| Diabetic kidney disease(DKD)is one of the most important microvascular complications of diabetes(DM).It is a clinical syndrome characterized by persistent proteinuria and progressive decline in renal function.The most common causes of chronic kidney disease(CKD)and end stage renal disease(ESRD).There is no record of the independent disease name of diabetic kidney diseases(DKD)in the ancient books of traditional Chinese medicine.It is mostly described as "diabetes of kidney","edema","turbid urine" and "Guan Ge"based on its pathogenesis and symptoms in DKD.It is believed that this disease is the result of diabetic disease that is long-term,and the damage to the five internal organs must reach the kidney.Traditional Chinese medicine believes that"long illness enters collaterals",the kidney is the place where the collaterals gather,and its pathology is often manifested as abnormal structure and function of the kidney collaterals,so DKD is a renal collateral pathology.Tongxinluo drugs are compound Chinese patent medicines developed under the guidance of the theory of collaterals.The characteristics of "Dispelling wind,dredging collaterals,removing collaterals and blood stasis"make it widely used in microvascular diseases.The early research on DKD of Tongxinluo focused on "Sunluo-Capillaries" and glomerular lesions."Sunluo-Xuanfu",as the smallest functional structural carrier of the traditional Chinese medicine collaterals for"promoting blood and qi and operating yin and yang",there is no report on the treatment of DKD from the perspective of "Sun Luo-Xuanfu".Therefore,this topic is based on the theory of qi collaterals and "Sun Luo-Xuanfu".The treatment of DKD and the study of the mechanism of the pathogenesis of the disease with renal tubular injury as the main point have new significance.Objective:This study expounds the pathogenesis of diabetic kidney disease(DKD)from the perspective of "Sun Luo-Xuanfu" theory of qi collaterals,and elaborates the new theoretical basis for the treatment of the disease with "Tongxuan and smooth collaterals" Chinese medicine Tongxinluo.In the experimental part,the DKD early animal model db/db mice and the HK-2 injury model induced by high glucose combined with a high palmitic acid were used to observe the protective effect of Tongxinluo on the kidneys of DKD mice and the renal tubules induced by high glucose combined with a high palmitic acid.Improve the experimental evidence of Tongxinluo in the treatment of DKD,and provide a basis for the consistency of the traditional Chinese medicine theory of treating different diseases with the same treatment and the advantages of traditional Chinese medicine compound multi-targets.Method:1.Theoretical discussion:The theoretical part of this research first systematically sorts out the names,pathogenesis and treatment of DKD in Chinese medicine.Secondly,it will explain the pathogenesis of DKD and the characteristics of treatment and medication under the guidance of the Qiluo theory "Sun Luo-Xuanfu"theory.2.Experimental research:Experiment 1:Select 6-week-old SPF male C57BL/Ks mice and male C57BL/Ks-LepRdb/LepRdb db/db mice,and after adaptive feeding for 2 weeks,randomly divide the db/db mice into 3 groups according to their body weight.Namely db/db group(model group),db/db+TXL group(Tongxinluo treatment group 0.75g·kg-1d-1),db/db+Met group(metformin treatment group 0.2g·kg-1d-1).After the animals are divided into groups,the mice will be administered by gavage,once a day,each time the experimental mice are given drugs before treatment,the mice are weighed with an electronic balance,and the volume of each mouse is 0.2mL/10g.Gavage was performed for 12 consecutive weeks.Both the C57BL/Ks mice and the db/db mice without treatment were given an equal volume of 0.5%sodium carboxymethylcellulose solution by gavage.From the 0th week of administration,every 4 weeks,use the mouse metabolic cage to collect the urine for 24 hours,then measure and record the urine volume,centrifuge to save the supernatant,and store it in the-80? refrigerator for the detection of urine albumin and urine creatinine levels.At the end of the 12-week experiment,the mice were put to death after anesthesia,and blood samples were collected to detect blood glucose(GLU),urea nitrogen(BUN),blood creatinine(Scr),cholesterol(CHO),triglycerides(TG),total protein(TP),alanine aminotransferase(ALT),aspartate aminotransferase(AST).The abdominal cavity of the mouse was opened,and both kidneys were removed for subsequent hematoxylin-eosin staining(H&E staining),periodic acid-Schiff staining(PAS)staining,tunel detection,and western blot.Experiment 2:Culture human proximal renal tubular epithelial