Profective Effects Of Ginkgo Biloba Extract On Bleomycin Induced Skin Fibrosis In Mice And Its Mechanism

Background and objectives Systemic sclerosis(SSc)is an autoimmune disease which typically results in tissue fibrosis of the skin as well as the internal organs.Skin fibrosis is the typical symptom of SSc,which causes significant cosmetic disabilities and irreversible structural and functional impairment.The annoying skin fibrosis of SSc can progress over the years and lead to significant mental health issuses.Although the etiology and pathogenesis of skin fibrosis remains unclear,the mechanism of the disease is believed to the activation of fibroblasts and fibroblasts-to-myofibroblast differentiation,which leads to the excessive accumulation of collagen and other extracellular matrix(ECM)components in skin tissues.It has been eluciated that epithelial-mesenchymal transition(EMT)is an important source of these mesenchymal cells including fibroblasts and myofibroblasts.There are a number of profibrotic cytokines implicated in skin fibrosis,but TGF-β1 is one of the most potent stimuli for cell proliferation and collagen synthesis.There are many previous reports which demonstrated that TGF-β1/Smad pathway activation is critical in the process of skin fibrosis and EMT.Ginkgo biloba extract(GbE)is an extract from leaves of the G.biloba tree,which contains 24% ginkgo flavonoids and 6% terpenelactones.It has been proved that GbE was able to exert anti-oxidant,anti-inflammatory,immune regulation and other activities.In recent years,GbE has shown anti-fibrosis characteristics in animal experiments and clinical studies.The aim of this study was to identify the effect of Ginkgo biloba extract(GbE)on skin fibrosis in bleomycin(BLM)-induced mouse model of SSc,exploring the relationship between the antifibrotic properties of Ginkgo biloba extract and oxidative stress and EMT.Meanwhile,the regulation of TGF-β1/Smad signaling pathway by Ginkgo biloba extract were also studied.Methods A total of 100 female BALB/c mice were randomly divided into five groups with twenty animals each: normal control group,skin fibrosis model group(BLM),GbE low dose group(BLM+GbE-L,50 mg/kg/d),GbE intermediate dose group(BLM+GbE-M,100 mg/kg/d),GbE high dose group(BLM+GbE-H,200 mg/kg/d).BLM was dissolved in phosphate buffered saline(PBS)to target dose(500 μg/ml).GbE was provided by Schwabe Pharmaceuticals(Karlsruhe,Germany)and dissolved in distilled water.The mice in the BLM,BLM+GbE-L,BLM+GbE-M and BLM+GbE-H groups received subcutaneously injection of BLM(100μl)and the mice in the control group received an equivalent volume of PBS.The mice in the BLM+GbE-L,BLM+GbE-M and BLM+GbE-H groups received an indicated dose of GbE by oral gavage 2 h after the BLM injection.The mice in the control and BLM groups were given an equivalent volume of distilled water.The above operations were performed once a day for 4 weeks.At the end of the treatment,all animals were killed by cervical dislocation.Skin specimens at the injection site were isolated for later analysis.(1)The general conditions of mice in each group,including mental state,activity,hair growth,diet,weight changes,etc were observed.The pathological changes of skin tissue at the injection site of each group of mice were examined by haematoxylin-eosin(HE)and Masson staining.Dermal thickness,which was defined as the linear distance between the dermal-epidermal junction and the dermal-subcutaneous fat junction,was measured in the HE sections.We calculated the collagen volume fraction(CVF)in the Masson sections and analyzed hydroxyproline content in the skin tissue samples.The mRNA levels of type Ⅰcollagen and type Ⅲ collagen in the skin from experimental mice of different groups were calculated by qRT-PCR.Type I collagen and type Ⅲ collagen protein expression levels were assessed by western blotting and analyzed by densitometry.(2)The MDA content,8-isoprostaglandin F2α(8-iso-PGF2α),and Glutathione peroxidise(GPx)activity in the skin tissue sample of different groups were measured by kits accordingto the manufacturer’s protocols.(3)The mRNA levels of E-cadherin,Vimentin andα-smooth muscle actin(α-SMA)in the skin tissue of mice of different groups were calculated by qRT-PCR.The protein expression levels of E-cadherin,Vimentin andα-SMA in the skin tissue were examined by immunohistochemical staining and western blotting.(4)qRT-PCR method was used to detect the mRNA levels of TGF-β1,Smad7,and Smad3 in skin tissue of mice in each group,and the protein expression levels of aboved mentioned were detected by western blotting and analyzed by densitometry.Results(1)Effects of GbE on skin fibrosis in BLM-induced scleroderma mice:Four weeks after the PBS injection,the mice from control group were normal in mental state,appetite,activity,hair growth and body weight.In comparison,after the the subcutaneous injections of BLM for 4 weeks,the mice showed the skin elasticity decreased,the skin thickness and hardness increased,the hair of skin at the injection site were difficult to growth.Compared with the control normal mice,there was a significant reduction in body weight of the mice in the model group(P<0.05).The weight change of the mice in the BLM+GbE-L group was similar to that of the model group.Compared with the model group,the mice in the medium-dose and high-dose of GbE groups had significantly increased in body weight(P<0.05).Histological changes in the skin tisssue were examined by H&E staining and Masson staining.The results showed that PBS-treated mice(control group)did not develop either sclerosis or fibrosis.The skin tissue structure was normal and no obvious inflammatory