The Retrospective Analysis Of Jiedu Tongluo Baoshen Formula In The Treatment Of Diabetic Kidney Disease And Its Protective Effect Via Regulation Of The Pyroptosis Signal Pathway

Objective:1.The objective of this study is to evaluate the effectiveness of Jiedu Tongluo Baoshen formula(JTB)in individuals diagnosed with Diabetic Kidney Disease(DKD)through a case-series research methodology,with a focus on self-comparisons,in order to establish a theoretical foundation for the clinical management of DKD.2.Network pharmacology and molecular docking techniques were utilized to elucidate the relationship between drug targets and disease targets.A comprehensive biological network of interactions among drug targets was constructed to illustrate the protective effects of JTB on DKD.This methodology seeks to explore the unique pharmacological properties of traditional Chinese medicine and provide a basis for future research and development.3.The aim of this study is to examine the potential protective effects of JTB and its active compounds,specifically loganin(LGN),on renal function in mice with DKD,and to elucidate the molecular mechanisms underlying these effects.4.The aim of this study is to examine the potential protective impact of LGN,the active constituents found in JTB,on pyroptosis within HK-2 cells.Methods:1.To gather data on patients diagnosed with diabetic kidney disease and treated with JTB at the Affiliated Hospital of Changchun University of Traditional Chinese Medicine from 2019 to 2023.Data collection will include basic patient information and relevant laboratory indicators such as urinary albumin-to-creatinine ratio(UACR),blood urea nitrogen(BUN),serum creatinine(Scr),cystatin C,glycated hemoglobin(Hb A1c),fasting plasma glucose(FPG),lipid levels,and markers of liver function safety.2.This study utilizes the Traditional Chinese Medicine systemic pharmacology database and computational simulations to examine and forecast the potential active components and pathways of JTB in the management of DKD.3.In vivo experiment: A total of 80 mice were provided with standard maintenance chow for a duration of one week in a laboratory with SPF grade conditions.Subsequently,10 mice were selected at random based on their body mass to form the Control group and were fed with clean grade chow.The remaining 70 mice were subjected to a high-fat diet for a period of 4 weeks,after which they were intraperitoneally injected with STZ at a dosage of 50 mg/kg for 5 consecutive days.The 60 mice were randomly divided into the following6 groups.Model group(DKD),positive control group(IRB),JTB-low dose group(JTB-L),JTB-high dose group(JTB-H),LGN-low dose group(LGN-L)and LGN-high dose group(LGN-H).The mice underwent an 8-week treatment via gavage,during which their body weights and fasting blood glucose levels were assessed biweekly.In weeks 4 and 8,the mice were individually housed in metabolic cages to facilitate urine collection,measurement of urine volume,and quantification of 24-hour urine protein levels.Following the completion of the 8-week drug intervention,24-hour urine samples were obtained from the mice.Blood samples were collected from the mice through eyeball blood sampling.Scr and BUN levels were quantified using a fully automated biochemical analyzer,while urine creatinine and urine microalbumin levels were determined following the instructions provided by the urine test kit.Subsequently,the UACR was calculated.The kidney tissues of mice were subjected to staining techniques such as H&E,PAS,and Masson staining in order to observe the pathomorphological alterations in the kidney tissues.Additionally,the expression levels of proteins and genes related to the pyroptosis signal pathway(NLRP3,Caspase-1,GSDMD,IL-1β,IL-18)and renal fibrosis(collagen I,α-SMA)were determined in the renal tissues of mice with DKD using RT-qPCR,immunohistochemistry,and Western blot methods.4.In vitro experiment: HK-2 cells were subjected to stimulation in a high glucose environment.The MTT method was utilized to determine the most effective concentration of high glucose for HK-2 cells and the intervention concentration of LGN.The degree of pyroptosis was assessed through the application of Calcein AM/PI staining.Semiquantitative analysis of ROS levels in HK-2 cells was conducted using the