| ObjectiveThe risk factors of cardiorenal syndrome(CRS)were analyzed by clinical observation and comparison between the patients with type Ⅱ and Ⅳ CRS and healthy subjects.According to the different stages of chronic kidney disease,the clinical indicators and traditional Chinese medicine(TCM)syndrome elements of patients with CRS were observed and studied,to analyze the characteristics of disease progression and TCM syndrome elements.To test whether artemisinin,an artemisinin derivative,has the effect of improving heart and kidney injury by treating doxorubicin mice with cardiotoxicity.To provide ideas for the diagnosis and treatment of CRS by clinical observation and animal experiment,and provide preliminary experimental basis for artemether treating the cardiorenal injury.MethodsPart 1:Clinical characteristics of patients with CRSSixty-nine patients with type Ⅱ and Ⅳ CRS were compared with 75 healthy people in Shenzhen Traditional Chinese Medicine Hospital,with the gender,age,blood pressure,Body Mass Index(BMI)and lifestyle habits of the two groups(CRS group and healthy group)were respectively collected and recorded,as well as the other indicators of Blood routine,electrolytes,renal function,cardiac function,liver function,blood lipid,serum Renalase,to analyze the epidemiological characteristics and risk factors of CRS.Part 2:Study on oxidation reaction and distribution of TCM syndrome elements in patients with CRSSixty-nine patients with type Ⅱ and Ⅳ CRS were divided into 3 groups(CKD3 group,CKD4 group,CKD5 group)according to eGFR level.Gender,age,blood pressure,BMI of patients were collected and recorded.Renal function,myocardial markers,liver function,blood lipid,serum Catalase and malondialdehyde(MDA)were detected.The TCM clinical symptoms and TCM syndrome elements of patients with CRS were collected and analyzed.Part 3:Effect and mechanism of artemether on doxorubicin induced cardiorenal injury in miceThe 6-week-old BALB/c male mice were randomly divided into three groups:control group,model group and artemether group.The mice of model group and artemether group induced by single injection of doxorubicin(10.4 mg/kg)through tail vein to construct cardiac and renal injury model.In three groups of mice,the artemether group was fed with artemether(0.3 g/kg)fodder,on the contrary,the other two groups were fed with standard fodder,and the treatment period was 4 weeks.After 4 weeks,24h total urine protein,the body weight,heart weight of mice were measured.Cardiac and renal related blood biochemical indicators were detected,including Lactate Dehydrogenase(LDH),Creatine Kinase(CK),Creatinine(Cr),Blood Urea Nitrogen(BUN),Total Protein(TP)and Albumin(ALB).Hematoxylineosin(HE)staining and Periodic Acid-Schiff(PAS)staining were used to observe the pathological changes of heart and kidney.The expression levels of proteins,including superoxide dismutase 1(SOD1),superoxide dismutase 2(SOD2),Catalase,Desmin and Renalase in heart and/or kidney tissues of mice in each group were detected by immunohistochemical method.Western Blot was used to detect the protein expression levels of SOD1,SOD2,Catalase,Renalase,ac-SOD2 K68,ac-SOD2 K122,Bak and Bcl-xL in the heart and kidney tissues of mice,and the expression levels of silent information regulator 3(Sirt3)protein in heart tissue and glutathione peroxidase 1(GPX1)protein in kidney tissue also were detected.The qRT-PCR was used to detect the mRNA levels of SOD1,SOD2,Catalase,GPX1 and Sirt3 in heart and kidney tissues,and the neutrophil gelatinase-associated lipocalin(NGAL)and kidney injury molecule-1(KIM-1)mRNA levels in kidney tissues.ResultPart 1:Clinical characteristics of patients with CRS1.The baseline results showed that age,smoking,drinking and systolic pressure were significantly increased in the CRS group compared with the healthy group,and were positively correlated with CRS(P<0.001).There was no significant difference in height,weight and BMI between the two groups(P>0.05),but the overweight patients in CRS group increased significantly.The results of blood routine showed that the CRS group had significantly more inflammation and anemia than the healthy group,and WBC,NEUT#,NEUT%,EO#,EO%,RDWSD,RDWCV were positively correlated with the occurrence of CRS,while LYM#,LYM%,BASO%,RBC,Hb,HCT and MCHC were negatively correlated with the incidence of CRS(P<0.05).Electrolytes kalium and phosphorus in CRS group were significantly higher than those in healthy group,and were positively correlated with the occurrence of CRS.Sodium,Chlorinum,Calcium and HCO3 were significantly decreased in CRS group and negatively correlated with the occurrence of CRS(P<0.05).2.Compared with the healthy group,the ALT,AST,TP and ALB were significantly decreased in the CRS group,and were negatively correlated with the occurrence of CRS(P<0.05),but AST/ALT and GGT were no significantly different,and had no correlation with the occurrence of disease(P>0.05).Compared with the healthy group,the TC,HDL-C and LDL-C of the CRS group were significantly decreased,and were negatively correlated with the occurrence of CRS(P<0.05).TG/HDL-C in CRS group was significantly higher than that in healthy group,which