cell infiltration and collagen deposition,whereas,subcutaneous injections of BLM for 4 weeks induced marked dermal sclerosis in mice of model group.The skin structure was disordered,as well as massive inflammatory cells infiltration,vascular walls thickening,collagen deposition and subcutaneous fat loss.Dermal thickness showed a significant increase in BLM-treated skin as compared with that in PBS-treated skin after 4 weeks(P<0.05).Compared with the model group,the medium-dose and high-dose of GbE markedly attenuated the histological changes induced by BLM and exhibited a significantly reduction in dermal thickness and collagen volume fraction(P<0.05).In the sclerotic skin induced by injections of BLM for 4 weeks,the content of hydroxyproline was significantly increased as compared with that in PBS-treated mice(P<0.05),while both the intermediate and high doses of GbE effectively reduced the content of hydroxyproline in BLM-stimulated mouse skin(P<0.05).The mRNA and protein expressions of type Ⅰ collagen and type Ⅲ collagen in skin tissue were also significantly elevated after exposure to BLM(P<0.05).The treatment with either intermediate dose or high dose of GbE markedly downregulated the mRNA and protein expressions of type Ⅰ collagen as well as type Ⅲ collagen(P<0.05).(2)Effects of GbE on skin oxidative stress in BLM-induced mice:The levels of MDA,8-iso-PGF2α and GPx activity in skin tissue sample of different groups as oxidative stress indicators were measured by kits according to the manufacturer’s protocols.The BLM exposure increased the content of MDA and8-iso-PGF2α and decreased GPx activity both dramatically(P<0.05),which indicate the imbalance of the oxidative and anti-oxidative states in the skin.GbE increased the GPx activity and decreased the MDA and 8-iso-PGF2α content in skin tissues.Particularly,the intermediate dose and high dose of GbE were more effective than low dose of GbE(P<0.05).(3)Effects of GbE on epithelial-mesenchymal transition in BLM-induced mice:The immunohistochemical staining and western blotting showed that the expressions of E-cadherin in the skin tissue was significantly decreased after BLM exposure(P<0.05).After treatment with intermediate and high doses of GbE,E-cadherin expression in the skin tissue was increased(P<0.05).In contrast,the expressions of Vimentin and α-SMA in the skin tissue were markedly increased after BLM exposure(P<0.05).Intermediate and high doses of GbE effectively reduced the expressions of Vimentin and α-SMA in BLM-stimulated mouse skin(P<0.05).The low dose of GbE had no effect on the expression of Vimentin and α-SMA as well as E-cadherin inBLM-stimulated mouse skin.The qRT-PCR results demonstrated that the mRNA expressions of E-cadherin in the skin tissue was significantly decreased after BLM exposure(P<0.05).After treatment with intermediate and high doses of GbE,the mRNA expression of E-cadherin in the skin tissue was increased(P<0.05).In contrast,the mRNA expressions of Vimentin and α-SMA in the skin tissue were markedly increased after BLM exposure(P<0.05).Intermediate and high doses of GbE effectively reduced the mRNA expressions of Vimentin and α-SMA in BLM-stimulated mouse skin(P<0.05).The low dose of GbE had no effect on the mRNA expression of Vimentin and α-SMA as well as E-cadherin in BLM-stimulated mouse skin.(4)Effects of GbE on TGF-β1/Smad signaling pathway:The qRT-PCR and western blotting demonstrated that the mRNA and protein expressions of TGF-β1 and Smad3 in the skin tissue were markedly increased after BLM exposure compared with those in PBS-treated mice(P<0.05).The high dose of GbE effectively reduced the mRNA and protein expressions of TGF-β1 in BLM-stimulated mouse skin(P<0.05).However,GbE had no effect on the mRNA and protein expression of Smad3 in BLM-stimulated mouse skin.The qRT-PCR and western blotting demonstrated that the mRNA and protein expressions of Smad7 in the skin tissue were markedly reduced after BLM exposure compared with those in PBS-treated mice(P<0.05).The high dose of GbE effectively elevated the mRNA and protein expressions of Smad7 in BLM-stimulated mouse skin(P<0.05).In addition,we examined the expressions of p-Smad3 in the skin tissue of different groups by western blotting.The results showed that the protein expressions of p-Smad3 in the skin tissue were markedly increased after BLM exposure compared with those in PBS-treated mice(P<0.05).The high dose of GbE effectively reduced the protein expressions of p-Smad3 in BLM-stimulated mouse skin(P<0.05).Conclusions(1)In this study,we have successfully established a mouse model of skin fibrosis by repeated local injections of bleomycin(BLM).Histologically,daily injection of BLM caused marked dermal sclerosis in mice with significant increased in dermal thickness,thickened and homogenous collagen bundles,thickening of vascular walls,and inflammatory infiltrates,which well mimicked the histological features of patients with clinical skin fibrosis.Additionally,the expressions of type Ⅰ collagen and type Ⅲcollagen were significantly increased.The present study demonstrated that BLM can induce oxidative stress and epithelial-mesenchymal transition(EMT)during the development of skin fibrosis.(2)Ginkgo biloba extract alleviated skin fibrosis induced by BLM via the attenuation of oxidative stress and it is related to the reduction of MDA and8-iso-PGF2α and the elevation of GPx activity.(3)GbE down-regulated the expressions of TGF-β1 and p-Smad3 and up-regulated the expression of Smad7.Our results demonstrated that GbE alleviated skin fibrosis by suppressing EMT,which is related to the inhibition of TGF-β1/Smad signaling pathway.