Study On The Mechanism By Which Hei Di Huang Wan Regulate Homing Of BMSCs And Apoptosis Of RTECs In 5/6 Nephrectomized Rats Based On IGF-1

Objective:1.To explore the characteristics of the prescription pattern of Hei Di Huang Wan(HDHW)in ancient and modern medical cases by using modern data mining statistical techniques and methods,and to provide ideas for the clinical application of HDHW.2.To explore the effect of HDHW on the proliferation and migration of bone marrow mesenchymal stem cells(BMSCs)based on insulin-like growth factor(IGF-1).3.To observe the effect of HDHW on the homing of transplanted BMSCs in the kidney of 5/6 nephrectomy model rats.4.To explore the effect and mechanism of HDHW on the apoptosis of renal tubular epithelial cells(RTECs)by regulating the migration of BMSCs based on IGF-1.Methods:1.Using"Hei Di Huang Wan","Hei Di Huang Tang"and"Di Huang Wan"as the search keywords,we searched the relevant literature through the Chinese medical dictionary software and the full-text database of journals on China Knowledge Network,and then The medical cases were supplemented by the ancient and modern medical case cloud platform and Wanfang database.The medical cases that met the inclusion criteria were entered into an Excel sheet to form a database,and data mining techniques and methods were used to conduct statistical analyses such as frequency,percentage,association,and clustering through the ancient and modern medical case cloud platform to explore the prescription patterns of the application of HDHW by ancient and modern medical practitioners in clinical evidence.2.Primary BMSCs were extracted by whole bone marrow cell apposition screening method,and then were identified.The effect of HDHW-containing serum on the proliferation of BMSCs was examined by MTT assay and screened for optimal action concentration.The effect of HDHW-containing serum on the regulation of migration of BMSCs was examined by scratch assay and Transwell migration assay,and the effect of HDHW on autocrine IGF-1 of BMSCs was examined by enzyme-linked immunosorbent assay(ELISA).The expression of IGF-1,CXC chemokine receptor 4(CXCR4),and stromal cell-derived factor-1(SDF-1)in BMSCs was examined by Western blot to explore the potential mechanisms by which HDHW promotes migration in BMSCs.3.Determine the optimal infection conditions for lentiviral transfection of rat primary BMSCs.Then the green fluorescent protein-labeled mNG-Luc-BMSCs stable transfection strain was constructed and its bioluminescence efficiency was detected by in vitro bioluminescence assay.A 5/6 nephrectomy model rat was constructed and divided into model group,BMSCs group,BMSCs+HDHW group,and another normal group.The distribution of mNG-Luc-BMSCs injected into the rats via tail vein was observed in vivo using a small animal live imaging system,and the number of mNG-Luc-BMSCs homing in the kidney tissue was observed by frozen section of rat kidney.Serology was also used to detect the renal function,including blood creatinine and urea nitrogen levels,in each group of rats.4.Primary rat RTECs were extracted and identified according to literature methods.A hypoxia/reoxygenation(H/R)method was used to construct an apoptosis model of HR-RTECs.BMSCs were co-cultured with HR-RTECs using the Transwell system,and HDHW-containing serum was used to interfere with BMSCs in the upper chamber of the co-culture system.After 48h of intervention,the number of BMSCs cells migration to HR-RTECs was detected by 1%crystalline violet staining solution,the apoptosis rate of RTECs in each group was detected by Annexin V-FITC Apoptosis Detection Kit,and the amount of IGF-1 in the supernatant of each cell culture was detected by ELISA.The expression levels of IGF-1 and CXCR4 in BMSCs,and apoptosis proteins Bax,Bcl-2,and Caspase3in RTECs were detected by Western blot.Results:1.Through data mining and analysis,the following medication patterns of HDHW were obtained:the etiology is mostly related to the weakness of the body after a long illness,lack of relaxation,diet,loss of treatment and mismanagement,and postpartum;the core symptoms are loose stools,cough,swelling of the lower limbs,shortness of breath,weakness,light or dark tongue,thin or white or greasy coating,sunken or fine or counted pulse;the main disease categories are Qi,blood and fluid disease,kidney system and lung system,and the main diseases include Deficiency labor,edema,spermatorrhea,cough,blood in stool,stroke,urinary turbidity,etc.;Chinese medicine symptoms are mostly spleen and kidney deficiency,spleen and kidney yang deficiency,kidney yin deficiency,and dampness obstruction is a common combined symptom of HDHW.When HDHW is used in clinical practice,there are few single prescriptions to treat the disease,and chemically tailored prescriptions are the most common.HDHW is also often used in combination with Buzhongyiqi Tang,Jinshuiliujun Jian,Guipi Wan,and Fuzilizhong Wan.The commonly used doses of the original HDHW decoction for internal use are Shu Di Huang18g,Cang Zhu10g,Gan Jiang6g,Da Zao6g(2 pieces),and Wu Wei Zi10g.Correlation analysis showed that when using HDHW for the treatment of deficiency labor disease,HDHW is often used with drugs that nourish the heart and spleen,benefits Qi,and nourish Yin.In the treatment of edema,HDHW is often used with drugs that warm the Yang,strengthen the spleen,and induce diuresis.In the treatment of seminal emission,HDHW is often used with sour and sweet astringent drugs to nourish the heart and spleen.When using HDHW for the treatment of spleen and kidney deficiency,HDHW can be supplemented with drugs that regulate qi and strengthen the spleen,such as Dang Shen,Fu Ling,Shan Yao,Huang Qi,Ze Xie,Chen Pi,etc.For the treatment of spleen-kidney-yang deficiency,Bai Zhu and Fu Zi were added