Study On The Mechanism Of Inflammatory Apoptosis Induced By High Glucose In Human Retinal Endothelial Cells

BackgroundDiabetic retinopathy(DR)is one of the most common microvascular complications of diabetes mellitus(DM).Retinal endothelial cells(RECs)which are important part of the blood-retinal barrier play many important functions in the retina.In the early stages of DR,RECs were dysfunctional and promoted the progression of DR.Studies have found that the inflammatory apoptosis changes in RECs play an indispensable role in the development of the disease.Under the stimulation of immune inflammatory response,cells undergo a series of changes including morphological changes,causing the expression of interleukin IL-1,IL-6,IL-8 and TNF and other inflammatory factors.The activation of the signal transduction pathway plays a biological role.Along with the severity of the disease worsens,inflammatory response of the retinal endothelial cells aggravate and eventually apoptosis occurs.The p53 signaling pathway and NF-?B signaling pathway are the two most important pathways for inducing apoptosis and inflammation.P53 is one of the important tumor suppressor genes in the body,which can lead to cell cycle arrest,induce apoptosis,and play an important role in inhibiting the occurrence of various tumors and tissue damage in the body.P53 transcribed protein molecules can play a potential role through the interaction of target genes P21,MDM2,and Bax related to apoptosis.It can induce the expression of pro-apoptotic genes such as Bax and PUMA,and can also inhibit the resistance.The expression of the degenerating gene Bcl-2 directly or indirectly leads to depolarization of the mitochondrial membrane and participates in the process of apoptosis.P53 can also be transferred to mitochondria,leading to depolarization of the mitochondrial membrane,release the pro-apoptotic factors and induction apoptosis.The P53 gene plays an important role in DNA repairing,cell cycle arrest,inhibition of angiogenesis,and promotion of non-programmed apoptosis.Our previous experiments have confirmed that the expression of pro-apoptotic proteins Bax and Cl-caspase-3 is increased in retinal endothelial cells cultured in high glucose while the expression of anti-apoptotic protein Bcl-2 is decreasing.Is this change induced by p53 regulation as the trend of apoptosis increases? We are waiting for our next experiment to confirm further.At present,many studies have confirmed that p53 has an important correlation with the occurrence and development of diabetes and its complications,but whether P53 has a synergistic effect with some signaling pathways or plays a separate role requires further study.NF-?B is a multi-directional function-regulating transcription factor whose main function is to regulate the expression of a variety of genes,including inflammatory response genes,virus-related genes and proto-oncogenes.NF-?B can be activated by a variety of factors,causing a general response to inflammatory factors such as IL-8,IL-6 and TNF-?.Our previous experiments have confirmed that high glucose-induced expression of IL-1,IL-6,IL-8 and tumor necrosis factor TNF-? in retinal endothelial cells increases with the increasing glucose concentration.And whether it further activates NF-?B and plays a key role in transcriptional regulation remains to be confirmed.Whether p53 and NF-?B signaling pathways play a collaboration effect in controlling the inflammatory and apoptotic processes of related diseases have attracted many researchers.The current research show that it exhibits mutual inhibition during the development of many cancer cells,but it shows synergy in the study of diabetes-related diseases.There is no similar report on the relationship between the two mechanisms of apoptosis in diabetic retinopathy.Based on this,inspired by the above research,this paper uses high concentration of glucose as an inflammatory factor to effectively simulate the occurrence and development of retinopathy.To provide a basis for subsequent research,we mainly detected the morphological changes,the function of the mitochondrial membrane,the apoptotic rate of RECs,and the intracellular inflammatory factors and the expression of apoptosis-related proteins.the first part was determined the cellular inflammatory effects and apoptosis,the second part attempts to explore the mechanism of apoptosis in retinal endothelial cells.And the role of the P53 signaling pathway and the cellular classical loop NF-?B in the process would be explored in order to give some inspiration to relevant research.ObjectiveTo explore the changes of inflammatory and apoptotic effects of human retinal endothelial cells induced by high glucose,paving the way for the study of inflammatory apoptosis mechanism.To study the expression mechanism and relationship of p53 and NF-?B cell pathway proteins in the apoptosis death manner of human retinal endothelial cell.Materials and MethodsPart one: Analysis of the effect of high glucose induced apoptosis of human retinal endothelial cellsHuman retinal endothelial cells were cultured in vitro for the log-growing period,and the research groups were set as the Control group(Control,5.5 mmol/L),the high-concentration glucose group(HG,30 mmol/L),and the mannitol as the hypertonic control group(Mannitol,24.4 mmol/L).In some experiments,different concentrations of glucose(15mmol/L,30mmol/L,60mmol/L,90mmol/L)were set according to different research contents.The medium of the above different components was added to the 6-well plate and cultured,and then detected.The specific test items were as follows: HE staining was used to detect the morphological changes of each group;transmission electron microscopy was used to detect the changes of cell organelles in each group;LDH method was used to detect cell membrane damage;DNA ploidy method was used to detect cell cycle;flow cytometry was used to detect apoptosis in each group;Western-blot method was to detect the expression of Bax,Bcl-2 and Cl-caspase-3.The expressions of IL-1,IL-6,IL-8 and tumor necrosis factor TNF-? were detected by ELISA.Finally do statistical analysis.Part