Clinical Features Of Diabetic Retinopathy Accompanied By Nephropathy And The Protective Effect Of Bushen Huoxue Prescription On The Retina And Kidney Of Db/db Mice Based On Pyroptosis

Objective:1.To summarize the TCM evidence patterns,evidence distribution patterns and common core etiology of Diabetic Retinopathy(DR)and Diabetic Nephropathy(DN)based on the literature.2.To visualize and analyze the research literature related to diabetes and cellular scorching based on the bibliometric approach.3.To observe and analyze the distribution of peripheral blood inflammatory indicators and optical coherence tomography(OCT)imaging biomarkers in patients with and without DN,in order to find clinical biomarkers that can predict the development of DR and DN.4.To observe whether the Bushen Huoxue prescription protects the retina and kidney tissues of db/db mice by regulating the expression of key proteins of pyroptosis and its related pro-inflammatory factors,thus delaying the progression of DR and DN,and to observe the difference in efficacy between the New Bushen Huoxue prescription and the Original prescription for the prevention and treatment of DR and DN.Methods:1.The research literature on TCM syndrome types and syndrome elements of DR And DN published in CNKI database,VIP Chinese Journal,Wanfang Medical Network and Chinese Traditional Medicine literature retrieval platform from January 2010 to April 2022 was searched,and the database was established by using Excel software in WPS office.The frequency and frequency of TCM syndrome type and disease location syndrome element were counted.2.Based on the web of science core collection database,this study comprehensively searched the literature in the field of diabetes and pyroptosis published from January 1985 to August 2022.The published research literature in this field was included,and the number of citations of the included literature,publication year,publication journal,author,research institution,country,Visual analysis was performed on research topics and other aspects.3.A total of 169 DR Patients with complete clinical data who were admitted to the Department of Ophthalmology,Affiliated Hospital of Chengdu University of Traditional Chinese Medicine from October 2020 to June 2022 were collected.DR Patients were divided into DR Group and DR/DN group according to whether they were complicated with DN,and then divided into NPDR group,PDR group,NPDR/DN group and PDR/DN group according to DR Stage.The distribution characteristics of peripheral blood inflammatory indexes(NLR and PLR),renal function indexes(CYS-C,Crea,UA and UACR)and OCT imaging indexes(HRF,DRIL,ORTs,CRT,RNFL and GCL)were observed and compared between the above groups.4.Ten18-week-old db/m mice were randomly set as the blank group(db/m),and 50 18-week-old db/db mice were randomly divided into model group(db/db),Yuanfang group(YF),Xinfang high-dose group(GJL),Xinfang medium-dose group(ZJL)and Xinfang low-dose group(DJL).After 8 weeks of intervention,the mice were sacrificed and the eyeball,retina,kidney and blood were collected for examination.The pathological changes of retina and kidney were observed by HE staining,trypsin vascular digestion and Masson staining.Immunofluorescence and Luminex multi-factor were used to detect the expression of pyroptosis key proteins NLRP3,GSDMD and pro-inflammatory factors IL-1β,IL-17 A,IL-6,TNF-α,VEGF-A,ANG-2,MCP-1,ICAM-1 in the retina,kidney and serum.Results:(1)A total of 320 articles were included,including 109 articles related to DR And211 articles related to DN,involving a total of 31652 patients,including 11108 patients with DR And 20544 patients with DN.After the normalization,24 TCM syndrome types of DR Were obtained,among which the top five high-frequency syndrome types were deficiency of Yin of both liver and kidney,deficiency of Qi-Yin,Yin deficiency and Heat dryness,deficiency of both Yin and Yang and blood stasis syndrome.There were 5 syndrome factors of disease location,the top three