracellular matrix, the extracellular matrix deposition andexcessive fibrous tissue repair, and these processes eventually cause pulmonaryinterstitial structure disorder, lung parenchymal damage, resulting in chronic pulmonaryfibrosis, collagen deposition, alveolar structural changes, and ultimately pulmonaryfibrosis.In recent years, more and more evidences also indicate that oxidative stress isclosely related to the formation and development process of idiopathic pulmonaryfibrosis, the anatomical structure of lung and its main physiological function determinesthat the lung is one of the most susceptible to oxidative stress target organs. OxidativeStress refers to an imbalance between oxidation and antioxidation, which tends tooxidation, resulting in neutrophil infiltration and protease, intermediate oxidationincreased.Under physiological conditions, the antioxidant enzyme system in the body,such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), can adjust and scavenge free radicals timely. But if there is excessive oxygen free radicals or thebody’s ability is too low not to remove oxygen free radicals, the activity of SOD andGSH-Px is reduced, which can cause lung tissue damage and cell membrane lipidperoxidation, and MDA as a main product of lipid peroxide can increase. So in thelaboratory the determination of the amount of MDA can be used to reflect the degree oflipid peroxidation in vivo, indirectly indicate the production of free radicals.Anotherkind of biomarkers of oxidative stress, myeloperoxidase, its level in BALF of patientswith IPF also has obviously increased. Under complex pathophysiological processes,the induced nitric oxide synthase is induced by a variety of stimulus factors,which leadto the concentration of nitric oxide significantly increase. At the same time, theabnormal increase of inflammatory mediators can make cyclooxygenase-2enhanced,then prostaglandin increase, especially PGE2, which can lead to inflammatory lesions.So many experts and scholars gradually accept that antioxidant can be a new way fortreating idiopathic pulmonary fibrosis.Myofibroblast phenotype transformation is an important pathological process ofpulmonary fibrosis, and transforming growth factor-beta1is the key factor of thepulmonary fibrosis progression. TGF-β1can promote myofibroblast phenotypetransformation, stimulate cytokines synthesis and secretion, and extracellular matrixaccumulation. TGF-β1signal mainly through its receptor and Smad2/3transductionpathways, and our previous research has also confirmed that activin receptor likekinase-5in the process of pulmonary fibrosis significantly increased, this suggestsALK5involved in the development of pulmonary fibrosis.Medical research on IPF treatment never stop, over the years, its treatment hasexperienced different direction of exploration, but IPF is a kind of disease of etiologyand pathogenesis is not very clear, idiopathic pulmonary fibrosis lack of effectivetreatment methods at present. The diagnosis and treatment guide of IPF in2011replacesthe ATS/ERS IPF consensus in2000, which changed the most treatment methods intodifferent strength of recommendations, only N-acetylcysteine, pirfenidone,anticoagulation drugs, etc. get weak recommendations, mainly because of their goodsecurity. And immunosuppressants such as corticosteroids and other drugs, because ofits adverse effects to the body, there is a lot of limitations for application in clinic.Therefore, developing an effective drug is of great significance for patients withpulmonary fibrosis. Modern pharmacology research also confirmed that a variety of traditional Chinese medicine monomer has some extent to inhibit the action of thepulmonary fibrosis. Salviae miltiorrhizae BGE. is a chinese traditional medicine.Tanshinone IIA is a kind of alcohol soluble ingredient, which extracted from salviaemiltiorrhizae BGE.. It has definite molecular structure, highest levels in Tanshinones,the most stable and effective active ingredient, most biological drug effect of salviaemiltiorrhizae BGE.. Recent studies have also shown tanshinone IIA has manypharmaceutical activities, including clear oxygen free radicals, anti-inflammatory,anti-tumor and lung protection. Wang et al. have shown that tanshinone IIA can reducepulmonary fibrosis in rats. However, its biological mechanism in pulmonary fibrosis isnot very clear.In this study, we adopt the rat model of bleomycin-induced pulmonary fibrosis toexplore protective effect of tanshinone IIA for pulmonary fibrosis, and to further studyits molecular mechanism.Part IThe effect of Tanshinone IIA on histomorphology of rats with pulmonary fibrosisObjective: To explore the effect