| Objective1.Analyze the clinical characteristics of hospitalized patients with type 2 diabetic kidney disease(DKD)through clinical research,to explore the correlation between circadian rhythm of blood pressure and DKD renal injury,as well as the clinical efficacy of Abelmoschus Manihot in the treatment of DKD.2.To explore the possible mechanism of Abelmoschus Manihot in the treatment of DKD based on network pharmacology.3.To validate the protect effect and mechanism of Abelmoschus Manihot against DKD through experimental research,to explore the effect of TFA on the expression of circadian genes and HIF-1α,VEGF in renal tissues of type 2 diabetic db/db mice.Methods1.Clinical research:(1)The clinical data of patients with type 2 diabetic kidney disease hospitalized in the Endocrinology Department of Affiliated Hospital of Nanjing University of Chinese Medicine between 2019 and 2020 were retrospectively analyzed to investigate the clinical features and characteristics of diagnosis and treatment.(2)Patients with DKD who underwent 24-hour ambulatory blood pressure monitoring(ABPM)during hospitalization were analyzed for control and circadian rhythm of blood pressure,and further divided into groups according to circadian blood pressure patterns,albuminuria(UACR)category,and glomerular filtration rate(eGFR)category.The clinicobiochemical indices and ABPM parameters were compared in each group.The correlations between circadian blood pressure patterns and renal function variables were calculated with Spearman’s correlation test.Multinominal logistic regression was peformed to analyze the possible influencing factors of renal injury in DKD patients.(3)DKD patients treated with Abelmoschus Manihot preparation during hospitalization were selected,and changes in renal function and urinary protein level were evaluated after treatment with different doses of Abelmoschus Manihot.2.Network pharmacology:The active components of Abelmoschus Manihot were collected from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)and literatures,and Swiss Target Prediction was employed to obtain the potential targets of these components.The related targets of DKD were searched from Therapeutic Target Database(TTD),Drugbank,and DisGeNET databases.The results were then mapped to obtain potential therapeutic targets.The component-target network and the protein-protein interaction(PPI)network were constructed by Cytoscape software,then topological parameters of nodes in network were calculated to find the key targets.Gene ontology(GO)and Reactome pathway analyses were performed using ClueGO plugin.3.Experimental research:Spontaneous type 2 diabetic db/db mice were used as DKD model,which were randomly divided into four groups:DKD group,low dose total flavone of Abelmoschl Manihot(TFA)group(LD-TFA group,100 mg/kg·d),high dose TFA group(HD-TFA group,200 mg/kg·d),and REV-ERB agonist group(REV-ERB group,SR9009,100 mg/kg),while db/m mice were used as normal control group.TFA was dissolved in double-distilled water and intragastrically administered once daily,while control group and DKD group were intragastrically given equal volume of double-distilled water.REV-ERB agonist SR9009 was administered by intraperitoneal injection once weekly.All groups were intervened for eight weeks.Body weight,food intake,blood glucose,serum creatinine(Scr),blood urea nitrogen(BUN),cystatin C(CysC)and 24-hour urinary total protein(UTP)levels were measured in each group of mice.Morphological changes of renal tissues were observed by hematoxylin-eosin(HE)staining,and quantitative real-time polymerase chain reaction(qRT-PCR)was used to assess the mRNA expression levels of circadian clock gene Bmal1,Clock,and HIF-1α,VEGF in renal tissues.Results1.Clinical research:(1)A total of 685 hospitalized patients with diabetic kidney disease(DKD)were included,and among these there was a male preponderance(67.4%).The mean age was(60.96±13.37)years,duration of diabetes was 13.0(7.0,20.0)years and the mean BMI was(25.46±3.79)kg/m2.Among the DKD patients,43.4%were complicated with