cells(HK-2)in vitro.First,the cells were divided into 8 groups,low-gluose group(5mmol/L D-Glucose,LG)and high-gluose group(25mmol/L D-Glucose,HG),high-gluose isotonic group(5mmol/L D-Glucose+20 mmol/L D-Mannitol,LGM),low-gluose combined with a low palmitic acid group(5mmol/L D-Glucose+300?mmol/L palmitic acid,LGLP),high-gluose combined with a low palmitic acid group(25mmol/L D-Glucose+300?mmol/L palmitic acid,HGLP),low-gluose combined with a high palmitic acid group(5mmol/L D-Glucose+500?mmol/L palmitic acid,LGHP),high-gluose combined with a high palmitic acid group(25mmol/L D-Glucose+500?mmol/L palmitic acid,HGHP),solvent control,use MTS detection method to study the effect of different concentrations of glucose and palmitic acid on the cell viability of HK-2 cells,and then determine the modeling conditions.Secondly,the MTS detection method was used to study the protective effect of different concentrations of Tongxinluo on HK-2 cells under glycolipid injury,and to determine the optimal concentration of Tongxinluo.Finally,the Western blot experiment was used to detect the effect of Tongxinluo on different modes of programmed cell death(apoptosis,necroptosis,pyroptosis,and ferroptosis)under glycolipid injury to clarify the Tongxinluo treatment target.Experiment 3:According to the target of Tongxinluo drug delivery determined inExperiment 2,exercise western blot experiment,go to the animal kidney of Experiment 1 to verify the target.Result:1.Theoretical research:DKD traditional Chinese medicine disease name should be called:Xiaoke disease nephropathy,the disease is located in the kidney,spleen and kidney deficiency-based,involving the heart,liver and lung,dryness and heat,phlegm and blood stasis,turbid toxin as the standard.In terms of treatment,ancient syndrome differentiation and treatment,pay attention to spleen and kidney,taking into account blood stasis;modern advocate staging combined with classification theory of treatment.Although the TCM disease name,etiology,pathogenesis,treatment principles and prescriptions of DKD have accumulated a relatively rich discussion,but the complex condition of diabetes nephropathy,deficiency and excess mixed,lingering difficult to cure the characteristics of(DKD)is still very difficult in treatment.Academician Wu Yiling systematically constructed the syndrome and treatment system of collaterals disease,formed two branches of qi theory and collaterals theory,and pointed out that collaterals disease showed a chronic pathological process from meridians into collaterals,from qi and blood,and from functional lesions to organic diseases.And most of them have the characteristics of long course of disease,repeated attacks of pain and difficult to cure.Long-term disease into collaterals,long-term disease and kidney,it is obvious that Xiaoke nephropathy belongs to the category of collateral disease research.Kidney collaterals circuitous and narrow,diabetes for a long time,dryness and heat,phlegm and blood stasis,turbid toxin invading kidney collaterals,then easy to stagnation and blood stasis,easy to accumulate,causing kidney collateral damage,causing diabetes nephropathy.Academician Wu Yiling took the lead in the study of treating nephropathy of thirst with collaterals for(DKD).He believed that deficiency of both qi and yin was the basis of DKD,stasis of collaterals,accumulation of fluid coagulation and phlegm were the main pathological links of DKD,and accumulation of collaterals was the main pathological changes of DKD.The previous studies of Academician Wu Yiling and his team on nephropathy(DKD)of Xiaoke disease mainly focused on its pathogenesis from the point of view of collaterals theory.This study further explained the pathogenesis and treatment of nephropathy(DKD)from the point of view of "Sun Luo-Xuanfu",and proposed that Tongxinluo can improve the opening and closing of water valley due to the stagnation of "Sun Luo-Xuanfu" in the development of DKD.Leakage is a symptom of turbid urine(albuminuria)."Tongxuan Changluo" is its treatment principle,we should pay attention to the kidney is the foundation of sealing,tonifying deficiency to help clear the kidney collaterals,on the basis of solid protection of the body fluid of essence and blood in the kidney,with the medicine of opening and opening Xuanfu stagnation,and pay attention to regulating the rise and fall of Zang-fu qi,always "pass" and "tonify" method,in order to restore the structure and opening and closing function of kidney "Sun Luo-Xuanfu",the opening and closing of "Sun Luo-Xuanfu" of kidney collaterals has a certain degree of opening and closing,then kidney collaterals are fluent..2.Experimental research:Experiment 