Study On The Distribution Characteristics Of TCM Syndromes Of Diabetic Nephropathy And The Intervention Mechanism Of "removing Symptoms And Dissipating Knots"

With worldwide epidemic of diabetes mellitus,diabetic kidney disease(DKD)which is one of the most frequent and severe microvascular complications has become a serious challenge for the socio-economic and public health development problem in China as well as the rest of the world.Over time,the current therapeutic measures of DKD are not adequate and lack of a breakthrough.The traditional Chinese medicine(TCM)’s knowledge of the disease etiology and pathogenesis has constantly enriched and deepened.TCM has accumulated a wealth of experience in the prevention and treatment of DKD.The miniature mass of renal collateral(Shen Luo Wei Xing Zheng Jia)is a classical microscopic syndrome differentiation of DKD.The treating methods of abating distension or fullness and disintegrating the mass(Xiao Zheng San Jie)which is based on the theory of the miniature mass of renal collateral,is effective in clinical practices and fundamental experiments.The first part of this study is based on the theory of the miniature mass of renal collateral to observe the disease characteristics and traditional Chinese Medicine(TCM)syndrome distribution among different stages of DKD,and to explore the evolution laws of TCM syndrome.As to further explain the macroscopic pathogenesis of the miniature mass of renal collateral and provide evidence for the curative effect of the treating methods of abating distension or fullness and disintegrating the mass(Xiao Zheng San Jie).The pathogenesis of DKD is complicated.Currently,the available treatments couldn’t prevent the disease progress to end stage renal disease(ESRD)effectively.The advantage and curative effect of TCM are desiderated to be confirmed.The mechanism of TCM is lack of further research.The second part of this study is based on the theory that Sphkl/S1P signaling pathway mediates DKD pathogenesis,to study the specific mechanism of Xiao Zheng San Jie formula for rat mesangial cell(MC)under the stimulation of high glucose.Objective:1.Based on the theory of the miniature mass of renal collateral to observe the disease characteristics and distribution of TCM syndrome among different stages of DKD,meanwhile to study the correlation between TCM syndrome and the laboratory examination results.To further explain the macroscopic pathogenesis of the miniature mass of renal collateral,and to analyze the macro performance of the theoretical system of the miniature mass of renal collateral.2.Based on the theory that Sphk1/S1P signaling pathway mediates DKD pathogenesis,to investigate the effects of SphK1/S1P signaling pathway on high glucose induced extracellular matrix(ECM)expression in glomerular MCs,and to research the specific mechanism of Xiao Zheng San Jie formula to the MCs as well as SphK1/S1P signaling pathway under the stimulation of high glucose.In order to provide a basis for further study of the efficacy of Xiao Zheng San Jie formula.Methods:1.The general information and syndrome data of 324 patients who were clinically diagnosed with DKD were obtained and the disease characteristics among different stages of DKD were analyzed.Based on the theory of the miniature mass of renal collateral,the TCM syndromes were classified as deficiency of qi,deficiency of yin,deficiency of both qi and yin,deficiency of both yin and yang,as well as qi stagnation,accumulation of heat,dampness-heat,turbidity toxin and blood stasis.According to the different stages of the disease,the correlation between TCM syndrome scores and laboratory indexes were investigated.As for important indicators,stepwise regression analyses were carried out.In addition,the expression of Sphk1 in normal subjects,patients with diabetes and patients with different stages of DKD were explored.2.The rat MC(HBZY-1)was cultivated by different concentrations of glucose(5.6～50mmol/L)for different times(24h、48h and 72h).The proliferation activities were tested by MTT assay and cell cycle changes were tested by FCM(flow cytometry).Further stimulated the rat MCs by high glucose of 30mmol/L for 24 hours and 48 hours respectively,and tested the expression of extracellular matrix(ECM)and Sphk1/S1P signal pathway at each time point by real-time PCR and western blot.3.Cultivated the rat’ MCs by serum containing Xiao Zheng San Jie formula in different concentration or serum containing irbesartan or S1PR2 inhibitor(JTE-013)respectively,along with high glucose(30mmol/L)for 48 hours.The proliferation activities were tested using MTT assay,the cell cycle changes were tested by FCM(flow cytometry),the cell hypertrophy was estimated by total protein content/total count of cells,the expression of ECM and Sphk1/S1P signal pathway were tested by both real-time PCR and western blot.Results:1.Most patients of DKD were elderly patients and visceral obesity was ubiquitous.The impairment of renal function and urinary protein levels were not paralleled in part of patients.the disease progress was