two: Mechanism of inflammatory apoptosis induced by high glucose in human retinal retinal endothelial cellsThe experimental cells were the same as the first part.The cells were grouped as follows: normal control group(Control,5.5 mmol/L),high-concentration glucose group(HG,30 mmol/L),high-concentration glucose + p53 inhibitor group(HG+pifithrin-?),and the p53 inhibitor group(pifithrin-?).The specific test items were as follows: morphological changes of cells were observed by transmission electron microscopy;Bax,Bcl-2 and Cl-caspase-3 protein concentrations were detected by Western-blot;P53,p-p53,p65,p-p65,I?B,p-I?B protein concentration were detected by western-blot;Immunofluorescence colocalization and expression of P53 and NF-?B p65 protein in retinal endothelial cells by immunofluorescence assay.The mRNA transcription levels of p53 and p65 proteins were detected by q-PCR.Finally do statistical analysis.ResultsPart one: Analysis of the effect of high glucose induced apoptosis of human retinal endothelial cells1.HE staining analysis: When the cells were cultured for 48 hours,significant morphological differences were observed under the light microscope.The normal control cells showed normal growth state,the cell shape was uniform,the mitosis was active,the order of magnitude was more,and the cell-to-cell fusion was better.With the prolonged culture time in high concentration glucose solution,it can be observed that the overall density of retinal vascular endothelial cells is low.We can see the clearance around the cells,and a large number of vacuoles and granules gradually appear in the cells.It could be observed significantly morphology changes.2.Transmission electron microscopy analysis: The cells in the high-concentration glucose group showed obvious bulging phenomenon.The mitochondria and Golgi were swollen in the cells,the mitochondria volume increased,and lipid precipitation was observed around the retinal endothelial cells.It could be observed the phenomenon of lipid precipitation accumulation,obvious vacuolization inside the cell,accompanied by a large number of primary lysosomes,secondary lysosomes and even phagosomes.3.LDH detection: LDH leakage rate of cells increased significantly after high glucose culture,suggesting cell membrane damage,the above changes in a concentration-dependent manner and time-dependent manner.4.DNA ploidy assay for cell cycle: After the high concentration of glucose solution is fully effective,the cell cycle arrest effect is obvious,mainly staying in the G2 phase,and the formation of DNA fragments is significantly increased,and a large number of apoptotic bodies are formed.5.Flow cytometry to detect apoptosis: High glucose effectively induced retinal endothelial cell apoptosis,the proportion of normal cells decreased significantly,the form of cell death was mainly apoptosis,the proportion of apoptotic cells increased significantly in the high glucose group.6.Western-blot results: The expression of Bax and caspase-3 proteins in the high-concentration glucose group increased significantly,and the inhibitory protein Bcl-2 was decreased.The results were significantly different.7.ELISA results: As the increase of glucose concentration,the expressions of IL-1,IL-6,IL-8 and tumor necrosis factor TNF-? increased in different degrees,and the difference was statistically significant.Part two: Mechanism of inflammatory apoptosis induced by high glucose in human retinal retinal endothelial cells1.Transmission electron microscopy: Retinal endothelial cells under high glucose culture showed obvious apoptotic morphological changes: vacuolation in mitochondria,chromatin condensation and degranulation of endoplasmic reticulum.The above changes were significantly alleviated in the high glucose + inhibitor group;2.Western-blot detection of apoptosis-related protein expression: After pifithrin-? inhibited the expression of p53 protein,the expression of pro-apoptotic proteins Bax and Cl-caspase-3 were decreased,and the expression of apoptosis-inhibiting protein Bcl-2 was increased.3.Western-blot detection of p53 and NF-?B pathway protein: in high glucose culture of RECs cells,p53,p-p53,p65,p-p65,I?B,p-I?B expression levels increased.When pifithrin-? inhibited p53 protein expression,p53 expression was significantly decreased,p-p53/p53 ratio was significantly increased,and NF-?B pathway protein p65 and I?B expression levels were significantly decreased,p-p65/p65 and p-I?B/I?B ratio was significantly reduced,the above results p<0.05,the difference was statistically significant.4.Immunofluorescence detection of p53 and NF-?B p65 protein expression co-localization: In the high glucose group,fluorescence signal of p53 and p65 showed strong positive,significantly enhanced compared with the normal control group.While in the high glucose + pifithrin-? group,the fluorescence signal of p53 and p65 decreased.The fluorescence signal of p53 protein was significantly decreased,and p65 fluorescence signal was partly decreased and still could be observed in the nucleus and cytoplasm.5.q-PCR results: The mRNA expression of p53 and p65 in high glucose group was significantly increased,P<0.05,the difference was statistically significant.The expression level of high glucose+pifithrin-? group was significantly lower,P<0.05,difference has statistical significane.ConclusionsHigh-concentration glucose solution can cause substantial damage to retinal endothelial cells,leading to its morphological changes in the early stage.High concentration of glucose activates retinal endothelial cell apoptosis pathway and inflammatory response pathway,leading to increased apoptosis rate and increased the expression of inflammatory factors.P53 signaling pathway is involved in the apoptosis process of retinal endothelial cells cultured in high glucose.High glucose can promote the activity of p53 protein and NF-?B pathway,promote cell inflammatory reaction and apoptosis;p53 inhibitor can inhibit p53 signaling pathway and NF-?B signaling pathway,which may play a synergistic role in the process of inflammatory apoptosis of retinal endothelial cells induced by high glucose.