1:The results of this part of the experiment confirm that db/db mice(BKS background)are an early DKD animal model.TXL can reduce the urine protein excretion of db/db mice,improve kidney morphological abnormalities,reduce the expression of Col I and Col ?,and inhibits the DNA fragmentation of the intrinsic cells of the kidney,but has no effect on lowering blood sugar and weight.It is suggested that the protective effect of TXL on DKD kidney may be the inhibition of programmed cell death of renal tubular epithelial cells.Experiment 2:In vitro experiments showed that high glucose(25mmol/L glucose,HG)had no effect on the survival activity of HK-2 cells,but when high glucose combined with high palmitic acid(25 mmol/L glucose+500 ?mmol/L palmitic acid,HGHP),the cell viability decreased significantly.The expression of HK-2 apoptosis-related protein pro-caspase-3,cleavedcaspase-3,Bax(pro-apoptotic factor)was significantly increased,while Bcl-2(anti-apoptotic factor)and Bcl-2/Bax ratio were significantly decreased under high glucose and high glucose combined with high palmitic acid.It is suggested that both HG and HGHP can induce apoptosis in HK-2 cells,but the degree of apoptosis induced by HGHP is higher than that in HG group.The protein expression of caspase-1,cleaved GSDMD and IL-1? in the high glucose intervention group was significantly higher than that in the NC group.However,there was no significant difference in the expression of other scorch-related proteins between the two groups.The expressions of NF-?B p-p65,NLRP3,ASC,pro-caspase-1,cleaved caspase-1,cleaved GSDMD and IL-1? were significantly increased under high glucose combined with high palmitic acid,which suggested that high glucose combined with high palmitic acid induced pyroptosis in HK-2 cells.TXL could inhibit the expression of these proteins,and there was significant difference compared with HGHP group(p<0.05).Although there were some changes in the expression of necroptosis-related proteins MLKL and RIP3 and ferroptosis-related proteins GPX-4 and FACL-4 under the intervention of HG and HGHP,there was no significant difference compared with NC group(p>0.05).Experiment 3:Western blot results showed that the expression of pro caspase-3,cleaved caspase-3 and Bax protein in the kidney of db/db mice in the model group was up-regulated,while the expression of Bcl-2 protein was down-regulated and Bcl-2/Bax was decreased.Compared with the normal C57BL/Ks group,the difference was statistically significant(p<0.05),indicating that apoptosis was activated.In the kidney of db/db mice treated with TXL,the expression of pro caspase-3,cleaved caspase-3 and Bax protein decreased,while the expression of Bcl-2 protein increased and Bcl-2/Bax increased,which was significantly different from that of db/db mice in the model group,suggesting that apoptosis was significantly inhibited.The expression of NF-?B p-p65,NLRP3,pro caspase-1,cleaved caspase-1 and IL-1? protein in kidney of db/db mice in model group was up-regulated,which was significantly higher than that in normal group(p<0.05),indicating that pyroptosis was activated.The expression of NF-?B p-p65,NLRP3,pro caspase-1,cleaved caspase-1 and IL-1?protein in kidney of db/db mice treated with TXL was significantly lower than that of db/db mice in model group(p<0.05),which suggested that the activation of pyroptosis was inhibited.Conclusions:1.This study sorted out the name,pathogenesis,treatment and treatment of DKD in Chinese medicine,and for the first time discussed the treatment of DKD from the perspective of"Sun Luo-Xuanfu" theory of qi collaterals,and considered DKD is a renal collateral disease,and its pathological changes are consistent with the pathological changes of the kidney"Sunluo-Xuanfu" due to stagnation and stagnation,phlegm,stasis,turbidity,and autism.It also puts forward the treatment method of "Tongxuan and smooth collaterals",the treatment is combined with eliminating Pathogenic and tonic,good use of pungent medicine,and the method of Tongxuan and Guxuan must be distinguished.Tongxinluo's whole prescription Xin San,Ganrun,and Wentong all play the power of Tongxinluo,taking into account the specimens.It is longer than Tongxuan Changluo and can cut the pathogenesis of DKD.2.Tongxinluo can significantly reduce the urinary protein excretion of db/db mice in the early animal model of DKD,improve the changes of kidney morphology,and protect renal function.Its role is to inhibit renal tubular epithelial cell apoptosis and pyroptosis in diabetic conditions.3.The lipotoxicity in the diabetic state is more worthy of attention than the gluose toxicity. |
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