were kidney,liver and spleen,and 9 syndrome factors of disease nature,the top four were Yin deficiency,Qi deficiency,blood stasis and Yang deficiency.There were 62 TCM syndrome types of DN,among which the top five high-frequency syndrome types were deficiency of Qi-Yin,blood stasis syndrome,deficiency of Qi-Yin and blood stasis,deficiency of both Yin and Yang.There were 5syndrome factors of disease location,the top three were kidney,spleen and liver,and 13 syndrome factors of disease nature,the top four were Qi deficiency,Yin deficiency,blood stasis and Yang deficiency.2.A total of 139 articles related to diabetes and pyroptosis from2011 to 2022 were retrieved.The total citation frequency was 3009 times,and the highest citation frequency of a single article was 255 times.Among the 20 publishing countries,China ranked first with 100 articles.Among the 222 published scientific research institutions,Harbin Medical University ranked first in the number of publications and influence institutions with 18 articles and 184 citations.Among the 980 authors,Chen,X from China ranked the first among the high-impact authors with 5 articles published and a total of 29 citations.Among the 98 journals,"CELL DEATH DISEASE" was cited 29 times in total,with4 articles published,and ranked first in the number of publications and high-impact journals.Among the 349 keywords,the cumulative frequency of "pyroptosis" was 65.In addition,the topic cluster analysis of literature in this field was divided into three categories: diabetes and pyroptosis,diabetes chronic complications and pyroptosis,diabetes combined with other diseases and pyroptosis.Since 2019,the number of publications related to diabetes and its chronic complications has increased rapidly.3.(1)General data: there were no significant differences in gender,family history of diabetes,duration of diabetes and BMI between DR Group and DR/DN group(P>0.05).The average age of DR/DN group was significantly lower than that of DR Group(P<0.05),and the proportion of hypertension in DR/DN group was significantly higher than that of DR Group(P<0.05).In subgroup analysis,there were no significant differences in gender,family history of diabetes,duration of diabetes,history of hypertension and BMI among the 4 groups(P>0.05),but the average age was significantly decreased in the trend of NPDR > PDR > NPDR/DN > PDR/DN(P<0.05).(2)There was no significant difference in Hb A1 c between the DR Group and the DR/DN group(P>0.05).HGB was significantly higher in the DR Group than in the DR/DN group(P<0.05),and NLR,PLR,Crea,UA and CYS-C were significantly higher in the DR/DN group than in the DR Group(P<0.05).Subgroup analysis showed that there were no significant differences in Hb A1 c,NLR,and UA among the four groups(P>0.05),while HGB decreased in the order of NPDR >PDR > NPDR/DN > PDR/DN(P<0.05).PLR,Crea and CYS-C increased significantly according to the trend of NPDR < PDR < NPDR/DN < PDR/DN(P<0.05).(3)OCT imaging indicators: There was no significant difference in the positive rate of HRF,DRIL,ORTs and CRT between the DR Group and the DR/DN group(P>0.05).The RNFL and GCL thickness in the DR/DN group were significantly lower than those in the DR Group(P<0.05).The positive rate of HRF increased in the order of NPDR < NPDR/DN < PDR/DN < PDR(P<0.05),and the positive rate of DRIL increased in the order of NPDR < NPDR/DN < PDR< PDR/DN(P<0.05).The GCL thickness decreased in the order of NPDR > PDR >NPDR/DN > PDR/DN(P<0.05).(4)Influencing factors: History of hypertension(OR=2.759),NLR(OR=1.316),PLR(OR=1.009),Crea(OR=1.018),UA(OR=1.004)and CYS-C(OR=3.742)were independent risk factors for DR/DN.Age(OR=0.951),HGB(OR=0.976),RNFL(OR=0.909)and GCL(OR=0.945)were independent protective factors for DR/DN.Subgroup analysis showed that CYS-C(OR=5.781),HRF(OR=1.322)and DRIL(OR=1.307)were independent risk factors for PDR,and CYS-C(OR=6.227)was an independent risk factor for NPDR/DN.CYS-C(OR=5.272)and UACR(OR=1.001)were independent risk factors for PDR/DN.RNFL(OR=0.899)and GCL(OR=0.935)were independent protective factors for NPDR/DN,and RNFL(OR=0.852)and GCL(OR=0.928)were independent protective factors for PDR/DN.