DCFH-DA probe.The levels of protein and gene expression associated with the pyroptosis signaling pathway(NLRP3,Caspase-1,GSDMD,IL-1β,IL-18)were assessed in renal tissues of mice with DKD through the utilization of RT-qPCR and Western blot techniques.Subsequently,the pyroptosis model was established using PPVI,an activator of pyroptosis,to validate the impact of LGN in modulating pyroptosis.Results:1.The incorporation of the JTB formula in the treatment regimen of DKD patients has been shown to enhance renal function markers such as Scr,BUN,urinary ACR,and lipid metabolism,while also lowering FBG levels.Furthermore,analysis of liver function indexes indicates that the JTB formula does not exhibit significant hepatotoxicity and is deemed safe(P < 0.01).2.LGN is identified as a potential active ingredient in JTB for the management of DKD,with its efficacy possibly being linked to the NLRP3/Caspase-1/GSDMD cellular pyroptosis signaling pathway.3.In vivo experiment: Following the implementation of streptozotocin(STZ)modeling,mice in the experimental groups exhibited weight gain,dull and unattractive fur,and delayed responsiveness compared to the Control group.Subsequent drug intervention resulted in varying degrees of symptom improvement in all groups receiving JTB intervention compared to the DKD group.Significant weight reduction was observed in the groups other than the Control group after successful modeling(P<0.01 or P<0.05),and a notable increase in weight was observed in the JTB-H-treated group compared to the DKD group starting from the sixth week(P<0.05).In comparison to the Control group,mice in the DKD group exhibited a significant increase in kidney weight(P<0.001),while mice in the JTB-H,JTB-L,LGN-H,and IRB groups demonstrated a decrease in kidney weight relative to the DKD group(P<0.01 or P<0.05).Additionally,the kidney weight coefficients of mice in the JTB-H,JTB-L,and LGN-H groups were significantly reduced compared to the DKD group(P<0.001,P<0.01,or P<0.05).Biochemical tests conducted on mice revealed a statistically significant increase in 24-hour urine protein quantification in the DKD group compared to the Control group(P<0.001).Additionally,there was a varying degree of decrease in 24-hour urine protein quantification in all treatment groups compared to the DKD group(P<0.001 or P<0.01).FBG levels in mice began to significantly decrease in the fourth week of treatment,with the most pronounced effect observed in the JTB-H group(P<0.001).Serum levels of BUN,Scr and UACR were found to be significantly elevated in mice within the DKD group compared to the Control group(P<0.001).Conversely,BUN levels decreased in mice within the JTBH,JTB-L,LGN-H,and IRB groups compared to the DKD group(P<0.05 or P<0.01).Scr levels decreased in the JTB-H,JTB-L,and LGN-H groups(P<0.01 or P<0.05),while UACR levels decreased in the JTB-H,JTB-L,LGN-H,and IRB groups(P<0.01 or P<0.001).In pathological examinations,H&E staining revealed swelling of renal tubular epithelial cells with detachment and vacuole-like deformation in the group with diabetic kidney disease(DKD).Additionally,the stroma of the glomerular mesangial area exhibited significant dilation,along with oedema and deformation of mesangial cells.In comparison to the DKD group,the groups treated with JTB-H and LGN-H demonstrated reduced swelling of renal tubular epithelial cells and less vacuole-like deformation,with statistical significance(P<0.05).PAS staining of mouse kidneys revealed that mice in the DKD group exhibited oedema and deformation of renal tubular epithelial cells,intracellular deposition of purplish-positive substances,and vacuolar degeneration of some renal tubular epithelial cells compared to the Control group(P<0.001).Additionally,the JTB-H-treated group demonstrated the most significant reduction in the area of positive staining compared to the DKD group(P<0.05).The immunohistochemical analysis revealed elevated expression levels of NLRP-3,Caspase-1,and GSDMD in the renal tissues of mice in the DKD group compared to the control group(P<0.001).Treatment with JTB-H or LGN-H resulted in a reduction of cellular pyroptosis-related proteins in the renal tissues,with the most significant decrease observed in NLRP3 expression(P<0.001).Western blot analysis revealed that the expression levels