Application of astilbin in stimulating mesenchymal stem cells in preparation of drugs for treating diabetic nephropathy

Abstract

The present application relates to the technical field of kidney disease treatment, and specifically provides application of astilbin in stimulating mesenchymal stem cells in preparation of a medicine for treating diabetic nephropathy. The present application provides application of mesenchymal stem cells in preparation of a medicine for treating kidney disease, wherein 5-20 μg / ml of astilbin is added to cell culture solution during subculture of the mesenchymal stem cells. The present application first discovers that the traditional Chinese medicine monomer astilbin can be used to pretreat bone marrow mesenchymal stem cells with differentiation capacity and potential for treating diabetes, greatly enhancing the treatment effect of bone marrow mesenchymal stem cells on diabetic nephropathy. Animal models also confirm that the astilbin can significantly reduce the urine protein level of a mouse model of diabetic nephropathy, improve kidney function, and significantly reduce the histopathological score of kidney tissue, thereby significantly reducing kidney damage and having a significant therapeutic effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of kidney disease treatment, and particularly relates to application of astilbin stimulating mesenchymal stem cells in preparation of a medicine for treating diabetic nephropathy. BACKGROUND

[0002] Diabetes is one of the most common and fastest growing diseases in the world, and is expected to affect 693 million adults by 2045, an increase of more than 50% compared with 2017. Macro- and micro-vascular complications (cardiovascular disease, diabetic nephropathy, diabetic retinopathy, and neuropathy) are the main causes of morbidity and mortality in diabetic patients, and have brought a huge economic burden to the country's medical expenditure (Nat Rev Nephrol.2020, 16(7):377-390.).

[0003] Nearly half of type 2 diabetes patients will develop diabetic kidney disease (DKD), which is one of the most common, most destructive, and most expensive diabetic complications, and is also one of the main causes of end-stage renal disease. And the number of people worldwide with DKD is expected to rise with the increasing incidence of diabetes. The definition of DKD is that diabetic patients have chronic kidney disease, which is characterized by a sustained increase in urinary albumin excretion (urinary albumin-to-creatinine ratio (UACR) > 30 mg / g) or a sustained decrease in glomerular filtration rate (eGFR) < 60 ml / min / 1.73 m 2 (Nat Rev Nephrol.2021, 17(4):227-244).

[0004] Under the influence of long-term blood glucose and high filtration, the pathological manifestations of diabetic nephropathy can include early glomerular hypertrophy, homogeneous thickening of the glomerular basement membrane, and later glomerular sclerosis with Kimmelstiel-Wilson nodules, microaneurysm formation, significant arterial hyalinization and fibrous cap formation, and renal cystic glassy degeneration (J Am Soc Nephrol.2010, 21(4):556-63).

[0005] Although strict blood glucose and blood pressure control can delay the occurrence of DKD, it cannot completely prevent the progression of DKD, and DKD will inevitably progress to end-stage renal disease. End-stage renal disease currently has only two ways, hemodialysis and kidney transplantation, and hemodialysis will significantly affect the quality of life of patients, while kidney transplantation has the problem of shortage of kidney sources. The pathogenesis of DKD is also very complex, therefore, exploring the pathogenesis of DKD and finding new potential therapeutic targets and treatment methods for DKD are hotspots of research at home and abroad.

[0006] Mesenchymal stem cells (MSCs) are an important member of the stem cell family. MSCs are plastic-adherent cells that can be expanded in vitro and have the ability to self-renew and differentiate. They also have unique immune regulatory and immune tolerance inducing functions. MSCs include bone marrow mesenchymal stem cells (BMMSCs), adipose-derived mesenchymal stem cells (ADMSCs), umbilical cord blood mesenchymal stem cells (UCMSCs), human placental-derived mesenchymal stem cells (PDMSCs), Wharton's jelly-derived MSCs (WJMSCs), and dental mesenchymal stem cells (DMSCs). The International Society for Cellular Therapy (ISCT) has proposed the minimum criteria for MSC definition, which include plasticity, trilineage and differentiation potential; positive expression of cell surface markers CD105, CD73, and CD90, and negative expression of CD45, CD34, CD14 or CD11b, CD79a or CD19, and HLA-DR; and the ability to differentiate into osteoblasts, adipocytes, and chondroblasts under in vitro culture conditions (Front Cell Dev Biol.2022, 10:910592).