diabetic retinopathy(DR),71.4%with diabetic peripheral neuropathy(DPN),72.3%with lower extremity atherosclerotic disease(LEAD),1.6%with diabetic foot(DF),and 1.3%with acute diabetic complications.The proportion of DKD patients with hypertension,stroke,coronary heart disease,fatty liver,osteoporosis/osteopenia,thyroid disease were 75.3%,40.6%,20.0%,43.8%,29.5%and 29.2%,respectively.13.6%of the patients had infection,and urinary tract infection,pulmonary infection,acute upper respiratory tract infection were the most common reasons.The mean HbA1c was(8.80±2.28)%,serum creatinine level was 82.60(62.90,124.30)μmol/L,cystatin C level was 1.27(0.95,1.84)mg/L,UACR level was 302.0(71.0,1410.0)mg/g,and UTP level was 433.5(156.5,1816.3)mg/24h.Urine protein electrophoresis showed that urinary albumin was the main component(98.50%),with a small amount of transferrin(0.70%),and IgG,IgA(0.60%).Renal ultrasound showed that the mean length,width and thickness of the left kidney amounted to(10.71±1.15)cm,(5.29±0.64)cm,and(4.99±0.66)cm,respectively.The right kidney measured(10.61±1.26)cm,(5.10±0.66)cm and(4.82±0.65)cm in mean length,width and thickness.The basic treatment of western medicine includes glycemic control,blood pressure control,lipid lowering,and so on.88.2%of DKD patients were treated with oral hypoglycemic agents,Dipeptidyl peptidase-4 inhibitor(DPP-4i),alpha-glycosidase inhibitor,sodium-glucose co-transporter 2 inhibitor(SGLT2i),and metformin were the most commonly used.While 75.0%of patients were treated with insulin,and 18.5%were treated with glucagon-like peptide-1 receptor agonist(GLP-1RA).The utilization rate of antihypertensive drugs was 73.6%,with calcium channel blocker,angiotensin converting enzyme inhibitor(ACEI)or angiotensin receptor blocker(ARB),beta-blockers,diuretics and alpha-blocker in descending order.92.4%of DKD patients were treated with traditional Chinese medicine prescription,and 82.6%used Abelmoschus Manihot preparation,of which the utilization rate of Jiahua tablets,Huangkui capsules,Qikui granules and Abelmoschus Manihot pieces were 35.8%,33.6%,52.3%,28.8%and,respectively.(2)①Blood pressure control:A total of 470 DKD inpatients were included.392(83.4%)patients had elevated office blood pressure,only 41.6%of which had their blood pressure controlled,and those with uncontrolled hypertension included sustained hypertension(31.4%),daytime hypertension(2.0%),nocturnal hypertension(23.7%),and white-coat hypertension(1.3%).78(16.6%)patients had normal office blood pressure,and 35.9%of which had masked hypertension.②Circadian rhythm of blood pressure:Among the 470 DKD patients,57(12.1%)had normal circadian blood pressure rhythm(dipping pattern),413(87.9%)had abnormal circadian rhythm,including 213(45.3%)with weakened circadian rhythm(non-dipping pattern)and 200(42.6%)with reversed circadian rhythm(reverse-dipping pattern).In DKD patients with elevated office blood pressure,the proportion of dipping,non-dipping and reversed-dipping pattern of blood pressure was 11.5%,44.6%and 43.9%respectively.While in DKD patients with normal office blood pressure,the proportion of dipping,non-dipping and reversed-dipping pattern of blood pressure was 15.4%,48.7%and 35.9%respectively.③Comparison among the groups:With the increase of abnormal degree of circadian rhythm,the levels of nighttime SBP,nighttime DBP,HbAlc,BUN,Scr,CysC,UACR and UTP gradually increased,while the levels of daytime DBP and eGFR gradually decreased(P<0.05).With advancing UACR stages,the proportion of patients with abnormal circadian rhythm of blood pressure increased,and the levels of office SBP,24-hour SBP,daytime SBP,nighttime SBP,nighttime DBP,TC,SUA,BUN and Scr increased gradually,while the level of eGFR decreased gradually(P<0.05).With advancing GFR stages,the levels of 24-hour SBP,daytime SBP,nighttime SBP,TC,SUA,BNU,Scr,CysC,UACR and UTP gradually increased,and the levels of office DBP,24-hour DBP,daytime DBP and daytime heart rate gradually decreased(P<0.05).