| Objective: Clinical study: Collecting clinical data of CKD patients with non-dialysis patients from stage 3 to 5,comprehensive analysis the distribution rule of TCM syndrome elements in CKD patients and its relationship with disease stage and laboratory indicators,to explore the basic pathogenesis and syndrome characteristics of CKD,and provide an objective basis for clinical syndrome differentiation.Experimental study: To investigate the effect of invigorating qi and activating blood circulation TCM Astragalus membranaceusthe root of red-rooted salvia effective monomer(isoflavones-tanshinone IIA)on angiogenesis of glomerular endothelial cells with ischemic injury in vitro and its regulation mechanism,to provide a theoretical basis for the use of invigorating qi and activating blood circulation to treat kidney disease.Methods:1.Formulate a questionnaire for TCM syndromes in patients with CKD,and involve in the CKD stage 3-5 non-dialysis hospitalized patients from December 2016 to October 2018 in the Department of Nephrology,Shandong Provincial Hospital of Traditional Chinese Medicine,collect general patient data,current medical history,disease stage,four-diagnosis information and major laboratory indicators,etc.SPSS22.0 software was used to analyze the basic data and syndrome distribution of patients.To clarify the distribution rule of TCM syndrome elements in CKD and its relationship with disease stage and laboratory indicators,and to explore the basic pathogenesis and syndrome characteristics of CKD.2.Protective effect of isoflavones,tanshinone IIA and its compatibility on HRGEC with ischemic injury: Human glomerular endothelial cells(HRGEC)were cultured in vitro,and the HRGEC ischemia model was induced by replacing the normal medium with Earle's balanced salt.Screening of the optimal drug concentration of isoflavones(0.1mg/L,1mg/L,5mg/L,10mg/L,15mg/L,20mg/L)and tanshinone IIA(0.001mg/L?0.01mg/L?0.05mg/L?0.1mg/L?0.5mg/L?1mg/L?10mg/L)to protect HRGEC with Earle's balanced salt-induced ischemic injury by MTT assay.The two drugs were divided into three groups: low,medium and high according to the optimal concentration of the selected drugs.design orthogonal experiment to Screen the optimal drug compatibility ratio of the two drugs by MTT assay.Using the optimal concentration of flavonoids,tanshinone IIA and its compatibility to intervene HRGEC with Earle's balanced salt-induced ischemic injury,MTT assay,apoptosis assay,adhesion assay and reactive oxygen species assay were used to observe the effects of isoflavones,tanshinone IIA and their compatibility on HRGEC survival rate,apoptosis,adhesion and reactive oxygen species.3.Study on the compatibility of isoflavones and tanshinone IIA on angiogenesis of HRGEC with Ischemic Injury: cell migration assay and Matrigel angiogenesis assay were used to observe the effects of compatibility of isoflavones and tanshinone IIA on angiogenesis of HRGEC with Ischemic Injury in vitro;Seahorse XF cell energy Metabolic analyzer was used to observe the effect of compatibility of isoflavones and tanshinone IIA on the mitochondrial respiratory function of HRGEC with Ischemic Injury.Transcriptome sequencing was used to detect the effects of compatibility of isoflavones and tanshinone IIA on HRGEC m RNA expression profile with Ischemic Injury,screen differential genes,RTPCR and Western Blot were used to verify the differential gene expression.4.The regulation of SGK1/TP53/SLC2A3 signaling pathway by compatibility of isoflavones and tanshinone IIA: SGK1 inhibitor(EMD638683)inhibited HRGEC SGK1 expression,Western Blot assay detected SGK1,TP53 and SLC2A3 protein expression.Results:1.A total of 185 non-dialysis patients with CKD stage 3 to 5 were enrolled,including 104 males and 81 females,with a male to female ratio of 1.28:1.The age ranged from 25 to 92 years with an average age of 54.68 ± 14.18 years.In the primary disease,chronic glomerulonephritis was the main cause,accounting for 36.76%,followed by diabetic nephropathy,accounting for 22.70%.Among the comorbidities and complications,hypertension was the most common,with 162 cases,accounting for 87.57%,followed by 96 cases of anemia,accounting for 51.89%.The frequency of symptoms of TCM is more than 20%,followed by fatigue,fatigue,dark complexion