CN115960091A - Traditional Chinese medicine monomer isoorientin for treating diabetic nephropathy - Google Patents

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and relates to a traditional Chinese medicine monomer isoorientin for treating diabetic nephropathy, which is a flavonoid compound containing a luteolin structure, is present in medicinal or edible parts of plants such as sow thistle, buckwheat, passion flower, fenugreek and the like, and has a molecular formula of C ₂₁ H ₂₀ O ₁₁ The traditional Chinese medicine monomer isoorientin is used for treating the diabetic nephropathy, has good curative effect, no toxic or side effect, wide and easily available isoorientin source and low cost.

Description

Traditional Chinese medicine monomer isoorientin for treating diabetic nephropathy

The technical field is as follows:

the invention belongs to the technical field of traditional Chinese medicines, and relates to a traditional Chinese medicine monomer isoorientin for treating diabetic nephropathy.

Background art:

diabetic Nephropathy (DN) is one of the most common and serious complications of diabetes. Recent data show that DN accounts for 1/3 of the total chronic kidney disease, and the incidence rate reaches 9.1%. And DN is also one of the major causes of end-stage renal disease. However, currently DN still lacks an effective therapeutic approach.

In clinical practice, we invented a Chinese medicinal formula for treating DN, named as 'jinhu formula for reinforcing kidney'. The formula has been used to treat more than 200 DN patients with renal insufficiency above stage III. It can significantly reduce the urinary microalbumin/creatinine ratio (UACR) and blood creatinine in patients with DN. Fenugreek is one of the important herbal monarch drugs in the formulation. According to ancient Chinese medicine records, fenugreek has long been used to treat the symptoms of renal insufficiency. Fenugreek is also used in modern chinese medicine for the treatment of chronic renal failure.

Podocyte depletion and morphological abnormalities are one of the major pathological changes of DN. Because podocytes are terminally differentiated cells that are unable to divide and self-renew, it is particularly important to protect podocytes from diabetic damage. Autophagy is an autophagy process that plays an important role in maintaining cell homeostasis. Meanwhile, autophagy is also very important for maintaining the structure, function and metabolic homeostasis of human podocytes. Under high-sugar conditions, autophagy can clear damaged mitochondria and protect cells from high-sugar damage. Autophagy defects have been shown to exacerbate diabetic kidney damage. Based on the important role of autophagy in the development of diabetic nephropathy, more than 40 active ingredients in the kidney-reinforcing prescription of the cucurbita pepo are screened to determine small-molecule compounds which can remarkably enhance autophagy and protect podocytes under the condition of high sugar. Among the screened compounds, isoorientin (ISO) performed best.

Isoorientin (ISO) is a flavonoid compound, also known as 3',4',5, 7-tetrahydroxyflavone-6-d-glucopyranoside or luteolin 6-C-glucoside, and is one of the effective components of fenugreek. Isoorientin (ISO) has been found to be useful in the prevention of metabolic syndromes such as hyperglycemia, hyperlipidemia and insulin resistance. According to the data, the therapeutic effect of Isoorientin (ISO) is attributed to its antioxidant and anti-inflammatory properties. However, the specific mechanism remains largely unclear.

The invention content is as follows:

the invention aims to overcome the defects in the prior art, provides the isoorientin serving as the traditional Chinese medicine monomer for treating the diabetic nephropathy, and discovers the potential value of the isoorientin for treating the diabetic nephropathy by verifying the hypothesis that the isoorientin reduces the damage of foot cells caused by high sugar and the related mechanism thereof.

In order to achieve the purpose, the chemical structural formula of the isoorientin is as follows:

isoorientin is a flavonoid compound containing luteolin structure, widely exists in medicinal or edible parts of herba Sonchi Oleracei, semen Fagopyri Esculenti, herba Passiflorae Caeruleae and semen Trigonellae, and has molecular formula C ₂₁ H ₂₀ O ₁₁ Can be used for treating diabetic nephropathy by enhancing mitochondrial phagocytosis and protecting podocyte mitochondria.