[0007] The most commonly used cell type for treating chronic kidney disease is BMMSC, as BMMSC has low expression of MHC I and MHC II, thus avoiding being attacked by allogeneic T cells. Currently, BMMSCs have been considered as an exploratory means for treating diabetic nephropathy, and experiments have shown that they have certain kidney protective effects on diabetic nephropathy: they can reduce the damage of high glucose to podocytes and reduce podocyte apoptosis; they can delay the progression of DKD by improving systemic and local inflammation in the kidney; and they can inhibit the apoptosis of renal tubular epithelial cells by reducing cellular oxidative stress, thus protecting the structure and function of the renal tubule. Studies have also shown that BMMSCs can differentiate into mesangial cells (Stem Cell Res Ther.2019;10(1):329;Semin Nephrol.2016, 36(3):174-88.). The application of stem cell technology in the treatment of experimental DKD has made great progress in animal models and can become one of the effective solutions for DKD treatment.

[0008] Traditional Chinese medicine is the treasure of the Chinese nation, and traditional Chinese medicine is our precious medical treasure. Traditional Chinese medicine is also an important source for the development of new drugs. Extracting traditional Chinese medicine monomers from natural traditional Chinese medicine plants and studying their effects on diseases are of great significance for further elucidating the mechanism of action of traditional Chinese medicine and expanding the influence of traditional Chinese medicine in life science and technology.

[0009] At present, the drug treatment of diabetes and diabetic nephropathy is still mainly through improving insulin resistance, enhancing insulin sensitivity and other methods, but long-term use of these drugs can cause edema, weight gain, and even liver damage in patients.

[0010] Current studies have shown that mesenchymal stem cells have a certain protective effect on diabetic nephropathy, can alleviate kidney cell damage in animal models of diabetic nephropathy, and improve microalbuminuria and renal pathological damage, and its mechanism may be through direct repair of kidney cell damage, regulation of inflammation and immune response, anti-fibrosis and through paracrine to achieve. Although bone marrow mesenchymal stem cells can improve the symptoms of diabetic nephropathy, when bone marrow mesenchymal stem cells are used alone, they have some problems and often fail to achieve the desired therapeutic effect, such as low survival rate of MSCs cultured in vitro after intravenous injection in the recipient, low homing rate of MSCs after transplantation, and insufficient MSCs to play a role in regeneration and repair and immune regulation.

[0011] And more and more views believe that diabetic nephropathy is an inflammatory disease caused by abnormal metabolic state, but the severe inflammatory response in the body of diabetic nephropathy affects the activity of MSCs, therefore, how to further improve the effect of MSC treatment and how to promote MSCs to better play the role of immune regulation are problems worthy of in-depth exploration.

[0012] Improving the function of MSCs by traditional Chinese medicine and traditional Chinese medicine monomers, and treating diabetic nephropathy by cell therapy, may become a therapy with less side effects, which is helpful for patients to accept, and has significant curative effect. In view of this, the present application is proposed. SUMMARY

[0013] Traditional Chinese medicine pretreatment of MSCs is a simple and rapid method, and different biological effects can be achieved by changing the concentration and action time. However, there is no research on whether rhodionin has immune regulation function on MSCs and whether MSCs pretreated by rhodionin can play a better therapeutic effect on diabetic nephropathy.

[0014] The application provides application of rhodionin-stimulated mesenchymal stem cells in preparation of a drug for treating diabetic nephropathy.

[0015] In the first aspect, the application provides application of mesenchymal stem cells in preparation of a drug for treating nephropathy, wherein 5-20 mu g / ml of rhodionin is added to the cell culture solution in the subculture process of the mesenchymal stem cells.

[0016] In the application of mesenchymal stem cells in preparation of a drug for treating nephropathy, the mesenchymal stem cells are bone marrow mesenchymal stem cells, adipose-derived mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, human placenta-derived mesenchymal stem cells and dental mesenchymal stem cells.

[0017] In the application, the mesenchymal stem cells are used in the preparation of a drug for treating kidney disease caused by diabetes complications.

[0018] In the application, the mesenchymal stem cells are used in the preparation of a drug for treating kidney disease caused by diabetes complications.

[0019] In the application, the mesenchymal stem cells are used in the preparation of a drug for treating kidney disease caused by diabetes complications.

[0020] In the application, the mesenchymal stem cells are used in the preparation of a drug for treating kidney disease caused by diabetes complications.

[0021] More specifically, the mesenchymal stem cells are bone marrow mesenchymal stem cells.

[0022] In the application, the mesenchymal stem cells are used in the preparation of a drug for treating kidney disease caused by diabetes complications.