accompanied by declined blood glucose,elevated blood pressure,declined kidney function,poor nutrition as well as decreased TT3 and FT3.2.The TCM syndromes of DKD varied in different stages of disease.Deficient syndromes in early and medium stages of DKD were mainly deficiency of both qi and yin,deficient syndromes in advanced stage of DKD were mainly deficiency of both yin and yang.The excessive syndromes were sophisticated.In the early stages of DKD,the TCM syndromes were deficiency of both qi and yin as primary syndrome with accompanying syndromes of qi stagnation,accumulation of heat and blood stasis.The accumulation of heat syndrome was positively correlated with the level of postprandial glucose and eGFR(r2hPBG=0.220,reGFR=0.193).In the medium stage of DKD,the TCM syndromes were deficiency of both qi and yin as primary syndrome with accompanying syndromes of qi stagnation,dampness-heat and blood stasis.The dampness-heat syndrome was positively correlated with the level of postprandial glucose as well as HbAlc(r2hPBG=0.268,rHbA1c=0.247).The relationship between dampness-heat syndrome integral and HbAlc level could be described as y=7.991+0.112x.In the advanced stage of DKD,the TCM syndromes were deficiency of both yin and yang as primary syndrome with accompanying syndromes of qi stagnation,turbidity toxin and blood stasis.The turbidity toxin syndrome was correlated with eGFR and 24-UTP(reGFR=-0.351,r24UTP=0.441).The relationship between the syndrome integral of turbidity toxin and the level of eGFR could be described as y=32.249-0.874x.The blood stasis syndrome was positively correlated with 24-UTP(r=0.373).The relationship between the syndrome integral of turbidity toxin and blood stasis as well as the level of 24-UTP could be described as y=1.392+0.104x,+0.093x2(y is the level of 24-UTP,X1 is the syndrome integral of turbidity toxin,X2 is the syndrome integral of blood stasis).3.Compared with normal subjects,the content of serum Sphk1 was elevated in patients with diabetes and declined in patients with DKD.In the early stages of DKD,the Sphk1 was negatively correlated with the level of serum albumin(r=-0.413).In the medium stage of DKD,the Sphk1 was positively correlated with the level of ESR(r=0.451).In the advanced stage of DKD,the Sphk1 was positively correlated with the level of diastolic blood pressure and hemoglobin(rDBP=0.376,rHGB=0.365),as well as negatively correlated with the level of HbAlc and hs-CRP(mbA1c=-0.463,rhs-CRP=-0.380).4.The effects of high glucose in MC proliferation and the abnormal changes of the cell cycle were related with glucose concentration and stimulation time length.In the early stage,the change of rat MCs was mainly cell proliferation and cell hypertrophy was mainly occurred in the later period.The excessive secretion of ECM in rat MCs mediated by high glucose might be caused by both cell proliferation and cell hypertrophy.5.The Sphk1/S1P signal pathway in rat MCs could be activated by high glucose(30mmol/L)along with the elevated expression of TGF-β land NF-κB(p65).Inhibition of S1PR2 activity could down-regulate the expressions of FN,ColIV,TGF-β 1 and NF-κB(p65)in rat MCs under high glucose.6.Cultivated the MCs by serum containing Xiao Zheng San Jie formula in different concentration and S1PR2 inhibitor(JTE-013)respectively,along with high glucose(30mmol/L)for 48 hours.Compared with high glucose group,the MCs in the Xiao Zheng San Jie formula group had lower proliferation activities,alleviative cell hypertrophy,with more cell cycle arrested in G 1 phase,as well as down-regulated expression of FN,ColIV,TGF-β1,NF-κB(p65),Sphk1 and S1PR2.The holistic intervention effect of Xiao Zheng San Jie formula is much better than JTE-013.Conclusions:1.The TCM syndromes of DKD varied in different stages of disease.As the disease progress,the deficient syndromes evolve from qi and yin deficiency to yin and yang deficiency,with the continuous and dynamic evolution of accompanying syndromes among qi stagnation,accumulation of heat,dampness-heat,turbidity toxin and blood stasis.They were both the microcosmic pathogenesis of the miniature mass of renal collateral(Shen Luo Wei Xing Zheng Jia)and the macro pathogenesis of DKD performed as the TCM syndromes.2.Compared with normal subjects,the content of serum Sphk1 was elevated in patients with diabetes and declined in patients with DKD.The correlation between Sphk1 and disease were diversed in different stages of the disease.3.Xiao Zheng San Jie formula could not only inhibit the high glucose-induced MC proliferation,cell hypertrophy and the abnormal changes of the cell cycle,but also downregulate the excessive expressions of FN,ColIV,TGF-β1,NF-κB(p65),Sphk1 and S1PR2 in rat MCs under high glucose.The Sphk1/S1P signal pathway might be one of the complex intervention mechanisms of Xiao Zheng San Jie formula in DKD.The treating method of abating distension or fullness and disintegrating the mass(Xiao Zheng San Jie)based on the theory of the miniature mass of renal collateral had superior efficiency and characteristic of multiple targets.