| Background:Interstitial lung diseases(ILDs)are a group of progressively deteriorating diseases,which seriously threaten the health all around the world.Because their etiology and pathogenesis are not completely clear,there are no specific drugs at present.The mortality rate is even higher than that of many malignant tumors,causing a serious social and economic burden.ILDs can be classified into those with known and unknown causes.Among them,the known causes of ILD may be more than 200 kinds,including genetics,tumors,drugs,treatments,and disease-related factors,and abnormal glucose metabolism is one of the important causes.Diabetes is a chronic and progressive metabolic disorder,and its incidence continues to increase year by year.According to the International Diabetes Federation,the number of diabetic patients worldwide will reach 600 million in 2035.Diabetes complications have always been a research hotspot and focus of diagnosis and treatment in scholars.Diabetes can induce a series of systemic pathological changes,increase the mortality and disability rate of diabetic patients,and has attracted wide attention from clinicians and patients.But people pay little attention to diabetic lung injury,especially the essence of diabetes complicated by lung injury.Due to the abundant reserves of the pulmonary capillary network,the respiratory system symptoms of diabetes-related ILD are not obvious at the beginning of the onset of diabetes,and it is easy to be confused with the manifestations of cardiovascular and cerebrovascular complications,and easily ignored by clinicians and patients.Therefore,once diabetes-related ILD are founded,symptoms are generally severe and progress rapidly,which seriously threatens the survival of diabetic patients.The preliminary work of our group confirmed that diabetes-related lung injury mainly manifested as pulmonary interstitial lesions,including inflammatory cell infiltration,increased collagen content,widening of alveolar septum and tissue fibrosis,etc.,which is similar to bleomycin-induced pulmonary fibrosis rat model.We expounded the relationship between diabetes and lung inflammation and fibrosis for the first time,and provided laboratory evidence that the lung is an important target organ for diabetes.However,the pathogenesis of diabetes-related lung injury has not yet been fully clarified,which involves inflammation,oxidative stress,and fibrosis.With the development of genomics and proteomics,we have a deep understanding of miRNAs.miRNAs are a type of small non-coding RNA(nc RNA)with a length of about 21-25 nucleotides,which cannot directly participate in protein synthesis.miRNAs have a post-transcriptional regulatory function and can regulate cell proliferation,differentiation and apoptosis as well.It is found that the expression of miR-21 is increased in diabetic model.Inhibiting the expression of miR-21 can reduce the complication of diabetic heart and kidney,which suggests that miR-21 plays an important role in the pathogenesis of diseases related to abnormal glucose metabolism.It is reported that miR-21 can target and regulate the expression of PDCD4.While PDCD4 can participate in the occurrence and development of various diseases through the JNK signaling pathway.However,there is no related report on the regulation of PDCD4/JNK signaling pathway by miR-21 in diabetes-related lung injury.Through to Jnk1 gene knockout technology and use of JNK inhibitors,this project intends to study the role of PDCD4/JNK signaling pathway in diabetic lung injury in diabetic mice.Meanwhile,we used miR-21 antagomir to intervene model mice in order to observe the effects of miR-21 inhibition on diabetic lung injury,and further to explore how miR-21 promotes inflammation and fibrosis and related signal pathways,which may provide a theoretical basis for the treatment of diabetes-related ILD in the future.Methods:1.Observing the effects of inhibiting JNK expression and activation on lung injury and miR-21 expression in diabetic mice by applying JNK inhibitors:we established a diabetes model with wild-type C57BL/6J mice,which conducted with small dose of continuous intraperitoneal injections of streptozotocin followed by JNK inhibitor(SP600125)for intervention.We used RT-PCR and Western Blot to detect the expression of inflammatory factors,pro-fibrosis factors,oxidative stress factors,antioxidant factors,miR-21,PDCD4,JNK,and p-JNK in the lung tissues of mice,in order to explore the effect of JNK inhibition on the expression of miR-21 in diabetic lung injury mice.2.Observing the effects of downregulating JNK expression on lung injury and miR-21 expression in diabetic mice by applying Jnk1 knockout mice: we selected Jnk1 knockout mice and diabetes model mice derived from wild-type C57BL/6J.Then used RT-PCR and Western Blot to detect the expression of inflammatory factors,pro-fibrosis factors,oxidative stress factors,and antioxidant factors,miR-21,PDCD4,JNK,p-JNK,in order to observe the effect of Jnk1 knockout on miR-21 expression in diabetic lung injury mice.3.Exploring the effect of miR-21 on lung injury in diabetic mice and its regulation on PDCD4/JNK signaling pathway by inhibiting miR-21 : we established a diabetes model with wild-type C57BL/6J mice,which conducted with miR-21 antagomir to inhibit the expression of miR-21.We used RT-PCR and Western Blot to detect the expression of miR-21,PDCD4,JNK,and p-JNK,in order to observe the expression of PDCD4 and JNK downstream of miR-21.Results:1.SP600125 can significantly down-regulate the inflammation and fibrosis indicators in the lung tissue of diabetic mice.At the same time,the expression of miR-21 in the lung tissue of diabetic mice is significantly increased.The intervention of SP600125 can further increase the expression of miR-21.2.The expression of inflammatory factors(TNF-? and IL-6)and pro-fibrosis factors(TGF-?1 and PAI-1)in the diabetes group of Jnk1 knockout mice was significantly lower than those in wild-type mice.At the same time,the expression of JNK and p-JNK was reduced,which was contrary to PDCD4 expression.Therefore,reducing the expression of JNK can reduce the degree of inflammation and fibrosis in the diabetic lung,while PDCD4 has a negative regulatory effect on the activation of JNK.3.Compared with the control group,the level of TNF-? and TGF-?1 in the lung tissue of the DM group,which decreased after the intervention of miR-21 antagomir,was significantly higher.At the same time,the expression level of miR-21 in the lung tissue of the DM group increased,while the expression level of PDCD4 decreased,which increased after antagomir intervention.Therefore,miR-21 may also have a negative regulatory effect on the expression of PDCD4 in the lung tissue of diabetic mice.Conclusions:1.The main pathological manifestations of lung injury in diabetic mice are inflammation and fibrosis.The inhibition of JNK signaling pathway or knocking out Jnk1 can reduce the degree of lung inflammation and fibrosis induced by diabetes to a certain extent.2.The expression of miR-21 is positively correlated with the level of diabetic lung inflammation and fibrosis in diabetic mice,and inhibiting the expression of miR-21 can reduce the degree of lung inflammation and fibrosis induced by diabetes.3.The increased expression of miR-21 may down-regulate PDCD4 followed by phosphorylation of JNK,leading to the occurrence and development of diabetic pulmonary inflammation and fibrosis(relevant signaling pathways need to be further verified by cell experiments).Therefore,inhibiting the expression of miR-21 may become a new target for the treatment of diabetes-related lung injury.Innovation:1.Animal experiments with signaling pathway related gene(Jnk1)knockout mice combined with JNK inhibitor(SP600125)have confirmed the effect of JNK activation on diabetic lung injury,making the results more reliable.2.We used miR-21 targeted inhibitors to intervene mice,confirmed the correlation between miR-21 and PDCD4/JNK signaling pathway,and further clarified the mechanism of miR-21's promotion of diabetic lung injury,providing new ideas for the treatment of diabetes-related ILD. |
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