as appropriate.Cluster analysis showed that cough,asthma,and coughing sputum were common symptom indications for the use of HDHW in the treatment of pulmonary pathologies,and blood in the stool,loose stools,and spontaneous sweating were common symptom indications for the use of HDHW in the treatment of qi,blood,and fluid pathologies.The common symptoms in HDHW cases include swelling of the lower extremities,abdominal fullness,weakness,and dullness,and the common symptoms in the treatment of kidney-related pathologies with HDHW,including Shu Di Huang and Wu Wei Zi to nourish the kidney and nourish Yin,Cang Zhu,Gan Jiang,Bai Zhu,and Fu Ling to warm Yang and promote water,and Mai Dong,Dang Shen,Fu Shen,and Shan Yao to benefit Qi and nourish Yin.2.The results of the MTT experiment suggested that HDHW could promote the proliferation of BMSCs,and the optimal intervention concentration was 5%,which was used as the intervention concentration for HDHW follow-up experiments.The results of the scratch assay showed that HDHW significantly promoted scratch healing(P<0.01);the results of the Transwell migration assay showed that HDHW significantly increased the migration number of BMSCs(P<0.01),while IGF-1R inhibitor significantly inhibited the pro-scratch healing and pro-migration effects of HDHW on BMSCs.The above results indicated that HDHW could promote the migration of BMSCs,and this process was regulated by IGF-1.ELISA results showed that HDHW significantly increased the IGF-1content in cell culture supernatants(P<0.01),and Western blot assay results showed that HDHW could upregulate the expression of IGF-1 in BMSCs(P<0.01),which in turn caused high expression of CXCR4,a chemokine on the surface of BMSCs(P<0.05),and downregulated the expression of SDF-1 in BMSCs(P<0.05).Simultaneous use of IGF-1R inhibitor validated the mechanism.Thus,HDHW can promote the responsiveness of BMSCs to SDF-1 by promoting autocrine IGF-1 in BMSCs,which in turn increases the expression of CXCR4,the surface antigen of BMSCs,and ultimately mobilizes the migration of BMSCs.3.The optimal infection ratio for lentiviral transfection of rat primary BMSCs was80.2ug/ml was the final screening concentration of puromycin and the duration of action was 72h.mNG-Luc-BMSCs fluorescence intensity was low within 48h of transfection,and the best fluorescence expression was achieved after 72h.The in vitro bioluminescence efficiency assay showed that the bioluminescence intensity of mNG-Luc-BMSCs increased with the increase of cell number,and there was a high linear correlation between the luminescence intensity and cell number(R~2>0.95).Small animal live imaging showed that HDHW promoted the homing of BMSCs injected into the model rats via tail vein.Fluorescence observation of frozen sections confirmed that HDHW promotes homing of BMSCs to damaged kidneys.The results of serological tests showed that both tail vein injection of BMSCs and tail vein injection of BMSCs combined with HDHW improved the renal function of the model rats,and the latter could reduce the urea nitrogen level more significantly(P<0.01).4.In this study,we successfully extracted rat primary RTECs and successfully constructed the HR-RTECs apoptosis model.After co-culture of BMSCs with HR-RTECs,the results of 1%crystalline violet staining showed that HDHW significantly promoted the migration of BMSCs to HR-RTECs(P<0.01);the results of Annexin V-FITC apoptosis assay showed that the live cell ratio of RTECs in the HDHW intervention group was significantly increased(P<0.01),while the total apoptosis rate was significantly decreased(P<0.01),with a more significant decrease in the rate of cells in the early apoptotic phase(P<0.01),suggesting that BMSCs after HDHW intervention could inhibit the apoptosis of RTECs by reversing the early apoptosis of RTECs.In contrast,IGF-1R inhibitor significantly inhibited the regulation of migration of BMSCs and apoptosis of RTECs by HDHW,suggesting that the regulation of migration of BMSCs and apoptosis of RTECs by HDHW is closely related to IGF-1.ELISA results confirmed that HDHW promoted IGF-1 secretion in BMSCs(P<0.01).Western blot assay results confirmed that HDHW increased IGF-1 expression in BMSCs(P<0.05)and upregulated the expression of CXCR4,the surface antigen of BMSCs(P<0.05).It was also able to down-regulate the expression of pro-apoptosis-related proteins Bax and Caspase3 in RTECs(P<0.05)and up-regulate the expression of anti-apoptosis-related protein Bcl-2(P<0.01).Thus,HDHW could promote the migration of BMSCs to apoptotic RTECs through the SDF-1/CXCR4 axis by increasing the autocrine IGF-1 level of BMSCs on the one hand;on the other hand,it could upregulate the expression of anti-apoptotic protein Bcl-2 and downregulate the expression of pro-apoptotic proteins Bax,Caspase3,reversing the early apoptosis of RTECs.Conclusion:1.The data of HDHW medical cases were analyzed by the ancient and modern medical case cloud platform,and the preliminary characteristics of the prescription pattern of HDHW were summarized to provide a reference for the clinical promotion and application of HDHW.2.HDHW can promote the responsiveness of BMSCs to SDF-1 by promoting the autocrine IGF-1 of BMSCs,which in turn increases the expression of CXCR4,the surface antigen of BMSCs,and thus mobilizes the migration of BMSCs.3.HDHW promotes the homing of transplanted BMSCs to the damaged kidneys of5/6 nephrectomized rats,which in turn improves renal function.4.The mechanism by which HDHW promotes the homing of BMSCs to improve renal injury may be that HDHW increases the autocrine IGF-1 of BMSCs and promotes the migration of BMSCs to damaged RTECs through IGF-1-mediated SDF-1/CXCR4 axis,while it may promote the paracrine secretion of IGF-1 from BMSCs to inhibit the apoptosis of RTECs.