④Spearman’s correlation analysis showed that the abnormal degree of blood pressure circadian rhythm was positively correlated with the levels of UACR,BUN,Scr and CysC,and negatively correlated with the level of eGFR(P<0.05).⑤Multinominal logistic regression analysis showed that reversed circadian rhythm of blood pressure may be a risk factor of renal injury in DKD patients(UACR stage:OR=1.88,95%CI 1.02-3.47,GFR stage:OR=1.86,95%CI 1.04-3.35).(3)A total of 240 DKD inpatients were included.After treatment with Abelmoschus Manihot preparation,the UACR level of the patients decreased significantly(P<0.05),and the level of UACR decreased more in the high-dose Abelmoschus Manihot group than the low-dose group(P<0.05).There was no significant change in serum creatinine level before and after treatment(P>0.05).2.Network pharmacology:A total of 13 active components and 155 predictive targets of Abelmoschus Manihot were screened,and 45 potential therapeutic targets were obtained after mapping with disease.The active components of Abelmoschus Manihot mainly acted on target proteins such as HIF-1α,VEGF,TNF,AKT1,MMP,and regulated inflammation,oxidative stress,circadian clock,and HIF-1α/VEGF pathway to play a role in the treatment of diabetic kidney disease.3.Experimental research:(1)Body weight and food intake:During the experiment,the body weight and average food intake of db/db mice were significantly higher than db/m mice of control group at the same time point,while the body weight and average food intake of db/db mice in each group were not significantly different.(2)Blood glucose:Compared with control group,the blood glucose level of mice in DKD group increased(P<0.05).There was no significant change in blood glucose level of db/db mice after the intervention of different doses of total flavone of Abelmoschl Manihot(TFA)(P>0.05).The blood glucose level of db/db mice decreased after the intervention of SR9009(P<0.05).(3)Renal function:Compared with control group,the serum creatinine level of mice in DKD group was significantly increased(P<0.05).After intervention with different doses of TFA and SR9009,the serum creatinine level in db/db mice showed a decreasing trend,but there was no statistically significant difference(P>0.05).(4)Urinary protein:Compared with control group,the level of urine protein in DKD group was significantly increased(P<0.05).After intervention of different doses of TFA and SR9009,the level of urine protein in db/db mice was significantly decreased,and the decrease in HD-TFA group was more obvious than that in LD-TFA group(P<0.05).(5)HE staining:Compared with control group,db/db mice in DKD group showed glomerular hypertrophy,basement membrane thickening,mesangial hyperplasia and renal tubular epithelial cell degeneration.Renal pathological changes in db/db mice were alleviated after the intervention of different doses of TFA.(6)Core circadian gene expression:Compared with control group,the expression level of Bmal1 mRNA in renal tissue of mice in DKD group was increased,and the expression level of Bmal1 mRNA was decreased after the intervention of TFA and SR9009(P<0.05).There was no statistically significant difference in the expression of Clock mRNA in each group of mice(P>0.05).(7)HIF-la and VEGF expression:Compared with control group,the expression levels of HIF-la and VEGF mRNA in renal tissue of mice in DKD group were increased,and the expression levels of HIF-1α and VEGF mRNA were decreased after the intervention of TFA,with statistically significant difference(P<0.05).Conclusion1.Circadian rhythm disorder of blood pressure is common in DKD patients,and is associated with renal injury in DKD.2.Both clinical and experimental research showed that Abelmoschus Manihot could ameliorate renal injury and reduce urinary protein level in DKD,and the efficacy is positively correlated with the dose.3.The prediction results of network pharmacology correspond to the results of experimental research,both indicate that Abelmoschus Manihot may regulate circadian clock,HIF-1α/VEGF pathways,and play a role in renal protection. |
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