Compared with the prior art, the invention adopts the traditional Chinese medicine monomer isoorientin to treat diabetic nephropathy, has good curative effect, no toxic or side effect, wide and easily-obtained sources of isoorientin and low cost.

Description of the drawings:

FIG. 1 is a flow chart of in vivo experiments in an embodiment of the present invention.

FIG. 2 is a flow chart of in vitro experimental work in an embodiment of the present invention.

Fig. 3 is an expression diagram of isoorientin ISO regulated SGLT-2 according to an embodiment of the present invention, wherein a is a result of detecting protein expression of SGLT-2, and B is a result of statistical analysis.

Figure 4 is a graph of the protection of podocytes from mercury induced injury by enhanced autophagy of Isoorientin (ISO) in an example of the invention, (a) exposure of podocytes to Isoorientin (ISO) molecules (0, 20, 40, 60, 80, 100, 120, 140 and 160 μ M) for 24, 48 and 72 hours and cell viability measured using the CCK-8 method; (B) Podocytes were exposed to HG and HG + ISO (10, 20, and 40 μ M), and cell viability was measured using the CCK-8 method 24-72 hours after treatment; (C-D) the level of apoptosis was assessed by flow cytometry (annexin V staining). (P < 0.05;. P < 0.01); (E) The number of spots showing LC3 distribution was counted in 100 to 150 cells per group and three independent experiments were performed; (F) MPC5 cells expressing GFP-LC3 were treated with glucose, HG + ISO and HG + ISO +3-MA and confocal images showing the distribution of LC3 in the cells were observed; scale: 25 μm (G-J), and detecting the expression of LC3 and p62 proteins by Western blotting; (K) MPC5 cells were treated with glucose, HG + ISO and HG + ISO +3-MA and then measured for absorbance P <0.01 using CCK-8; (L-R) is the statistical result of experimental analysis.

FIG. 5 is a graph showing ISO protects podocyte mitochondria by enhancing mitochondrial phagocytosis in the examples of the present invention, (A) immunofluorescent labeling of podocyte mitochondrial proteins TOM20 and GFP-LC3 in normal, HG, ISO and 3-MA groups; (B-C) randomly analyzing 100-150 cells using ImageJ plug for co-localization threshold and scatter, and obtaining co-localization images; (D) fluorescence distribution of LC3 and TOM20 on random line segments; (E-H) statistical analysis of co-localization coefficients such as PCC, overlap coefficient and MCC was performed using ImageJ plug-in JACoP to randomly analyze 100-150 cells. Quantitative statistics of mitochondria; (J) Statistics show the number of mitochondria co-localized with punctate LC3, # P <0.05, # P <0.01; (K) immunoblotting to detect TIM23 and TOM20; statistical analysis of grey values found (L-M) · P <0.0; (N) damaged and depolarized mitochondria were detected by JC-10 staining with a fluorescent probe. Green and red fluorescence represent mitochondria depolarization and those with normal membrane potential, respectively, scale: 50 μm; (O) shows the ratio of green fluorescence intensity to total fluorescence intensity for statistical analysis, data expressed as mean ± SEM values of independent experiments, one-way analysis of variance (ANOVA) for multiple group comparisons, × P <0.01.

FIG. 6 shows that ISO intervention can reverse mercury-induced abnormal phosphorylation of PI3K Tyr458 and Tyr199, AKT Thr308, TSC2 Ser939 and mTOR Ser2448 sites in the example of the present invention, (A-B) improved proteomics and Wien graph results; (C) from top to bottom: internal parameters of phosphorylated TSC2 (pTSC 2), total TSC2 (tTSC 2), phosphorylated PI3K (pPI 3K), total PI3K (tPI 3K), phosphorylated AKT (pAKT), total AKT (tAKT), phosphorylated mTOR (pmTOR), total mTOR (tmTOR), and β -actin; (D-G) the ratios of pTSC2, pPI3K, pAKT and pmTOR to β -actin, the ratio of total TSC2, PI3K, AKT and mTOR to β -actin, and the ratio of pTSC2, pPI3K, pAKT and pmTOR to tTSC2 were analyzed, and tPI3K, tAKT and tmTOR P <0.01.

