| Purpose:In this study,animal experiments were conducted to observe the effects of Yitang kang,a traditional Chinese medicine compound,on the body weight、blood glucose and pathological changes of the retinal tissue structure and function of in DR rats.Mean while,the expression of key proteins in the TLR4/MyD88/NF-κB signaling pathway and NLRP3/ASC/Pro-Caspase-1 inflammasome pathway were detected.Observe the effect of medicated serum on the morphology and cell viability of HMEC-1 cells induced by high glucose through cell experiments,and detect the expression of key proteins in the TLR4 / MyD88 /nf-κB signaling pathway and NLRP3/ASC/pro-Caspase-1 inflammasome pathway.Analyses the effect of traditional Chinese medicine compound Yitan gkang to DR,and discusses its treatment mechanism.This study is committed to provide reliable experimental basis and to find new therapeutic targets for clinical therapy of DR.Material and method:Paper one:After adaptive feeding,80 SPF male SD rats were divided into normal control group(20rats)and high-fat group(60 rats)by random number table method based on body weight.The rats in the normal control group were fed with ordinary feed,and the rats in the high-fat group were fed with high-fat feed.After 8 weeks,fasting for 12 hours,intraperitoneal injection of streptozotocin(STZ)was used to construct a DM rat model.After the establishment of the DM model(blood glucose concentration ≥ 16.7mmol/L),the surviving rats were randomly divided into the model control group(16 rats),the Yitangkang group(17 rats),and the metformin group(17 rats)according to the blood glucose level of the rats.Gavage was started on the second day after the model was successfully established.The normal control group and the model control group were given an equal volume of 0.9% saline.The Yitangkang group was given 9.45ml/kg/d Yitangkang decoction and the calcium hydroxybenzene sulfonate group was given 135mg/kg/d calcium hydroxybenzene sulfonate solution,gavage once daily,for 12 weeks.During this period,the body weight and blood glucose were measured every 3weeks,until the end of the experiment,the rats were sacrificed and the samples were taken for testing.The pathological changes of the retina of each group of rats were studied through three aspects: EB penetration experiment,retinal ultrastructure,and retinal tissue HE staining,so as to explore the preventive and therapeutic effects of Yitangkang on the retina and blood-retinal barrier of diabetic DR rats.Paper two:The western-blot method was used to detect the expression levels of TLR4,Myd88,NF-κB,NLRP3,pro-Caspase-1,and Caspase-1 protein in the retinal tissues of each group of rats,and the Elisa method was used to detect the expression of inflammatory factors IL-1βand IL-18 in the serum.Immunohistochemistry method was used to detect NF-κB and pro-Caspase-1 proteins in retinal tissue sections of DR rats to observe the expression degree of positive staining and analyze the average optical density value of the reaction color intensity.Immunofluorescence method was used to detect expression degree of NLRP3 protein and Caspase-1 protein on retinal slices in each group.Paper three:Twenty SPF male SD rats were divided into blank group(10 rats)and Yitangkang group(10 rats)by random number table method.Rats in the Yitangkang group were gavaged twice daily,for 7 days using 4 concentration dose of normal experimental animals;the normal control group was given 20ml/kg body weight with normal saline.And 1h after the last administration,the rats were anesthetized and blood was collected to prepare medicated serum.1h after the last administration,the rats were anesthetized and blood was collected to prepare medicated serum.HMEC-1 cells were divided into 5 groups: blank group,model group,Yitangkang group,NF-κB inhibitor group,and pro-Caspase-1 inhibitor group.The corresponding medicated serum and inhibitor were used for intervention.Use an inverted microscope to observe cell morphology changes,CCK-8 method to detect cell viability,western-blot method to detect the expression levels of TLR4,Myd88,NF-κB,NLRP3,pro-Caspase-1,and Caspase-1 proteins in cells,and Elisa method to detect The expression level of IL-1β and IL-18,immunofluorescence method was used to detect the expression of NF-κB and pro-Caspase-1,so as to determine whether the traditional Chinese medicine compound Yitangkang passed the TLR4/MyD88/NF-κB signaling pathway and NLRP3/ASC/pro-Caspase-1 inflammasome pathway protects HMEC-1 cells induced by high glucose.Results:Paper one:1.Yitangkang,a traditional Chinese medicine compound,has the function of maintaining weight and blood glucose stability in DM rats,and alleviates the symptoms of water intake increased,food intake increased,urine output increased and weight loss.2.Before drug intervention,compared with the normal control group,the weight of the rats in the high-fat group increased significantly(P<0.01).After drug intervention,the body weight of rats in the normal control group still showed a gradual increase in 12 weeks,while the body weight of rats in the model control group,Yitangkang group,and calcium hydroxybenzene sulfonate group showed a downward trend.In the 3th week of drug intervention,compared with the model