(5)ROC curve analysis showed that the area under the curve(AUC)of CYS-C,PLR,Crea,UA and the combination of the four indicators for predicting DR/DN were 0.717,0.625,0.647,0.616 and 0.717,respectively.The AUC of CYS-C,Crea,UACR and the combination of the three indicators for predicting PDR/DN were 0.787,0.683,0.783 and 0.795,respectively.The AUC of GCL for predicting DR/DN,NPDR/DN and PDR/DN was 0.642,0.605 and 0.642,respectively,and the AUC of RNFL for predicting PDR/DN was 0.636.4.(4)Animal study(1)The changes of fasting blood glucose and body weight: from 18 weeks old,the body weight and fasting blood glucose of db/db mice were significantly higher than those of db/m mice(P<0.05).After 8 weeks of treatment,neither Bushen Huoxue Yuan Decoction nor Xinfang treatment significantly improved hyperglycemia and obesity(P>0.05).(2)Retinal pathomorphological changes: There was no obvious abnormality in retinal structure and morphology in db/m group.Compared with db/m group,the retinal ganglion cells and inner nuclear layer cells were significantly reduced and lost,the cells of inner nuclear layer and outer plexiform layer were disordered,telangiectasia and microthrombi appeared in each layer of retina,and the thickness of retinal GCL was significantly reduced in db/db group(P<0.05).Compared with db/db group,the above retinal pathological changes were improved in YF,GJL,ZJL and DJL groups,and the GCL thickness was significantly increased in ZJL group(P<0.05).(3)Retinal trypsin digestion: retinal capillary network in db/m group was normal.Compared with db/m group,the retinal capillary arrangement was disordered,distorted,endothelial cell proliferation,pericyte reduction,E/P value and the number of acellular vessels were significantly increased in db/db group(P<0.05).Compared with db/db group,the pathological changes of retinal capillaries in YF,GJL,ZJL and DJL groups were alleviated,the number of cell-free vessels was significantly decreased(P<0.05),and the E/P value in YF and ZJL groups was significantly decreased(P<0.05).(4)Retinal immunofluorescence: compared with db/m group,the expression levels of NLRP3,GSDMD and VEGF-A in db/db group were significantly increased(P<0.05).Compared with db/db group,the expression levels of NLRP3,GSDMD and VEGF-A in YF,GJL,ZJL and DJL groups were significantly decreased(P< 0.05).(5)Compared with db/m group,the expression levels of IL-1β,IL-17 A,IL-6,TNF-α,ANG-2,VEGF-A,MCP-1 and ICAM-1 in db/db group were significantly increased(P<0.05).Compared with db/db group,the expression levels of IL-17 A,TNF-α and ANG-2 in YF group were significantly decreased(P<0.05),and the expression levels of IL-17 A,TNF-α and MCP-1 in GJL group were significantly decreased(P<0.05).The expression levels of IL-1β,IL-17 A,IL-6,TNF-α,ANG-2,VEGF-A and ICAM-1 were significantly decreased in ZJL group(P<0.05),and the expression levels of IL-1β,IL-17 A and TNF-αwere significantly decreased in DJL group(P<0.05).(6)Renal pathomorphological changes: there were no obvious abnormalities in glomeruli,tubules and renal interstitium in db/m group.Compared with db/m group,the renal pathological changes in db/db group showed glomerular hypertrophy,cystic cavity stenosis and cyst wall thickening,glomerular mesangium and basement membrane thickening,renal tubular dilatation,degeneration and necrosis of renal tubular epithelial cells,inflammatory cell infiltration and fibrous tissue proliferation in renal interstitium,and significant increase in renal tubulointerstitial fibrosis area(P<0.05).Compared with db/db group,the above pathological changes were improved in YF,GJL,ZJL and DJL groups,and the area of renal tubulointerstitial fibrosis was significantly reduced in ZJL and DJL groups(P<0.05).