Antifibrotic Effect And Mechanism Of Curcumin In SD Rat Model Of Oral Submucous Fibrosis

Background and ObjectiveOral submucous fibrosis (OSF) is a chronic and concealed disease of oral mucosa. It is closely related to betel quid (BQ) chewing habit and has been defined by WHO as one of the precancerous conditions. The same as other fibrotic diseases, there is still no definitive and effective treatment for OSF in the moment. The shortage of satisfied animal model of OSF has become a bottleneck slowing down the researches on the pathogeny, pathogenesis and clinical treatment of OSF.Curcumin is the primary active substance isolated from Curcuma Longa L. rhizome, and has been reported as an anti-fibrotic drug. Regard this as the starting point, we expected to create a stable and reliable animal model of OSF by locally injecting bleomycin (BLM), and then explored the feasibility of treatment with curcumin for OSF combined with in vivo and vitro experiment.MethodsFirstly,100Sprague-Dawley (SD) rats were randomly divided into3groups. Rats in the experimental group were injected BLM diluent to the bilateral buccal mucous membrane, once a day for8weeks, when they were under Isoflurane (inhalation) anesthesia. Rats in the control group were injected PBS instead of BLM diluents. Rats in the normal group did not receive any intervention. After the rats were executed, HE staining, masson staining, the soda hydrolysis in the measurement of hydroxyproline, immunohistochemistry, western blotting and transmission electron microscope were used to detect the changes of the histopathological features, levels of collagens, myofibroblast (MFB), TGF-β1,IFN-γ and ultrastructure.Secondly,35SD rats were randomly divided into4groups. Rats in the normal group did not receive any intervention. Rats in the control group, high curcumin group and low curcumin group were injected BLM diluent to the bilateral buccal mucous membrane, once a day for4weeks. Then curcumin solution of different dosages was given to the30rats by gavages, once a day for4weeks. After all the rats were executed, HE staining, immunohistochemistry and western blotting were used to detect the changes of the histopathological features, levels of collagens, MFB, TGF-β1,IFN-γ and IL-13.Thirdly, MFB were generated by incubating fibroblasts, obtained from human oral mucosa, with TGF-β1. MTT, PI staining, and FACS assays were used to investigate curcumin's effect on proliferation and cell cycle of fibroblast (FB) and MFB. Annexin V/PI binding and FACS assays were used to examine apoptosis of MFB, western blotting to determine the levels of Bcl-2and Bax, and enzyme-linked immunosorbant assay was employed to examine the levels of collagen type Ⅰ and Ⅲ in the supernatants of myofibroblasts.Results1,Local injection of1mg/ml BLM diluent to the bilateral buccal mucous membrane of SD rats, once a day for8weeks, can create similar changes with human OSF in many ways of clinical symptoms and histopathological changes.2,The higher doses of curcumin decreased the expression of TGF-β1and IL-13, increased the expression of IFN-y and reduced MFB in the bucal mucous of the rats which were injected BLM diluents for4weeks. The levels of type Ⅰ,Ⅲ collagens had no significant change.3,Curcumin of2.5-40μmol/L inhibited proliferation of FB and MFB; MFB are more sensitive to curcumin than FB. Curcumin of10-40μmol/L also disturbed the cell cycle, induced apoptosis and decreases the generation of collagen type I and III in MFB. Curcumin induces apoptosis in MFB by down-regulating Bcl-2/Bax ratio.Conclusions1,Local injection of lmg/ml BLM diluent to the bilateral buccal mucous membrane of SD rats, once a day for8weeks, can create an experimental rat model of OSF. This model was of good repeatability and can be conveniently made. But there were still some defects in it.2,Curcumin can improve the mouth opening degree of the model rats, decreases the expression of TGF-β1, and prevent the further development of fibrosis.3. Curcumin play an anti-fibrosis role in vitro by inhibitting MFB proliferation, inducing apoptosis and decreasing the generation of collagen type Ⅰ and Ⅲ.