| Objective: By observing the retinal thickness and vascular density(VD)changes in patients with type 2 diabetes mellitus(DM)without clinically visible retinopathy(DR0 stage),the possibility of early damage to the retinal neurovascular unit(NVU)in DM patients without clinically visible retinopathy was identified.The "Luo Bing Luo Zhi" theory was used as a guide to investigate the effect of Dahuang Zhechong capsules on non-proliferative diabetic retinopathy(NPR)based on the pro NGF-p75 NTR signaling axis.The protective effect and mechanism of Dahuang Zhechong capsules on NVU in the retina of rats with non-proliferative diabetic retinopathy(NPDR)was investigated based on the pro NGF-p75 NTR signaling axis.Methods:Clinical observation: A case-control study was set up to quantitatively analyse and compare the mean thickness of the peripapillary retinal nerve fiber layer(p RNFL),retinal thickness and vascular density(VD)in the macula on a 3 × 3 mm scan in healthy subjects with type 2 DM using optical coherence tomography(OCT)and optical coherence tomography angiography(OCTA)techniques.The correlation between the thickness of the retinal layers and the VD was statistically analysed;the possible correlates of retinal damage in early DM and those associated with the course of DM were screened by regression analysis,and ROC curves were plotted to determine the joint diagnostic indicators of retinal damage in DR-free diabetic patients and those associated with the course of DM.Experimental study: Sixty 7-8 week olds SPF grade Wistar male rats were selected and randomly divided into blank group,model group,and Dahuang Zhechong capsule low,medium and high dose groups.All groups,except the blank group,were induced with STZ intraperitoneal injection into the diabetic retinopathy(DR)rat model,and the Dahuang Zhechong capsules were gavaged with the corresponding concentration of Dahuang Zhechong capsules for 8 weeks after successful DR modelling.The relative expression of serum pro NGF,p75 NTR,NGF and Trk A were measured by ELISA;the relative expression of pro NGF,sortilin,p75 NTR,NGF,Trk A and PI3 K m RNA in rat retinal tissues were measured by RT-PCR;the relative expression of retinal m RNA was measured by Western Blot.The relative expression of sortilin,p75 NTR,NGF and PI3 K in the rat retina was measured by Western Blot;PAS staining of paraffin sections and immunohistochemical staining of sortilin were also performed.Results:Clinical observations:(1)Statistical analysis of the differences showed that the mean thickness of patients in the p RNFL thickness index in all four subdivisions,TI,NS,N and NI,was significantly lower in the DM group than in normal subjects(P<0.05);the mean thickness of each retinal layer in the macula showed that the NFL-inferior,NFL-central,GCL-superior,GCL-inferior and ONL-nasal in the DM patients group significantly lower than the control group(P<0.05),and no significant differences were observed in IPL,INL and OPL thicknesses.(2)Correlation analysis showed that age,duration of DM disease and some of the p RNFL thickness,macular-NFL thickness,macular-GCL thickness,macular-ONL thickness and VD showed a negative correlation trend;however,there was a positive correlation trend between the duration of DM disease and the thickness of some of the macular INL;VD of the macular area showed a moderate typical correlation trend with NFL,GCL,INL and ONL thickness,and positive correlation trend between the central thickness of NFL and the thickness of two subdivisions of SVC-VD and DVC-VD;positive correlation trend between the thickness of two subdivisions of GCL-nasal and GCL-central of GCL and DVC-VD;positive correlation trend between the thickness of INL-nasal subdivision of INL and DVC-VD of VD,while the two subdivisions of INL-temporal and INL-inferior of INL The thickness of INL-temporal and INL-inferior subdivisions of INL showed a negative correlation with the DVC-VD of VD;the thickness of ONL-superior and ONL-inferior subdivisions of ONL showed a positive correlation with the DVC-VD and DVC-AC of VD,while the thickness of ONL-temporal subdivisions of ONL showed a negative correlation with the DVC-VD and DVC-AC of VD.p RNFL thickness showed a positive correlation with the VD of macular area.The trend of correlation was positive between p RNFL thickness and VD of macula.(3)The prediction model of retinal damage in DR-free diabetic patients was developed by logistic regression analysis according to whether they had DM or not,and the final multivariate regression analysis screened out three variables,p RNFL-TI,p RNFL-TS and GCL-superior thickness,with AUCs of 0.742,0.668 and 0.668,respectively.these three indicators were included in the combined diagnostic test.The AUC of the combined diagnostic prediction could reach 0.843.