Protective Effect And Mechanisms Of Linagliptin On Pulmonary Fibrosis Associated With Systemic Sclerosis

BackgroundSystemic sclerosis（SSc）,a rare autoimmune-mediated connective tissue disease often causes progressive skin and organ fibrosis.Pulmonary fibrosis is the most common and leading cause of SSc mortality.Glucocorticoid and immunosuppressor remain the major treatment.However,the therapeutic regimen for SSc-related pulmonary fibrosis is limited.Therefore,development of novel therapeutic approaches is an urgent need to improve the clinical benefit and quality of life of patients with SSc-related pulmonary fibrosis.Dipeptidyl peptidase 4（DPP4）,a serine protease,is a type II transmembrane glycoprotein,wihch inactivates incretin hormones such as neuropeptide,chemokine,glucagon-like peptides,and glucose-dependent insulinotropic peptide.DPP4 inhibitor has been widely used for the treatment of type 2 diabetes because of its activity against DPP4.DPP4 has also been found to be involved in fibrotic diseases.Previous investigations have demonstrated its anti-fibrotic property in various animal models of fibrosis,but the relevant mechanism has not been fully clarified.ObjectiveTo investigate the effects of linagliptin,a highly specific DPP4 inhibitor,on pulmonary fibrosis in SSc mouse model and potential underlying mechanisms.Part I The establishment of mouse model of pulmonary fibrosis associated with systemic sclerosisMethodsThirty female C57BL/6 mice were randomly divided into 5 groups with 6 mice in each group: control group,1.25mg/kg group,2.5mg/kg group,5mg/kg and 10mg/kg group.In the control group,0.1m L phosphate buffer saline（PBS）was injected subcutaneously into the central area of the back of mice with sterile injection needle,and the other four groups were injected with bleomycin（BLM）at the corresponding dose diluted with PBS,once a day for 4 weeks.The general status,weight and survival of mice in each group were recorded.At the end of the experiment,the back skin and left lung tissue were collected and stained with HE and Masson.And the pulmonary fibrosis was further evaluated by Ashcroft score.Results1.With the increase of BLM dosage,the general status of mice and weight loss became worse gradually.And the mortality was also gradually increased.2.HE staining of skin showed that there were no significant structural changes in skin epidermis,dermis,fat layer and muscle layer in control group.In 1.25mg/kg group,the dermis was slightly thickened,the collagen fibers were slightly thicker.The skin dermis and collagen fibers began to thicken significantly in 2.5mg/kg group.With the further increase of BLM dosage,the above changes became further aggravated,and the subcutaneous fat layer became thinner or even disappeared.3.Masson staining of skin showed that a small amount of collagen fibers could be seen in the control group.With the increase of BLM dosage,collagen fibers began to increase.When the dose reached 2.5mg/kg,collagen fibers increased significantly compared with the control group.With the further increase of dose,collagen fibers further increased.4.HE staining of lungs showed that with the increase of BLM dosage,the destruction and thickening of alveolar septum gradually increased,and the range of pulmonary fibrosis gradually increased.When the dose reached 2.5mg/kg,local fibrosis consolidation were observed.At the BLM dose of 5mg/kg and 10mg/kg,extensive lung consolidation appeared.When the BLM dose reached at least 2.5mg/kg,the Ashcroft scores were different significantly between the control and other groups.5.Masson staining showed that the degree of collagen deposition was positively correlated with the dose of bleomycin.When the dose of bleomycin was 5mg/kg and10mg/kg,a large amount of blue collagen fibers could be seen in the lung tissue.Both dosages can successfully induce obvious pulmonary fibrosis.Part II Protective effect of linagliptin on pulmonary fibrosis in systemic sclerosis miand its preliminary mechanismMethodsThirty-two 6-8 weeks female C57BL/6 mice,weighing 20-22 g,were randomly divided into four groups（n=8 each）: control,BLM,BLM+linagliptin and BLM+rapamycin.The mice in the control group were subcutaneously injected with 0.1m L PBS with a 4.5-gauge needle.The mice in the other groups were injected with 0.1m L of BLM solution（1 mg/m L）daily for 4 weeks.After the first week of injections,the mice in the control,BLM+linagliptin and BLM+rapamycin groups were orally administrated daily with PBS,linagliptin（10 mg/kg）and rapamycin（1.5 mg/kg）,respectively.The general status,weight and death of mice in each group were recorded.At the end of the experiments,Hydroxyproline,superoxide dismutase（SOD）,3,4-methylenedioxyamphetamine（MDA）in lung tissues and tumor necrosis factor（TNF）-and interleukin（IL）-6 in bronchoalveolar lavage fluid（BALF）were measured.HE and Masson staining were