of tanshinone IIA on pulmonary fibrosis in rats, inorder to make clear whether tanshinone IIA is beneficial for pulmonary fibrosis.Methods: Sprague-Dawley rats, weighing200-220g, male and female unlimited, weredivided into four groups by random number table method: control group (N group),tanshinone IIA group, fibrosis group(model group), tanshinone IIA+fibrosis group,with six rats in each group.28days after the intervention, collected alveolar lavage fluidof each group, then put to death all the rats, and collected lung tissue. We calculated andcompared the histopathology and wet/dry weight ratio of lung among groups.Results: The wet/dry weight ratio of tanshinone IIA+fibrosis group was significantlylower than that in the model group, the histopathology showed that fiber foci in alveolarseptum, lesions, structural damage to the lung parenchyma, total collagen content wereless.Conclusion: Tanshinone IIA could reduce the content of extracellular matrix ofbleomycin-induced pulmonary fibrosis, lower the degree of fibrosis, which play theprevention and treatment role for pulmonary fibrosis. Part IIThe effects of Tanshinone IIA on inflammation and oxidative stress of rats withpulmonary fibrosisObjective: To observe the effects of Tanshinone IIA on inflammation and oxidativestress of rats with pulmonary fibrosis, and to further explore the mechanism oftanshinone IIA on pulmonary fibrosis.Methods: Sprague-Dawley rats, weighing200-220g, male and female unlimited, weredivided into four groups by random number table method: control group (N group),tanshinone IIA group, fibrosis group(model group), tanshinone IIA+fibrosis group,with six rats in each group.28days after the intervention, collected alveolar lavage fluidof each group, then put to death all the rats, and collected lung tissue. The total proteincontent and cell number, the neutrophils, macrophages count in BALF were detected,the expression of TNF-α, IL-1β and IL-6in BALF of each group was detected byELISA to explore the effect of tanshinone IIA on inflammatory cells and cytokines, thecontent of MDA, NO and PGE2and the activity of MPO were detected by kits, theiNOS and cox-2expression level were detected by real-time fluorescent quantitativePCR and western blot.Results:28days after the bleomycin administration, the total protein content and thenumber of neutrophils, macrophages in BALF obviously increased, and in the meantimethe expression of TNF-α, IL-1β and IL-6in BALF also increased significantly, thecontent of MDA and the activity of MPO in lung tissue obviously increased or enhanced,compared with control group, also accompanied by the generation of NO and PGE2increasing. Western blot and real-time fluorescent quantitative PCR results showed thatthe expression of iNOS and cox-2also increased significantly at the protein level andmRNA level. But in tanshinone IIA-treated group, the inflammatory cells and cytokinesreduced obviously, which could further explain the protection mechanism of tanshinoneIIA on pulmonary fibrosis.Conclusion: Tanshinone IIA can relieve pulmonary fibrosis by reducing theinflammatory cells and cytokines, mitigating oxidative stress and reducing theexpression of iNOS and cox-2in the process of pulmonary fibrosis. Part IIIThe effects of Tanshinone IIA on TGF-β1/ALK5/smad2/3signal transductionpathway of rats with pulmonary fibrosisObjective: To further explore whether the protective effect of tanshinone IIA wasrelated to its inhibition of TGF-β1/ALK5/smad2/3signal transduction pathway, andprovide more theoretical basis for application of tanshinone IIA in treatment ofpulmonary fibrosis.Methods: Sprague-Dawley rats, weighing200-220g, male and female unlimited, weredivided into four groups by random number table method: control group (N group),tanshinone IIA group, fibrosis group(model group), tanshinone IIA+fibrosis group,with six rats in each group.28days after the intervention, collected alveolar lavage fluidof each group, then put to death all the rats, and collected lung tissue. the expressionlevel of serum TGF-β1,lung ALK5and Smad2/3were detected by ELISA and westernblotting.Results:28d after the endotracheal bleomycin administration in model group, weobserved that the level of serum TGF-β1,lung ALK5and Smad2/3increasedsignificantly, compared with control group, there was significant difference(P<0.05),but after tanshinone IIA interventional therapy, the serum TGF-β1level decreasedsignificantly, and the level of lung ALK5and Smad2/3decreased,the difference wasstatistically significant compared with model group (P<0.05).Conclusion: Tanshinone IIA can inhibit TGF-β1/ALK5/smad2/3signal transductionpathway of rats with pulmonary fibrosis.