(4)The development of the retinal damage prediction model related to the duration of DM disease,according to the duration of DM disease within 5years and more than 5 years,after logistic regression analysis,the model included 3indicators,DVC-VD,p RNFL-N thickness and ONL-nasal thickness,and the AUCs were0.776,0.880,and 0.899.Combining the two indicators for diagnosis resulted in an AUC of0.983.Experimental studies:(1)ELISA: Compared with the blank group,serum pro NGF and p75 NTR expression were significantly increased(P<0.05)and NGF and Trk A expression were significantly decreased(P<0.05)in the model group;compared with the model group,Dahuang Zhechong capsule medium and high dose groups could significantly decrease serum pro NGF,p75 NTR Compared with the model group,both the medium and high dose groups of Dahuang Zhechong capsules significantly reduced serum pro NGF and p75 NTR levels(P<0.05),while the low,medium and high dose groups of Dahuang Zhechong capsules significantly increased serum NGF levels(P<0.05),and the medium dose group significantly increased serum Trk A levels(P<0.05);compared with the low dose group of Dahuang Zhechong capsules,the medium dose group significantly reduced serum pro NGF and p75 NTR levels were significantly lowered in the medium-dose group compared with the low-dose group of Dahuang Zhechong capsules(P<0.05),and serum NGF levels were significantly increased in both the medium and high-dose groups(P<0.05);serum p75 NTR levels were significantly lowered in the medium-dose group compared with the high-dose group of Dahuang Zhechong capsules(P<0.05).(2)RT-PCR: compared with the blank group,the expression of pro NGF,sortilin and p75 NTR m RNA in the retinal tissues of rats in the model group were significantly increased(P<0.05),and the expression of NGF,Trk A and PI3 K m RNA were significantly decreased(P<0.05);compared with the model group,the medium and high dose groups of Dahuang Zhechong capsules significantly decreased the level of p75 NTR in the retinal tissues of rats(P<0.05).Compared with the model group,the medium and high dose groups of Dahuang Zhechong capsules significantly reduced the level of pro NGF m RNA in retinal tissues(P<0.05),and the low,medium and high dose groups significantly reduced the level of sortilin m RNA in retinal tissues(P<0.05),while the medium dose group significantly increased the level of NGF and PI3 K m RNA in retinal tissues(P<0.05).Compared with the low-dose group of Dahuang Zhechong capsules,both the medium and high-dose groups could significantly reduce the level of pro NGF m RNA in retinal tissues(P<0.05),and both the medium-dose groups could significantly increase the level of PI3 K m RNA in retinal tissues(P<0.05).(3)Western Blot: compared with the blank group,the expression of sortilin and p75 NTR in the retinal tissues of rats in the model group was significantly increased(P<0.05),and the expression of NGF and PI3 K in the model group was significantly decreased(P<0.05);compared with the model group,the medium and high doses of Dahuang Zhechong capsule significantly decreased the expression of sortilin and p75 NTR in the retinal tissues of rats in the model group.Compared with the model group,both the medium and high dose groups of Dahuang Zhechong capsules significantly reduced retinal sortilin and p75 NTR protein levels(P<0.05),and both the medium and high dose groups significantly increased NGF protein levels(P<0.05),and the medium dose group significantly increased PI3 K protein levels in retinal tissues(P<0.05);compared with the low dose group of Dahuang Zhechong capsules,the medium dose group significantly reduced retinal tissue p75 NTR protein levels(P<0.05).Compared with the low-dose group of Dahuang Zhechong capsules,the medium-dose group significantly decreased the level of p75 NTR protein in retinal tissues(P<0.05),and the medium-dose group significantly increased the level of NGF protein in retinal tissues(P<0.05).(4)Immunohistochemical staining: a small amount of sortilin protein was expressed in normal rat retinal tissues;sortilin was strongly positively expressed in the ONL and GCL of the model group;the expression of sortilin protein in the Dahuang Zhechong capsule group was between the blank group and the model group,and the expression of sortilin protein in the GCL was weakest in the medium-dose group.The expression of sortilin protein in the GCL was the weakest in the middle dose group.(5)PAS staining: The retinal structure of the blank group was neat,uniform and tightly arranged;the cells in each layer of the model group were sparse and the interlayer arrangement was not tight;compared with the model group,the retinal layers of the Dahuang Zhechong capsule group were more closely arranged,among which the retinal structure of the Dahuang Zhechong capsule medium-dose group was clearer and the cells were more closely arranged.Conclusions:(1)The retinal NVU of DM patients without retinopathy may have been damaged and the nerve and glial damage may have occurred before the microvascular damage.(2)Dahuang Zhechong capsule can activate blood circulation and attack blood stasis by regulating the expression of pro NGF-p75 NTR axis,and it can repair the retinal NVU damage in DR. |
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