FIG. 7 is a graph of DN animal models analyzed in the present examples demonstrating that ISO inhibits diabetic renal damage, (A-F) from left to right: ALB, UACR, scr, BUN, AU and Ccr, { P > <0.05 }, { P > <0.01 }, { Ccr (L/24 hours) = (urinary creatinine concentration/blood creatinine concentration) × 24 hours urine volume; (G-M) from left to right: blood glucose, CHO, TG, FFA, LDL, HDL, and body weight; (N) black and orange arrows in HE and Masson stained images indicate glomeruli; among representative images obtained using transmission electron microscopy are basement membrane, foot processes, mitochondria and mitochondria phagocytosed by autophagosomes; (P-R) statistical analysis of mean foot process spacing, maximum foot process spacing and mean basal membrane thickness in the top-to-bottom field of view; arrows indicate mitochondria P <0.05 and P <0.01.

FIG. 8 is an analysis of primary podocytes in an example of the invention demonstrating that ISO protects podocytes from DN mice because it enhances mitotic phagocytosis, (A) immunofluorescent labeling and sorting of podocytes by confocal laser imaging; (B) identifying genes of nephrin and podocin by agarose gel electrophoresis; (C) Performing immunofluorescence labeling LC3 laser confocal imaging on primary podocytes of a normal group, a DN group and an ISO group; (D) statistical plots of LC3 points in 50 cells. (E) immunoblotting to detect LC3 expression; (F) statistical analysis of LC3 grey scale values; (G) quantitative statistics of mitochondria. (H) Immunofluorescence labeling mitochondria of normal, DN and ISO groups; (I-K) detecting the changes of the TOM20 and TIM23 levels and gray statistics by Western blotting; (L-M) ImageJ plug for co-localization threshold and scatter was used for random analysis of 100-150 cells to obtain co-localization images; (N) fluorescence distribution of LC3 and TOM20 on random line segments; (O) observing immunofluorescent-labeled primary podocytes of the normal, DN, and ISO groups by confocal laser imaging; (P) punctate LC3 co-localisation of mitochondria; (Q-T) ImageJ plug-in JACoP was used for random analysis of 100-150 cells to obtain statistics of co-localization analysis coefficients, such as PCC, overlap coefficient and MCC; (U) fluorescent probe JC-10 stain to detect mitochondrial damage and depolarization, scale: 40 μm; (V) statistical analysis of the ratio of green to red fluorescence, data expressed as mean ± SEM values of independent experiments, and analysis of variance was used to compare groups P <0.05 and P <0.01.

FIG. 9 analysis of primary podocytes in this example of the invention to verify that ISO can reverse DN-induced changes in PI3K/AKT/TSC2/mTOR pathways, (A) from top to bottom: pAKT, total AKT, pTSC2, total AKT, total TSC2 and β -actin; (B-E) analyzing the ratio of pTSC2, pPI3K, pAKT and pmTOR to β -actin, the ratio of total TSC2, total PI3K, total AKT and total mTOR to β -actin, and the ratio of pTSC2, pPI3K, pAKT and pmTOR to total TSC2, total PI3K, total AKT and total mTOR,. P <0.01.

Figure 10 is a model graph of the mechanism studied in the examples of the invention, under diabetic conditions, PI3K-AKT-TSC2-mTOR pathway is over-activated in podocytes, resulting in inhibition of autophagy, ISO decreases the activity of this pathway, thereby restoring autophagy,. P <0.01.

The specific implementation mode is as follows:

the invention is further illustrated by the following examples in conjunction with the accompanying drawings.