of cellular pyroptosis-related proteins NLRP-3,Caspase-1,and GSDMD in kidney tissues of mice in the DKD group were markedly elevated compared to those in the Control group(P<0.001).Treatment with JTB-H or LGN-H resulted in a significant reduction in NLRP3(P<0.001)and Caspase-1(P<0.01 or P<0.05).Additionally,the expression of cellular inflammatory factors IL-1β and IL-18 was notably increased in renal tissues of mice in the DKD group(P<0.001).Treatment with JTB-H or LGN-H significantly decreased the expression of inflammatory factors in renal tissues(P<0.001,P<0.01 or P<0.05).The expression levels of fibrosisrelated proteins in renal tissues of mice in the DKD group exhibited significantly elevated levels of Collagen I and α-SMA compared to the Control group(P<0.05 or P<0.01).Collagen I demonstrated a decreasing trend across all groups,with the most pronounced decrease observed in the JTB-H group(P<0.05)when compared to the DKD group.Additionally,α-SMA protein exhibited a significant down-regulation in the JTB-H and LGN-H groups compared to the DKD group(P<0.001 or P<0.01).The results of RT-qPCR analysis indicated significantly higher expression levels of Nlrp3,Caspase-1,Gsdmd,Il-1b,and Il-18 in the renal tissues of mice in the DKD group compared to the Control group(P<0.001).Furthermore,the down-regulation of Nlrp3 and Il-1b was most pronounced in all treatment groups compared to the DKD group(P<0.001,P<0.01,or P<0.05),with a notable inhibitory effect observed on the Caspase-1 gene in the JTB-H,JTB-L,and LGN-H groups(P<0.001,P<0.05,or P<0.01).Additionally,the JTB-H,JTB-L,LGN-H,and IRB groups exhibited down-regulation of the Gsdmd gene(P<0.001 or P<0.01).The Il-18 gene exhibited down-regulation in the JTB-L,LGN-H,LGN-L,and IRB groups(P<0.001 or P<0.01).The expression levels of Collagen I and Acta2 were significantly elevated in the renal tissues of mice in the DKD group compared to those in the Control group(P<0.01 or P<0.05).Following treatment,the expression levels of the Collagen I gene were down-regulated in the JTB-H,JTB-L,and LGN-H groups compared to the DKD group(P<0.05 or P<0.01).Similarly,the Acta2 gene expression levels were down-regulated in the JTB-H,JTB-L,LGN-H,and IRB groups(P<0.01 or P<0.05)relative to the DKD group.The concentrations of cytokines IL-1β and IL-18 in the serum of mice in the end-stage DKD group were found to be significantly elevated as determined by ELISA(P<0.01).In comparison to the DKD group,the JTB-H,JTB-L,and LGN-H groups exhibited a notable decrease in the levels of IL-1β and IL-18 in the serum of mice,indicating a significant down-regulation of these inflammatory factors(P<0.01 or P<0.05).4.In vitro experiment: Compared to the Control group,the high-glucose cell model(HG)group and PPVI-activated cellular pyroptosis model(PPVI)group exhibited a significantly higher percentage of cellular pyroptosis(P<0.001)and increased levels of reactive oxygen species(ROS)in the cells of the high-glucose cell model(HG)group(P<0.01).Western blot analysis revealed significant up-regulation of inflammatory factorassociated proteins NLRP3,Caspase-1,GSDMD,IL-1β,and IL-18(P<0.001,P<0.01,or P<0.05).The m RNA expression levels of cellular pyroptosis and inflammatory factorrelated genes NLRP3,CASP-1,GSDMD,IL-1B,and IL-18 were significantly increased as determined by RT-qPCR analysis(P<0.001,P<0.01 or P<0.05).In comparison to the HG group,the protein expression levels of cellular pyroptosis and inflammatory factor-related proteins NLRP3,Caspase-1,GSDMD,IL-1β,and IL-18 were notably decreased following treatment with LGN20 intervention,as confirmed by Western blot analysis(P<0.001,P<0.01,or P<0.05).RT-qPCR also identified the down-regulation of cellular pyroptosis and inflammatory factor-related genes,including NLRP3,CASP-1,GSDMD,IL-1B,and IL-18,at statistically significant levels(P<0.001,P<0.01,or P<0.05).Conclusion:1.The incorporation of the JTB formula has been shown to enhance renal function and lipid metabolism in patients with DKD,resulting in decreased FBG levels and UACR.Furthermore,assessments of liver function parameters suggest that the JTB formula is linked to a favorable safety profile.2.The JTB formula and its active compound,loganin,exhibit a protective effect against DKD through the regulation of the NLRP3/Caspase-1/GSDMD pathway.