[0023] In the application, the mesenchymal stem cells are used in the preparation of a drug for treating kidney disease caused by diabetes complications.

[0024] In the application, the mesenchymal stem cells are used in the preparation of a drug for treating kidney disease caused by diabetes complications.

[0025] The application has the following advantages:

[0026] The application first discovers and proposes that a Chinese medicine monomer, astilbin, is used to pre-treat bone marrow mesenchymal stem cells which have differentiation ability and potential for treating diabetes, so that the treatment effect of the bone marrow mesenchymal stem cells on diabetic nephropathy is greatly enhanced. It is also proved by an animal model that the urine protein level of a mouse model of diabetic nephropathy can be obviously reduced, the kidney function is improved, the histopathological score of the kidney tissue is obviously decreased, and the kidney damage is obviously reduced, so that the application has a significant curative effect.

[0027] The present application first found that the concentration of astilbin is 20 μg / ml to culture MSC for 48h, which has a significant promoting effect on the growth of MSC. Moreover, the effect of MSC treated by astilbin with the concentration of 20 μg / ml on treating diabetic nephropathy is better. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 is the cell growth condition of MSC under different concentrations of astilbin detected by real-time cell analysis system in the present application.

[0029] Figure 2 is the cell activity of MSC treated by different concentrations of astilbin for 48h detected by CCK8 method in the present application.

[0030] Figure 3 is the result graph of the urine microalbumin (ACR) of diabetic nephropathy mice obviously reduced by astilbin pretreated bone marrow mesenchymal stem cells.

[0031] Figure 4 is the result graph of the blood creatinine reduced by astilbin pretreated bone marrow mesenchymal stem cells compared with the MSC group without astilbin treatment.

[0032] Figure 5 is the result graph of the urea nitrogen reduced by astilbin pretreated bone marrow mesenchymal stem cells compared with the MSC group without astilbin treatment.

[0033] Figure 6 is the result graph of the 24h urine protein quantification reduced by astilbin pretreated bone marrow mesenchymal stem cells compared with the MSC group without astilbin treatment.

[0034] Figure 7 is the PAS staining graph of kidney tissue section detection of different experimental groups. DETAILED DESCRIPTION

[0035] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be described clearly and completely below in combination with the drawings in the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0036] Embodiment 1

[0037] The present application provides the culture of mouse bone marrow mesenchymal stem cells (BMMSC).

[0038] BMMSC were purchased from Sanyei (Guangzhou) Biotechnology Co., Ltd. and cultured in mouse bone marrow mesenchymal stem cell complete medium containing 10% premium fetal bovine serum and penicillin / streptomycin in a 37°C incubator (5% CO2) with medium changed once a day. When the cell density reached 80%-90%, the cells were digested with 0.25% trypsin and subcultured at a ratio of 1:3-4. The cells were subcultured according to the above method and inoculated according to the required cell concentration for detection.

[0039] Example 2

[0040] This example provides the effect of astilbin on MSC growth.

[0041] MSC cells were monitored for cell proliferation using a real-time cell analysis (RTCA) system (ACEA Biosciences, USA).

[0042] 1) First, 50 μL of cell complete medium was added to a real-time microelectronic detection plate, and then the detection plate was placed in an RTCA instrument to measure the baseline of the reaction.

[0043] 2) BMMSC in the logarithmic growth phase were digested with trypsin for 3 min, then collected in a sterile centrifuge tube, and the reaction was terminated by adding serum-containing medium. After centrifugation, the cells were collected and counted.

[0044] 3) BMMSC were inoculated in the real-time microelectronic detection plate at a cell amount of 2 x 10 3 cells / 100 μL.

[0045] 4) After the cells were incubated for 30 min, the real-time microelectronic detection plate was placed in the RTCA instrument to record the growth index of the cells in real time.

[0046] 5) After the cells adhered, different concentrations of astilbin (0, 5, 10, 20, 40, 80) μg / ml were added to the real-time microelectronic detection plate. After incubation for 30 min, the RTCA instrument was used to continue recording the growth index of BMMSC treated with different concentrations of astilbin in real time.

[0047] It was found through control detection that astilbin at a concentration of 5 μg / ml or 10 μg / ml promoted the growth of MSC slightly, and astilbin at a concentration of 20 μg / ml promoted the growth of MSC significantly after culturing MSC for 48 h, as shown in Figure 1 .

[0048] Moreover, MSC treated with astilbin at a concentration of 20 μg / ml had a better effect on treating diabetic nephropathy, as shown in Example 4.