or dullness,edema,limbs,heavy complexion or chlorosis,pale lips,less food,less stool,softer stools,less gas,and urination.Long or nocturia,abdominal fullness,dry mouth and bitterness,cold and cold limbs,pale complexion,dry oropharyngeal,nausea and vomiting,limb numbness,activity is not good,and the urine is short.The composition of TCM syndrome elements is the most common in the case of false and real inclusions,a total of 149 cases,accounting for 80.54% of the total number of cases,simple deficiency syndrome only accounted for 11.89%,simple evidence only accounted for 7.57%;four virtual syndrome elements in the air deficiency The most common syndromes were 123 cases,accounting for 66.49% of the total cases,followed by blood deficiency syndrome,accounting for 45.95%,yang deficiency syndrome accounting for 31.89%,and yin deficiency syndrome accounting for 25.41%;four solid syndrome elements Among the most common blood stasis syndromes,102 cases,accounting for 55.14% of the total number of cases,followed by damp heat certificate,accounting for 45.41%,wet turbidity card accounted for 17.84%,water wet syndrome accounted for the least,accounting for 14.05%;Among the elements of false and mixed syndromes,the combination of the top ten syndrome elements in frequency is qi deficiency syndrome + blood stasis syndrome + damp heat syndrome,qi deficiency syndrome + blood stasis syndrome,qi deficiency syndrome + blood deficiency syndrome + dampness heat syndrome,qi deficiency syndrome + damp heat Syndrome,qi deficiency syndrome + blood stasis syndrome + wet turbidity syndrome,qi deficiency syndrome + blood deficiency syndrome + blood stasis syndrome + wet turbid syndrome,blood deficiency syndrome + dampness heat syndrome,qi deficiency syndrome + blood stasis syndrome + water dampness syndrome,qi deficiency syndrome + yang Deficiency syndrome + blood stasis syndrome + water dampness syndrome,qi deficiency syndrome + blood deficiency syndrome + blood stasis syndrome + damp heat syndrome.patients with different stages of CKD frequency distribution of syndrome elements,imaginary syndrome elements: CKD3 stage: qi deficiency syndrome > yin deficiency syndrome > blood deficiency syndrome > yang deficiency syndrome;CKD4 phase in order: qi deficiency syndrome > blood deficiency syndrome > yang deficiency syndrome > yin The deficiency of CKD is as follows: qi deficiency syndrome > blood deficiency syndrome > yang deficiency syndrome > yin deficiency syndrome;qi deficiency syndrome is evenly distributed in each stage of CKD(P>0.05),blood deficiency syndrome,yin deficiency syndrome and yang deficiency syndrome in CKD The distribution of each period was uneven(P<0.05).The elements of imaginary syndrome: CKD3 stage is: blood stasis syndrome> damp heat syndrome> water dampness syndrome> wet turbidity syndrome;CKD4 phase is: blood stasis syndrome> damp heat syndrome> wet turbid syndrome> water dampness syndrome;CKD5 phase is : Blood stasis syndrome> Damp heat syndrome> Wet turbidity syndrome> Water wetness syndrome.The blood stasis syndrome,damp heat syndrome and water-wet syndrome were balanced in all stages of CKD(P>0.05);the wet turbidity syndrome was unevenly distributed in each stage of CKD(P<0.05).The relationship between TCM syndrome elements and laboratory indicators,the difference of hemoglobin,serum albumin and serum creatinine between the syndrome elements was statistically significant(P<0.05).There was no statistically significant difference in 24 h urine protein quantitation(P>0.05).The serum hemoglobin level of patients with blood deficiency syndrome and yang deficiency syndrome was significantly lower than that of patients with qi deficiency syndrome and yin deficiency syndrome(P<0.05).The albumin level of patients with qi deficiency syndrome was significantly lower than that of patients with blood deficiency syndrome,yin deficiency syndrome and yang deficiency syndrome(P< 0.05);patients with blood deficiency syndrome and yang deficiency syndrome were significantly higher than those with qi deficiency syndrome and yin deficiency syndrome(P<0.05).There were no significant differences in the quantitative characteristics of hemoglobin,serum albumin,serum creatinine and 24 h urine protein between the actual syndrome elements(P>0.05).2.Protective