was positively correlated with the occurrence of disease(P<0.05).3.BUN,Cr and UA in CRS group were significantly higher than those in healthy group,and were positively correlated with CRS(P<0.05).LDH and α-HBDH in CRS group were significantly higher than those in healthy group,and were positively correlated with the occurrence of disease(P<0.05).There was no significant difference in CK between CRS group and healthy group,and was no correlation in disease occurrence(P>0.05).4.Serum Renalase in CRS group was significantly higher than that in healthy group,and was positively correlated with CRS(P<0.05).Part 2:Study on oxidation reaction and distribution of TCM syndrome elements in patients with CRS1.There were no significant difference in age,systolic pressure,and diastolic pressure between CKD3 group,CKD4 group,and CKD5 group(P>0.05).Compared with CKD4 group,CKD5 group showed a significant decrease in BMI(P<0.05).Cr and BUN were significantly increased among the three groups(P<0.05),but UA was no significantly different(P>0.05).The myocardial marker BNP was significantly increased in CKD4 and CKD5 groups compared with CKD3 group(P<0.001),while there was no significant difference in CK-MB among the three groups(P>0.05).The hsTnI of CKD5 group was significantly higher than that of CKD3 group and CKD4 group(P<0.001).The level of MY O was significantly increased in CKD4 and CKD5 groups compared with CKD3 group(P<0.05).In terms of liver function and blood lipid,the ALT and AST in CKD5 group were significantly lower than those in CKD3 group and CKD4 group(P<0.05),but there were no significant differences in AST/ALT,GGT,TG and TC among the three groups(P>0.05).2.Compared with CKD3 group,the serum Catalase showed decreased trend and MDA showed increased trend in CKD4 group(P>0.05),while Catalase decreased significantly and MDA increased significantly in CKD5 group(P<0.05).There was no significant difference in serum Renalase level among the three groups(P>0.05).3.The common clinical symptoms of 69 patients with CRS were chest discomfort,fatigue,poor appetite,insomnia and dreaminess,soreness and weakness of waist and knees,lumbago,shortness of breath,palpitation,less urine,no urine,dry stool,etc.The main tongue and pulse manifestation are dark tongue,red tongue,white tongue coating,greasy tongue coating,thin pulse,string-like pulse,slippery pluse,etc.The clinical symptoms of patients can be divided into three categories by systematic clustering,which mainly involve heart,kidney,lung and spleen,and all of those were with excess and deficiency syndromes.Deficiency syndromes were mainly Qi deficiency,Yin deficiency and Yang deficiency,and the most common excess syndrome was blood stasis,followed by phlegm dampness,dampness heat and water retention.The common compound syndrome elements of diseases are Qi deficiency and blood stasis,Yin deficiency and blood stasis,Yang deficiency and blood stasis,Qi and Yin deficiency combined with blood stasis,Qi deficiency and phlegm dampness,Yin deficiency and phlegm dampness.Yang deficiency and phlegm dampness.Part 3:Effect and mechanism of artemether on doxorubicin induced cardiorenal injury in mice1.The body weight and heart weight in doxorubicin model group were significantly decreased compared with control group(P<0.05),while artemether group had an increasing trend compared with model group(P>0.05).Compared with control group,LDH of cardiac function in model group was significantly increased(P<0.05),and the artemether group just showed a downward trend compared with model group,while CK among the three groups had no statistical difference(P>0.05).The pathology of mice heart in model group showed myocardial fiber disorder,rupture and vacuolar degeneration,by contrast,myocardial cell structure in the artemether group was improved.2.Cardiac immunohistochemical staining showed lower expression of proteins,including SOD 1,SOD2,Catalase and Renalase in the model group,and increased expression after artemether treatment,with strong expression in the cytoplasm.Desmin antibody staining also showed that the expression of protein in model group decreased,while the expression was enhanced and evenly distributed after artemether treatment.Western Blot analysis of heart tissue showed that SOD1,SOD2 and Catalase protein expressions in model group were significantly decreased compared with control group,and artemether group was significantly increased compared with model group(P<0.05).The protein of ac-SOD2 K68 showed no statistical difference among three groups.The ac-SOD2 K122 showed the expression in model group were increased trend compared with control group,and decreased trend in artemether group compared with model group(P>0.05).Compared with control group,Sirt3 protein expression in model group had a downward trend(P>0.05),while that in arteinether group was significantly increased compared with model group(P<0.05).The expression of Renalase protein in model group was significantly decreased compared with control group,and that in artemether group was significantly increased compared with model group(P<0.05).Compared with control group,Bcl-xL protein decreased and Bak protein increased trend