[0049] Example 3

[0050] This example uses CCK8 method to measure cell viability, detection steps and results as follows:

[0051] BMMSCs growing to about 90% were digested into sterile centrifuge tubes with trypsin, and the reaction was terminated by adding serum-containing medium. After centrifugation, the cells were collected and counted.

[0052] Six concentration gradients were set according to 0, 5, 10, 20, 40, 80 μg / ml, 5 replicate wells were set for each concentration, and cells were inoculated at a cell density of 2 x 10 3 cells per well, and the final volume was 200 μL / well.

[0053] 200 μL of sterile PBS was added around the experimental wells to prevent edge effects as much as possible.

[0054] 4) After the cells were attached and grew, the culture medium in the original wells was discarded, and the wells were washed 3 times with sterile PBS. Then different concentration gradients of astilbin were added to the culture medium for continuous culture for 48 h.

[0055] 5) The culture medium in the wells was discarded, and the wells were washed 3 times with sterile PBS. Then 100 μL of culture medium and 10 μL of CCK8 reagent were added to each well, and the cells were cultured in the cell incubator for 4 h.

[0056] 6) The absorbance of each well was measured at 490 nm using an enzyme marker.

[0057] The CCK8 method further confirmed that low concentrations (5, 10, 20 μg / ml) of astilbin had no significant effect on MSC growth for 48 h, while 40, 80 μg / ml astilbin inhibited MSC growth for 48 h Figure 2 ). Therefore, the drug concentration and action time for selecting astilbin to pretreat MSCs were 20 μg / ml and 48 h, respectively.

[0058] Example 4

[0059] This example provides experimental results of mesenchymal stem cells pretreated with astilbin for the treatment of diabetic nephropathy.

[0060] (1) Animal feeding

[0061] 30 male db / db mice of 6-8 weeks old and 10 db / m mice of the same age were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The body weight range was 15-35 g. The mice were fed in a specific animal feeding room, and the room temperature environment was maintained at about 25°C, and the relative humidity was about 45%. After adaptive feeding for 1 week, the test was ready to start.

[0062] (2) Animal random grouping and administration of intervention treatment observation

[0063] The experimental animals were randomly divided into 4 groups, namely, a control group, a model group, an MSC treatment group, and an AST-MSC treatment group, each group having 10 animals.

[0064] Among them, the control group is db / m mice; the experimental objects of the model group are db / db mice that can spontaneously develop diabetes and diabetic nephropathy, and are not treated; the experimental objects of the MSC treatment group are db / db mice, and are treated by tail vein injection of bone marrow mesenchymal stem cells, once a week; the experimental objects of the AST-MSC treatment group are db / db mice, and are treated by tail vein injection of mesenchymal stem cells pretreated with astilbin, once a week.

[0065] MSC tail vein injection cell amount: 2x10 6 General condition of the mice was monitored and samples were collected, fixed, and frozen.

[0066] (3) Collection of samples and sacrifice of animals

[0067] From the 8th week, urine of the mice was collected every 2 weeks for monitoring of urine ACR (protein / creatinine ratio), and at the 14th week, the mice in step (2) of the experiment were sacrificed, 12h fasting was performed before sacrifice, and 24h urine was collected, urine volume was measured, and was frozen for subsequent detection; body weight of the mice in each group was measured, blood glucose was measured, and data were recorded; eyeball was extracted for blood collection to obtain serum for freezing and subsequent monitoring of indexes. After sacrifice, bilateral kidneys were collected, and were fixed and frozen.

[0068] (4) Comparison of urine ACR of mice in each group

[0069] Significant level P<0.05 compared with the db / db model group is indicated by “#”, and significant level P<0.05 compared with the simple MSC treatment group is indicated by “*”.

[0070] From the detection results in Table 1, at the 14th week, compared with the model group db / db mice: urine ACR of the MSC group and the AST-MSC group mice was significantly reduced, as shown in Figure 3 , and urine ACR value of the AST-MSC group mice was also significantly reduced compared with the simple MSC group.