effect of isoflavone,tanshinone IIA and its compatibility on HRGEC of ischemic injury: the optimal concentration of isoflavones is 10mg/L,the optimal concentration of tanshinone IIA is 0.05mg/L,and the isoflavones and tanshinone IIA The optimal concentration of the ratio is 10mg/L+0.025mg/L;the HRGEC ischemic injury induced by Earle's balanced salt reduces the survival rate,the apoptosis rate increases,the adhesion ability decreases,and the intracellular reactive oxygen species level increases(P <0.05);the isoflavones and tanshinone IIA can increase the survival rate of HRGEC,reduce apoptosis and decrease the level of intracellular reactive oxygen species(P<0.05).The compatibility of the two drugs is better than that of single drugs(P<0.05),and tanshinone IIA can improve adhesion.The ability(P<0.05),hairy isoflavones did not improve the adhesion ability(P>0.05).3.The combination of vermiculone and tanshinone IIA in the treatment of HRGEC angiogenesis in ischemic injury: Earle's balanced salt-induced HRGEC ischemic injury reduced migration ability and decreased angiogenic ability(P<0.05).The compatibility of veratium isoflavone and tanshinone IIA improved.HRGEC migration and angiogenesis were inhibited by ischemic injury(P<0.05);Earle's balanced salt-induced ischemic injury significantly inhibited HRGEC mitochondrial respiration,reduced basal respiration,ATP production capacity,and respiratory reserve,and isoflavones Tanshinone IIA compatibility can significantly improve the mitochondrial respiration ability of HRGEC in ischemic injury,improve basal respiration,ATP production capacity and respiratory reserve;the effect of veratium isoflavone and tanshinone IIA on HRGEC gene expression profile of ischemic injury: model group compared with normal group A total of 3464 differential m RNAs were screened out,of which 2273 were up-regulated and 1191 were down-regulated(qvalue?0.05).Compared with the model group,the bristles of flavonoids and tanshinone IIA were screened for 642 differential m RNAs,of which 299 were up-regulated.343 downregulation(q-value?0.05);Select relevant genes for verification,q-RT-PCR was used to detect the expression of SGK1,TP53,MAPK14,SLC2A3,VEGFA and CS m RNA,Western Blot was used to detect the expression of VEGFA and CS protein.The verification results show that the expression changes of each gene are basically the same as the sequencing results,suggesting that the sequencing results are true and reliable.4.The regulation of SGK1/TP53/SLC2A3 signaling pathway by the combination of veratium isoflavone and tanshinone IIA: The expression of SGK1 and SLC2A3 protein was down-regulated and the expression of TP53 protein was significantly up-regulated by HRGEC ischemic injury induced by Earle's balanced salt(P<0.05).The expression of SGK1 and SLC2A3 protein was significantly up-regulated after flavonoids and tanshinone IIA intervention,and the expression of TP53 protein was significantly down-regulated(P<0.05).After inhibiting the expression of SGK1 by EMD638683,the expression of SGK1 and SLC2A3 protein was further down-regulated,and the expression of TP53 protein was further up-regulated(P<0.05).Conclusion: Clinical research: CKD pathogenesis is complicated,qi deficiency syndrome and blood stasis syndrome are common in all stages of CKD.Qi deficiency and blood stasis are the basic pathogenesis of CKD;blood deficiency syndrome and yang deficiency syndrome have certain correlation with hemoglobin and serum creatinine,qi deficiency There is a certain correlation between serum and serum albumin.Experimental study: Isoflavones and tanshinone IIA have significant protective effects on HRGECinduced ischemic injury induced by Earle's balanced salt,which can improve cell survival rate,reduce apoptosis and reduce intracellular reactive oxygen species.The two drugs are superior to single drug,tanshinone IIA.Can improve cell adhesion.The combination of veratium isoflavone and tanshinone IIA has a significant pro-angiogenic effect,which can enhance the migration ability and angiogenic ability of HRGEC in ischemic injury.The mechanism may regulate the expression of CS,VEGFA,SGK1,TP53 and SLC2A3,promote cell survival and inhibit apoptosis.Enhance mitochondrial respiration and glycolysis. |
PDF Full Text Request