Example (b):

this embodiment has verified the operating mechanism that isoorientin is used for treating diabetic nephropathy through concrete experiment, including in vivo experiment and in vitro experiment, and concrete process is:

1. in vivo experiments

(1) Constructing a DKD model and intervening medicaments:

selecting male C57BL/6J mice of 5-6 weeks old, randomly dividing the mice into a normal group and a sugared kidney model group (DN group), feeding the mice with 60 percent high-fat feed for 4 weeks by the sugared kidney model group, continuously injecting streptozotocin (40 mg/kg) for 5 times into the abdominal cavity, measuring the random blood sugar of the mice after 1 week, wherein the random blood sugar is more than 16.7mmol/l, the model building of the diabetes model of the mice is successful, and measuring the urine trace protein/creatinine of the mice after continuously feeding the mice for 2 months, and the urine trace protein/creatinine is more than 30mg/kg, the model building of the diabetes nephropathy is successful; then, the mice successfully molded are randomly divided into a non-intervention group and an isoorientin molecule intervention group (ISO group): the low dose treatment group (10 mg/kg), the medium dose treatment group (20 mg/kg), and the high dose treatment group (40 mg/kg) were administered by intraperitoneal injection, and for the normal group and the DN group, the mice received the same amount of solvent (0.2% sodium carboxymethylcellulose and 0.1% tween 80) once a day, and were continuously administered for 8 weeks, general conditions, body weight, and blood sugar of the mice were monitored, and after 8 weeks of administration, 24 urine of the mice was collected again using a metabolic cage, microalbuminuria and urinary creatinine levels of the mice were detected, and after anesthesia, blood was taken and serum was separated for renal function evaluation; kidney tissue will be used for podocyte sorting (performed immediately after removal), morphological analysis, RNA and protein analysis;

(2) Sorting podocytes:

and (3) sorting the mouse kidney podocytes by a magnetic bead sorting method by using antibodies of marker proteins such as Nephrin, distributed on the podocytes on the cell surface: cutting a freshly obtained kidney, digesting the kidney with type II collagenase and neutral enzyme, removing red blood cells by using a red blood cell lysate, separating a single cell suspension by using a 25-micron filter, firstly removing vascular endothelial cells by using magnetic beads of a cross-linked CD31 antibody, then separating podocytes by using PE-labeled Nephrin antibody and magnetic beads of the cross-linked PE antibody, and detecting the purity of the sorted podocytes by using flow cytometry, wherein the method is used for successfully sorting high-purity podocytes, and the like, and in order to improve the sorting purity of the podocytes, the following method for sorting the podocytes is adopted in the embodiment: (a) Injecting 2% sodium pentobarbital into abdominal cavity to kill the mouse, disinfecting with alcohol, taking out the kidney tissue of the mouse, soaking and washing in precooled sterile PBS buffer solution containing double antibodies, and stripping to remove the kidney capsule; (b) After the kidney tissues are mechanically dissociated, preheating mixed enzyme liquid for digestion, and mixing completely culture solution to stop digestion; (c) Obtaining kidney podocytes by adopting a differential screening method, and inoculating the podocytes into a treated cell bottle for culture; (d) Digesting the cells cultured for 3d by pancreatin, filtering by a screen again, centrifuging, discarding supernatant, and re-suspending and inoculating in a treated cell bottle; (e) identifying the renal foot process cells by cellular immunofluorescence; (the method is invented by Jiangsu Qishi Biotechnology Co., ltd., and the isolated mouse kidney foot process cells have high adherence rate, the survival rate of more than 98 percent and the cell purity of more than 97 percent);

(3) The immunoblotting experiment carries out relative quantitative analysis on the content and phosphorylation level of target protein in podocytes: comprising detecting AKT, TSC2 and their corresponding phosphorylated forms; detecting LC3-I and LC3-II to assess autophagy levels; mitochondrial marker proteins such as TOM20 and TIM23 are detected to compare the number of mitochondria, mitochondrial autophagy key proteins such as Parkin, PINK1, BNIP3 are detected, and proteins with significant differences in phosphoproteomics are screened out as shown in table 1:

table 1: screening 54 proteins with significant differences (p value less than 0.05) by modified proteomics

