Mechanism Of Curcumin Regulating Autophagy Through JNK Signaling Pathway In Prevention And Treatment Of Diabetic Retinopathy

Background:Diabetic retinopathy(DR)is one of the complications caused by microvascular changes in patients with diabetes mellitus(DM).DR is the main cause of blindness among working age people in the world.The pathogenesis of DR is very complex and not completely clear,involving hyperglycemia,hyperlipidemia,inflammatory response,oxidative stress and so on.The treatment of DR adopts different methods according to different stages of the disease.It mainly includes retinal laser photocoagulation,intraocular injection of anti vascular endothelial growth factor drugs,and vitrectomy.It has always been a hot and difficult issue to find research on the pathogenesis of DR and new treatment methods.China has accumulated rich experience in using Chinese herbal medicine to prevent and treat diseases.In the theory of traditional Chinese medicine,curcuma longa has the functions of promoting blood circulation and relieving pain.Domestic and foreign research results show that curcumin,the main component of curcuma longa,has many biological activities,including anti-inflammatory,antioxidant and promoting autophagy.It is considered as an effective natural compound for treating diabetic complications.JNK is a member of mitogen activated protein kinase family,which is involved in the regulation of inflammation,apoptosis,autophagy and other processes.Its activity in diabetic animal models is significantly higher,and may be a therapeutic target for diabetes and its complications.However,whether curcumin plays a therapeutic role on DR by regulating JNK signaling pathway and its mechanism have not been fully clarified.Purpose:To observe the regulation of curcumin on JNK signaling pathway,autophagy,inflammation and other activities in the retinas of C57 diabetic mice and human retinal endothelial cell(HREC)cultured with high glucose in vitro.To explore the protective effect and mechanism of curcumin on DR.Method:1.Protective effect of curcumin on diabetic retinopathy in mice by JNK-mediated autophagy.Wild-type C57 male mice aged 6-8 weeks were randomly selected to establish type 1 diabetic mouse model by multiple abdominal injection of Streptozotocin(STZ),and the control group was given multiple abdominal injection of equal volume of sodium citrate buffer.Type 1 diabetic mice were given curcumin or sodium carboxymethyl cellulose at a dose of 200 mg/kg/d by intragastric administration.Blood glucose and body weight were measured every two weeks,and mice were killed at 12 weeks.The body weight and blood glucose of the mice were measured before death.Then,the eyeball was removed and total RNA was extracted from the mouse retinal tissue.The mRNA levels of JNK,Beclin-1,LC3,ICAM-1 and TNF-α were detected by RT-q PCR.Paraffin sections were made from the eyeballs of mice,and the pathological changes of retina were observed by HE staining,and the activation of JNK protein in retina was detected by immunohistochemical staining.2.The fuction of JNK signaling and autophagy in HREC injury induced by high glucose cultureThe negative control small interfering RNA(siRNA)and JNK siRNA were transfected into HREC cultured with high glucose in vitro.The mRNA and protein expressions of Beclin-1,LC3,ICAM-1 and TNF-α were detected by RT-q PCR and Western blot.The effect of JNK siRNA on cell migration was observed by wound-healing test.The changes of JNK siRNA on cell permeability were observed by monolayer cell permeability experiment.3.Protective effect of curcumin on HREC in high glucose culture by regulating autophagy through JNK pathwayHREC was pretreated with curcumin at different concentrations and cultured with high glucose.MTS was used to detect the cell activity of each group.The mRNA and protein expressions of Beclin-1,LC3,ICAM-1 and TNF-α were detected by RT-q PCR and Western blot.The effect of curcumin on HREC migration in vitro was observed by wound-healing test.The effect of curcumin on HREC permeability was observed by monolayer cell permeability experiment.Result:1.After multiple injections of STZ at low doses in C57 mice,fasting blood glucose met the diagnostic criteria for diabetes,and the model of type 1 diabetic mice was successfully established.HE staining showed vacuolar changes in the retinal nerve fiber layer in diabetic mice,and the retinal structure was chaotic.Curcumin can alleviate the retinal pathological damage in diabetic mice.Immunohistochemical staining results showed that p-JNK protein was diffused in all retinal layers of mice,and the expression of p-JNK in all retinal layers of mice in DM group was significantly increased.Ganglion cell layer(GCL),inner plexiform layer(IPL)and outer plexiform layer(OPL)are the main layers.RT-q PCR results showed that the level of JNK,autophagy related proteins Beclin-1,LC3,inflammatory factors ICAM-1,TNF-α mRNA were increased in the retina of DM mice.Curcumin can reduce the mRNA levels of JNK and inflammatory factors,and further increase the mRNA levels of autophagy related proteins.These results suggest that JNK signaling pathway plays a role in diabetic retinal injury,and curcumin can control JNK activation for therapeutic effect.2.JNK mRNA level and p-JNK protein expression level were significantly increased in HREC cultured with high glucose.High glucose can cause abnormal increase of HREC migration ability and enhanced cell permeability.After transfection with JNK siRNA,the expression level of JNK was significantly inhibited.Meanwhile,the mRNA and protein levels of Beclin-1 and LC3 were significantly increased,while the expressions of ICAM-1 and TNF-α were significantly decreased.After transfection with JNK siRNA,the cell migration ability and permeability of monolayer cells were significantly reduced,and the function of HREC was restored.3.In HREC cultured with high glucose,the cells were pretreated with curcumin at different concentrations,MTS test showed that the activity of HREC in high glucose cultured group decreased.After the cells were pretreated with 10μM curcumin,the cell activity recovered and was closest to the level of the control group.This concentration was used for subsequent experiments.After pretreatment of HREC with curcumin,the expression level of JNK was inhibited,the mRNA and protein levels of Beclin-1 and LC3 were significantly increased.The expression of ICAM-1 and TNF-α were decreased significantly.At the same time,the application of curcumin can effectively control the abnormal increase of HREC migration ability and monolayer cell permeability caused by high glucose,and the function of HREC can be restored.Conclusion:1.In the DM mouse model,curcumin can inhibit the activation of the retinal JNK pathway,enhances the autophagy level,controls the inflammatory response,and alleviates the retinopathological damage caused by diabetes,thus playing a role in the prevention and treatment of diabetic retinopathy.2.In HREC cultured with high glucose,the activation of JNK was enhanced,which aggravated the cellular inflammatory response and affected the permeability and migration of HREC.3.Curcumin or JNK siRNA transfection treatment of HREC cultured in vitro with high glucose can inhibit JNK signaling pathway,improve autophagy level,reduce inflammatory response,and reduce abnormal cell permeability and migration ability caused by high glucose,thus protecting HREC cultured in vitro with high glucose.