(7)Renal immunofluorescence: compared with db/m group,the expression levels of NLRP3,GSDMD and VEGF-A in db/db group were significantly increased(P<0.05).Compared with db/db group,the expression levels of GSDMD in YF,GJL,ZJL and DJL groups were significantly decreased(P<0.05),the expression levels of NLRP3 in GJL and ZJL groups were significantly decreased(P<0.05),and the expression level of VEGF-A in ZJL group was significantly decreased(P<0.05).(8)Compared with db/m group,the expressions of IL-1β,IL-17 A,IL-6,TNF-α,VEGF-A,ANG-2,MCP-1 and ICAM-1 in db/db group were significantly increased(P<0.05).Compared with db/db group,the expression levels of IL-1β,IL-17 A,IL-6,TNF-α,VEGF-A,ANG-2,MCP-1 and ICAM-1 were significantly decreased in YF,ZJL and DJL groups(P<0.05).The expression levels of IL-1β,IL-17 A,IL-6,TNF-α,VEGF-A and ANG-2 in GJL group were significantly lower than those in control group(P<0.05).(9)Compared with db/m group,the expression of IL-1β,IL-17 A,IL-6,TNF-α,VEGF-A,ANG-2,MCP-1 and ICAM-1 in db/db group were significantly increased(P< 0.05).Compared with db/db group,the expression levels of IL-1β,IL-17 A,IL-6,TNF-α,VEGF-A,ANG-2 and MCP-1 in YF,ZJL and DJL groups were significantly decreased(P<0.05),and the expression level of ICAM-1 in ZJL group was significantly decreased(P<0.05).The expression levels of IL-1β,IL-17 A,IL-6,TNF-α,VEGF-A and ANG-2 in GJL group were significantly lower than those in control group(P<0.05).(10)Comparison of proinflammatory cytokines expression in retina,kidney and serum of db/db mice: The expression levels of IL-1β,TNF-α,VEGF-A,MCP-1 and ICAM-1 in the retina were significantly lower than those in the kidney(P<0.05),and the expression levels of IL-6,TNF-α,ANG-2,MCP-1 and ICAM-1 were significantly lower than those in the serum(P<0.05).The expression levels of IL-1β and VEGF-A in the kidney were significantly higher than those in the serum(P<0.05),but the expression levels of IL-6,TNF-α and ANG-2 were significantly lower than those in the serum(P<0.05).Conclusion:1.The common characteristics of DR And DN are deficiency of the essence and excess of the standard,deficiency and excess,and the disease location is mainly in the kidney,which is closely related to the liver and spleen.The syndrome factors of deficiency are mainly qi deficiency,Yin deficiency and Yang deficiency,and the syndrome factors of solid disease are mainly blood stasis.2.Based on the comprehensive analysis of the literature in the field of diabetes and pyroptosis from 2011 to 2022,the visual analysis of the research that has important and outstanding contributions to the field is realized,so as to construct and show the development and hot trend of the field.At the same time,it provides research data and direction for future research on diabetes and pyroptosis.3.(1)Young DR Patients with hypertension have a high risk of DN.HGB,NLR,PLR,CYS-C,Crea and UA may be used as serum biomarkers for predicting DN in DR Patients.PLR,CYS-C,Crea,UA and the combination of the four indicators can be used for the risk assessment and auxiliary diagnosis of DR Combined with DN.(2)The thickness of RNFL and GCL in the temporal fovea may be used as imaging biomarkers for DR To predict DN.At the same time,GCL thickness has important value in the risk prediction and diagnosis of DR Combined with DN.4.(1)Bushen Huoxue prescription may inhibit the expression of NLRP3,GSDMD and its related pro-inflammatory factors IL-1 β,IL-17 A,IL-6,TNF-α,VEGF-A,ANG-2,MCP-1 and ICAM-1.It can improve the degeneration of retinal nerve tissue,microangiopathy,and renal tissue lesions such as glomerular and renal tubules,reduce the inflammatory response of retinal and renal tissue,and delay the occurrence and development of early DR And DN in db/db mice.(2)The changes in the dosage and combination of Bushen Huoxue prescription affected the types of pro-inflammatory cytokines,the degree of inflammatory response,and the site of action regulated,and the medium dosage of the Bushen Huoxue new prescription may be the optimal dose-effect ratio for the prevention and treatment of DR and DN.