performed on lung tissues.And the pulmonary fibrosis was further evaluated by Ashcroft score.Results1.The BLM-treated mice showed a significant body weight loss and worse general status compared with the control mice,both of which were reversed by linagliptin or rapamycin.2.HE staining of lung sections showed that subcutaneous injection of BLM resulted in destruction of normal lung structures and marked thickening of alveolar septa.Most alveolar space disappeared,and replaced by large fibrous foci.The Ashcroft score in BLM group was significantly higher than that in control group.These alterations were reversed by linagliptin or rapamycin.3.Masson’s trichrome staining showed a significant increase in collagen deposition in the lungs of BLM-treated mice.However,both linagliptin and rapamycin significantly alleviated these pathological changes and reduced collagen production.Consistently,linagliptin or rapamycin treatment reduced the BLM-induced increased levels of hydroxyproline,though still higher than that in control group.4.BLM administration induced increased levels of TNF-α and IL-6 in the BALF,and that these levels were reduced by linagliptin or rapamycin treatments.5.The MDA content was increased and the activity of SOD was significantly decreased in the lungs of BLM-treated mice.In contrast,linagliptin or rapamycin treatment remarkably suppressed the BLM-induced alterations in SOD and MDA.Part III Linagliptin ameliorates pulmonary fibrosis in systemic sclerosis mouse model via inhibition of endothelial to mesenchymal transitionMethods1.In vitro experiment: CCK-8 kits were used to determine the appropriate concentrations of BLM,linagliptin and rapamycin.The human umbilical vein endothelial cells（HUVECs）were seeded in 6-well plates and treated as follows: control, BLM（2 μ g/m L）,BLM（2 μ g/m L）+linagliptin（100 n M）and BLM（2 μ g/m L）+rapamycin（200 n M）.After 48 h of treatment,the cells were harvested for further analysis.The expression of marker genes,proteins and transcription factors related to endothelial mesenchymal transformation（End MT）in different groups were detected by western blotting,RT-q PCR and immunofluorescence staining.Changes in cell morphology were also observed and photographed.Cell migration ability in different groups were evaluated by scratch test and cell migration test.2.In vivo experiment: The grouping and treatment of in vivo experiment are the same as that in part II.The changes of End MT marker proteins and transcription factors in different groups of mice were detected by tissue immunofluorescence double staining,immunohistochemistry and western blotting.The expression of Akt,P-Akt,m TOR and P-m TOR in different groups were also detected.Results1.When the concentrations of BLM,linagliptin and rapamycin reached 4μg/ml,200 n M and 400 n M,respectively,obvious influences on the viability of HUVECs were observed.2.BLM treatment for 48 h induced HUVECs to transform from a cobblestone-like shape to an elongated,spindle shape.Immunofluorescence staining showed that the cytoskeleton was reconstructed and typical fibrous structure appeared.These changes were attenuated by linagliptin or rapamycin treatment.3.Western blotting and RT-qPCR results revealed that BLM induced decreased endothelial markers（CD31 and VE-Cadherin）and increased levels of mesenchymal marker（α-SMA and Collagen I）in HUVECs at the molecular and gene levels.End MT related transcription factors,such as Snail,Slug and Twist,were also up-regulated.However,these alterations were suppressed by linagliptin or rapamycin treatments.Immunofluorescence analysis further confirmed these results.4.BLM treatment significantly enhanced the migration ability of HUVECs,which was attenuated by linagliptin or rapamycin treatment.5.More CD31/α-SMA double-positive cells localized at the intima of the capillary vessels were observed in BLM group compared with the control group.Immunohistochemistry analysis also confirmed the results.Western blotting results showed upregulation of total VE-Cadherin and donwregulation of collagen I increased in BLM-treated mice.Expression of Slug in lungs was also increased.All of the alterations were inhibited by linagliptin or rapamycin treatment.6.Western blotting analysis revealed that BLM significantly upregulated P-Akt and P-m TOR levels in SSc mice,which were reversed by linagliptin or rapamycin treatment.Conclusions1.Subcutaneous injection of BLM at a dose of 5mg/kg can successfully induce the mouse model of systemic sclerosis,accompanied by obvious pulmonary fibrosis with a relatively low mortality.2.Linagliptin significantly alleviated pulmonary fibrosis in SSc mouse model,which may be attributed to its inhibitory effects on inflammation and oxidative stress.3.Linagliptin ameliorated pulmonary fibrosis in SSc mouse model via inhibition of Mdn MT,by inhibiting Akt/m TOR signaling pathway.