control group,the weight of rats in the Yitangkang group increased significantly,and the downward trend appeared to be significantly slower.The difference was statistically significant(P <0.05).In the 6th,9th and 12 th week,the weight of the rats in the model control group decreased more significantly than that in the Yitangkang group,while the weight of the rats in the Yitangkang group gradually stabilized,and the difference was statistically significant(P < 0.01).3.Before drug intervention,compared with the normal control group,the fasting blood glucose level of the model control group,Yitangkang group,and calcium hydroxybenzene sulfonate group increased significantly(P < 0.01);at the third week of drug intervention,compared with the model control group the fasting blood glucose of the Yitangkang group decreased significantly,and the difference was statistically significant(P < 0.01).At the 6,9,and 12 weeks of intervention,the blood glucose of the Yitangkang group gradually stabilized.4.Compared with the normal control group,the average value of standardized EB penetration in the model control group,Yitangkang group,and calcium hydroxybenzene sulfonate group increased,and the difference was statistically significant(P < 0.01).Compared with the model control group,the Yitangkang group and the calcium hydroxybenzene sulfonate group showed a significant reduction in the average value of standardized EB permeation,the difference was statistically significant(P<0.01,P<0.01),but compared with the calcium hydroxybenzene sulfonate group and the Yitangkang group,there was no significant difference between the groups(P > 0.05).5.According to the results of HE staining of rat retinal tissue,it is found that the retinal tissue of DR rats has undergone visible pathological changes.The ganglion cells in the retinal tissue of the model control group are reduced and arranged disorderly;the inner and outer nuclear layer cells are arranged disorderly;The nerve fiber layer and the ganglion cell layer have dilated capillaries,thickened the lumen,and thinned the retina.Some red blood cells can be seen to leak into the tissues outside the blood vessels.Compared with the model control group,the pathological changes of each layer of the retina in the Yitangkang group were lighter,the cell nucleus arrangement was more regular,and the retinal edema was lighter.6.Transmission electron microscopy(sem)results showed that compared with the normal control group the retinal endothelial cells of the model control group showed obvious pathological changes,such as cell swelling,finger-like protrusions,decreased tight junctions,thickened basement membrane.Compared with the model control group,the Yitangkang group had significantly reduced pathological changes.Paper two:1.Western-blot test results: Compared with the normal control group,the protein expression levels of TLR4,MyD88,NF-κB,NLRP3,pro-Caspase-1,and Caspase-1 in the retina tissue of the model control group increased significantly(P<0.01);Compared with the model control group,the protein levels of TLR4,MyD88,NF-κB,NLRP3,pro-Caspase-1,and Caspase-1 in the Yitangkang group and the calcium hydroxybenzene sulfonate group were significantly reduced(P<0.01,P<0.01),and there was no difference between two groups(P > 0.05).2.Results of immunohistochemistry: by labeling NF-κB and pro-Caspase-1 proteins in retinal tissue sections of DR rats to observe the expression degree of positive staining and analyze the average optical density value of the reaction color intensity.The results indicate that :compared with the normal control group,the brown-yellow staining was widely expressed in the retina slices of the model control group,and the positive staining expression increased,the difference was statistically significant(P < 0.01);compared with the model control group,the staining of Yitangkang group and calcium hydroxybenzene sulfonate group were relatively shallow,and the positive staining expression were less,the differences were statistically significant(P < 0.01).3.Immunofluorescence test results:(1)After fluorescent staining of the NLRP3 protein on the retina slices of each group,compared with the normal control group,the rat retina slices of the model control group showed obvious green fluorescence-like expression,indicating that the expression of NLRP3 was significantly increased.After statistical analysis of positive cell count,the difference was statistically significant(P <0.01).Compared with the model control group,the expression of green fluorescence in the retina slices of rats in the Yitangkang group and the calcium hydroxybenzene sulfonate group was significantly reduced.After statistical analysis of positive cell counts,the difference was statistically significant(P < 0.01).