in model group,while compared with model group,the Bcl-xL protein appeared increased and Bak protein decreased trend in artemether group(P>0.05).The results of qRT-PCR in heart tissue showed that the expression of SOD1 and Sirt3 mRNA in the model group was significantly decreased compared withthe control group(P<0.05).and the expression of artemether group was increased compared with the model group(P>0.05).The expression of SOD2and Catalase mRNA in the model group showed a downward trend compared with the control group,and the artemether group showed an increasing trend compared with the model group(P>0.05).The expression of GPX1 mRNA in the model group was significantly decreased compared with the control group,and artemether group was significantly increased compared with the model group(P<0.05).3.The 24h urinary protein quantification in doxorubicin model group was significantly higher than that in control group,while that in artemether group was significantly lower than that in model group(P<0.05).The level of blood serum ALB in model group was significantly decreased compared with control group,and artemether group was significantly increased compared with model group(P<0.05).There was no statistical difference in TP level among the three groups(P>0.05).Compared with control group,Cr and BUN of renal function in model group were significantly increased,and artemether treatment could significantly decrease Cr and BUN of renal function in model group(P<0.05).PAS staining of kidney tissue have showed obvious glomerulosclerosis and tubule segmental dilatation in the model group.Compared with the model group,the glomerular structure was recovered and the cast of renal tubule was reduced in the artemether group.4.The immunohistochemical staining of Kidney tissue have showed that the expression of SOD 1,SOD2 and Renalase proteins in the model group were decreased compared with that in the control group,and the expression of those proteins were obviously increased after artemether treatment.The three antibodies all expressed in cytoplasm,and showed more strongly expressing in renal tubules than in glomerulus.The results of Western Blot in renal tissue have showed that SOD1 and SOD2 in model group were significantly decreased compared with control group,and artemether group was significantly increased compared with model group(P<0.05).Compared with the control group,GPX1 in the model group showed decreasing trend(P>0.05),and that in artemether group increased significantly(P<0.05).Catalase protein expression in model group was significantly decreased compared with the control group(P<0.01),and Catalase protein showed an increasing trend after artemether treatment(P>0.05).Compared with control group,ac-SOD2 K68 and ac-SOD2 K122 came out a trend of increasing in model group,and artemether expression showed a trend of decreasing in the model group(P>0.05).The expression of Renalase protein in model group was significantly decreased compared with control group,while the expression of Renalase protein in artemether group was significantly increased compared with model group(P<0.05).There was no statistical difference in Bak protein expression in model group compared with control group,and the artemether group showed a trend of decrease compared with model group(P>0.05).Compared with the control group,the expression of Bcl-xL protein in model group showed decreased trend(P>0.05),while the expression increased significantly after artemether treatment(P<0.05).The results of qRT-PCR in renal tissue showed that the expressions of SOD1.SOD2,Catalase,GPX1 and Sirt3 mRNA in the model group were significantly decreased compared with control group,and the expressions of artemether group were significantly increased compared with the model group(P<0.05).The expressions of NGAL and KIM-1 mRNA in the model group both were significantly increased compared with the control group(P<0.05),and NGAL was significantly decreased in the artemether group compared with the model group(P<0.05),while KIM-1 showed a downward trend(P>0.05).Conclusion1.A preliminary understanding of the clinical characteristics of CRS and the distribution of TCM syndromes is of certain guiding significance for the prevention and treatment of clinical risk factors and TCM syndrome differentiation.2.Artemether could alleviate the myocardial injury,as well as ameliorate the damage of glomeruli and renal tubules of the mice induced by doxorubicin,which also suggests that the artemether might protect the cardiac and renal function in mice.Besides,through our experimental results we supposed that the molecular mechanism of aremether ameliorating cardiac and renal injury might be related to the regulation of oxidative stress,Renalase protein expression and apoptosis related signaling pathway.3.Finally,the results of this study provides a further understanding of the clinical manifestations and the TCM syndrome distribution of CRS,the TCM syndrome distribution,and the mechanism of artemether alleviating cardiorenal injury,which could provide new ideas for clinical diagnosis and treatment and experimental research. |
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