[0071] Table 1 Comparison of urine ACR of mice

[0072] Group Control Model MSC group AST-MSC group 8 weeks 19.12±3.05 42.98±5.34 46.87±3.96 44.23±3.54 10 weeks 22.02±3.32 84.21±5.93 70.12±4.88 59.89±4.72 12 weeks 20.21±5.12 106.12±8.02 90.12±6.42 76.22±6.28 14 weeks 21.30±3.98 137.89±8.43 113.41±6.87# 85.05±6.53#*

[0073] (5) Comparison of BUN and Scr of mice in each group

[0074] Measurement results of BUN and Scr of mice in each group are shown in Figure 4 andFigure 5 As shown in Table 2 below:

[0075] Table 2 Comparison of BUN and Scr of mice in each group

[0076] Group Control Model MSC group AST-MSC group Scr 0.599±0.12 2.691±0.22 1.876±0.15# 1.53±0.13#* BUN 46.14±3.97 117.89±9.86 96.03±8.93# 84.11±7.03#*

[0077] In the table, "#" represents a significant level P < 0.05 compared with the db / db model group, and "*" represents a significant level P < 0.05 compared with the simple MSC treatment group.

[0078] From the detection results in Table 2, compared with the model group db / db mice: the BUN and Scr of the MSC group and the AST-MSC group mice were significantly reduced; and the BUN and Scr values of the AST-MSC group mice were also significantly reduced compared with the simple MSC group.

[0079] (6) Comparison of 24h urine protein quantification of mice in each group

[0080] The test results of 24h urine protein quantification of mice in each group are shown in Table 3. Figure 6 As shown in Table 3:

[0081] Table 3 Comparison of 24h urine protein quantification of animals in each group (x ± s)

[0082] Group Control Model MSC group AST-MSC group 24h urine protein quantification 48.87±9.32 280.24±30.03 229.96 ± 26.12 # ]] 180.21 ± 17.27 # *]]

[0083] In the table, "#" represents a significant level P < 0.05 compared with the db / db model group, and "*" represents a significant level P < 0.05 compared with the simple MSC treatment group.

[0084] From the detection results in Table 3, compared with the model group db / db mice: the 24h urine protein quantification of the MSC group and the AST-MSC group mice was significantly reduced; and the 24h urine protein quantification of the AST-MSC group mice was also significantly reduced compared with the simple MSC group.

[0085] (7) PAS staining for kidney morphological pathology examination

[0086] According to the following steps: paraffin section deparaffinization to water → distilled water washing → iodine alcohol 10 min → tap water flushing 10 min → Schiff's solution 10 min → running water flushing 5 min → using Harris hematoxylin or Meyer hematoxylin to stain the nucleus 3 min → hydrochloric acid alcohol differentiation 1 s → running water flushing 5 min → conventional dehydration, transparency → fixation → light microscope observation, kidney morphological pathology detection was performed on mice in each group, and the detection results are shown in Table 4. Figure 7

[0087] Among them, the db / m mouse control group: no exudation and adhesion in the glomerular capsule, no increase of PAS positive substances in the mesangial area, and no proliferation of mesangial cells.​

[0088] The db / db mouse model group: visible mesangial cell proliferation, increased mesangial area PAS positive material, uneven red staining, partial capillary compression collapse.

[0089] The MSC treatment group and the AST-MSC treatment group: glomerular mesangial cells have no obvious proliferation, and the PAS positive material is significantly reduced compared with the model group, and most of the capillaries are open, and the improvement of the AST-MSC treatment group is obviously better than that of the single MSC treatment group.

[0090] The kidney tissue section detects the PAS staining diagram of different experimental groups; the pathological changes of the kidney are observed by PAS staining, and it is observed that the model group shows obvious glomerular hypertrophy and glomerular mesangial matrix expansion in histology, it is found that the mesangial volume fraction of the mouse is significantly reduced after MSC treatment and AST-MSC treatment, and the reduction of the AST-MSC treatment is more obvious.

[0091] According to the test results of (4)-(7) above, in the present application, the 24-hour urine protein quantification, renal function and kidney structure of the mouse in the simple MSC treatment group and the AST-MSC treatment group are improved to a certain extent, and compared with the simple MSC group, the AST-MSC treatment group is obviously better in improving the renal function and reducing the kidney injury.

[0092] Finally, it should be pointed out that: the above examples are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. The application of bone marrow mesenchymal stem cells in the preparation of drugs for treating kidney disease, characterized in that, During the passage culture of the bone marrow mesenchymal stem cells, 5-20 μg / ml of astilbene was added to the cell culture medium and the cells were cultured for 30-50 hours. The kidney disease mentioned is caused by complications of diabetes.

2. A drug for treating kidney disease, characterized in that, The drug contains bone marrow mesenchymal stem cells. During the passage culture of the bone marrow mesenchymal stem cells, 5-20 μg / ml of astilbene is added to the cell culture medium and the cells are cultured for 30-50 hours. The kidney disease mentioned is caused by complications of diabetes.

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