(2)After fluorescence staining of Caspase-1 protein on retinal slices in each group,compared with the normal control group,the rat retinal slices in the model control group showed obvious green fluorescence-like expression.After statistical analysis of positive cell count,the difference was statistically significant(P <0.01).Compared with the model control group,the expression of green fluorescence in the retina slices of rats in the Yitangkang group and the calcium hydroxybenzene sulfonate group was significantly reduced.After statistical analysis of the positive cell count,the difference was statistically significant(P < 0.01).Paper three:1.The CCK-8 method was used to detect the cell viability of HMEC-1.The results showed that compared with the blank group,the cell viability of the model group and the Yitangkang group were significantly reduced(P < 0.01).Compared with the model group,the cell activity of the Yitangkang group was significantly higher(P < 0.01).2.Western-blot method to detect the expression of TLR4,MyD88,NF-κB protein in HMEC-1 cellsThe results indicated that compared with the blank group,the expression of TLR4,MyD88,and NF-κB protein in the HMEC-1 cells of the model group were significantly increased(P<0.01);compared with the model group,the Yitangkang group significantly reduced TLR4,MyD88,NF-κB protein expression(P<0.01).Comparing the NF-κB inhibitor group with the model group,there was no significant difference in TLR4,MyD88 protein expression(P(29)0.05),and NF-κB protein expression was significantly reduced(P<0.01);compared with Yitangkang group,TLR4,MyD88 protein The expression was significantly increased(P<0.01),and the expression of NF-κB protein was significantly reduced,which was statistically significant(P<0.01).Compared with the blank group,model group,and Yitangkang group,the pro-Caspase-1 inhibitor group showed a significant increase in TLR4,MyD88,and NF-κB protein expression(P<0.01).3.Western-blot method to detect the expression of NLRP3,pro-Caspase-1,Caspase-1protein in HMEC-1 cellsThe results indicate that compared with the blank group,the expression of NLRP3,pro-Caspase-1,and Caspase-1 protein in HMEC-1 cells of the model group increased significantly(P<0.01);compared with the model group,the Yitangkang group significantly reduced NLRP3,Pro-Caspase-1,Caspase-1 protein expression(P < 0.01).Compared with the model group and the Yitangkang group,the expression of NLRP3,pro-Caspase-1 and Caspase-1 in the NF-κB inhibitor group were significantly reduced(P<0.01).Compared with the Yitangkang group,the NF-κB inhibitor The inhibitory effect of the group on downstream factors was more significant than that of the Yitangkang group.Compared with the model group,the pro-Caspase-1 inhibitor group showed no significant difference in the expression of NLRP3 protein(P(29)0.05),while the expression of pro-Caspase-1 and Caspase-1 proteins were significantly reduced(P <0.01);Compared with the Yitangkang group,the expression of NLRP3 protein in the pro-Caspase-1 inhibitor group was significantly increased(P <0.01),and the expression of pro-Caspase-1 and Caspase-1 proteins were significantly decreased(P<0.01),and pro-Caspase-1 was inhibited The inhibitory effect of the drug on downstream factors was more significant than that of the Yitangkang group.4.Immunofluorescence detection results:(1)NF-κB: Compared with the normal group,the expression level of NF-κB in the model group was significantly increased,showing a broad red fluorescence distribution,and the difference was statistically significant(P <0.01).Compared with the model group,the NF-κB expression level of the Yitangkang group and the NF-κB inhibitor group decreased,and the red fluorescence distribution decreased,and the difference was statistically significant(P <0.01).Compared with the Yitangkang group,the NF-κB expression level in the NF-κB inhibitor group decreased more significantly,and the difference was statistically significant(P <0.01).The pro-Caspase-1 inhibitor group had a lower ability to inhibit its upstream factor NF-κB,and there was no statistical difference compared with the model group(P(29)0.05).(2)pro-Caspase-1: Compared with the normal group,the expression level of ro-Caspase-1 in the model group was significantly increased,showing a broad red fluorescence distribution,and the difference was statistically significant(P <0.01).Compared with the model group,the expression level of pro-Caspase-1 in the Yitangkang group,the NF-κB inhibitor group,and the pro-Caspase-1 inhibitor group decreased and the red fluorescence distribution decreased.The difference was statistically significant(P < 0.01).Compared with the Yitangkang group,the expression levels of pro-Caspase-1 in the NF-κB inhibitor group and the pro-Caspase-1 inhibitor group decreased more significantly,and the difference was statistically significant(P < 0.01)Conclusion:1.The traditional Chinese medicine compound Yitangkang can improve the blood-retinal barrier function of diabetic retinopathy rats,reduce the damage of blood-retinal barrier,and delay the pathological changes of the retina tissue of diabetic retinopathy rats.2.The traditional Chinese medicine compound Yitangkang may exert anti-inflammatory and anti-pyrolysis effects by inhibiting TLR4/Myd88/NF-κB signaling pathway and NL RP3/ASC/pro-Caspase-1 inflammasome pathway.3.The traditional Chinese medicine compound Yitangkang may protect HMEC-1 cells by regulating the TLR4/ MyD88/ NF-κB signaling pathway and the NLRP3/ASC/pro-Caspase-1inflammasome pathway.4.As possible targets,NF-κB and pro-Caspase-1 play an important regulatory role in protecting HMEC-1 cells induced by high glucose. |
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