(4) Detecting the morphology of kidney tissues:

the approximate structures of glomeruli and renal tubules are observed by HE staining, the structure of glomeruli, the morphology of podocytes and mitochondria and autophagosomes in the podocytes are observed by a transmission electron microscope, and the renal interstitial fibrosis is detected by Masson staining;

(5) In situ observation of podocyte autophagy

Adopting an immunofluorescence labeling experiment to observe autophagy conditions in the podocyte in situ, carrying out immunolabeling by using an antibody of a podocyte marker protein to display the position of the podocyte, and carrying out immunofluorescence labeling by using an LC3 antibody to display an autophagosome;

2. in vitro experiments:

(1) Culture and high sugar and drug treatment of podocyte line MPC 5:

culturing and intervening with drugs under the same conditions as in vivo experiment, wherein the glucose concentration of the normal group is 5.5mM as compared with that of the culture medium, the glucose concentration adopted in the high-sugar treatment is 30mM, and isoorientin molecules are added into the ISO treatment group for 40 μ M on the basis of high sugar, and the ISO treatment time is 48 hours;

(2) Stable RNA interference:

introducing shRNA into podocytes by adopting a transfection or lentivirus mediated mode, and screening resistance genes carried by plasmids to obtain podocyte strains with stable RNA interference as shown in table 1;

(3) Gene knock-out and gene knock-in:

gene knockout: constructing a gene knockout MPC5 strain by adopting a Cas9 gene editing technology, designing sgRNA by using CHOP online software, connecting the sgRNA to a Lenti-Cas9-v2 vector, transfecting podocytes, and screening through a resistance gene carried by a plasmid to obtain a gene knockout cell for research;

gene knock-in: constructing gene knock-in MPC5 cell strains with two phosphorylation site mutations of TSC 2S 939A and S939E, firstly designing sgRNA, connecting to a Lenti-Cas9-v2 vector, then constructing a donor vector carrying the mutation with the same sense mutation of the PAM sequence, and introducing the mutation by the cutting action of Cas9 nuclease and the homologous recombination of a homologous arm;

(4) Detection of autophagy:

immunoblotting assays the ratios of LC3-I to LC3-II, LC3-II/LC3-I reflect the level of autophagy in the cells. In addition, the GFP-LC3 expression plasmid is transfected, and the GFP-LC3 distributed in a dot shape in the cells is counted by adopting laser confocal microscope imaging;

(5) Detection of mitochondrial autophagy:

detecting the number of mitochondria, carrying out relative quantitative analysis on the content of mitochondria in cells by detecting mitochondrial markers, and analyzing various markers in order to avoid influence of processing factors on the markers, wherein the content of mitochondrial proteins TOM20 and TIM23 and the content of mitochondrial DNA are detected, and mitochondrial autophagy is observed by a transmission electron microscope; then detecting the co-localization of mitochondria and autophagosomes, and detecting the co-localization of mitochondrial marker proteins TOM20 and TIM23 (marker bus mitochondria) and non-dispersive LC3 (activated IC3, namely LC3-II presents punctate or bubble distribution) by adopting immunofluorescence labeling; the number of damaged but not autophagy-cleared mitochondria within the cell is then analyzed: marking mitochondria with MitoTracker under the state of living cells, selecting MitoTracker (such as MitoTracker Red CMXRos) which is bound with mitochondria and depends on mitochondrial membrane potential and still keeps fluorescence after being fixed by paraformaldehyde, staining with MitoTracker to be normal mitochondria, marking TOM20 (showing all mitochondria) by immunofluorescence, wherein TOM20 signals positively, and mitochondria with MitoTracker signals negatively are damaged mitochondria;

(5) Biacore experiments search for ISO targets of action:

ISO is connected to a sensor chip of Biacore, prepared podocyte lysate is injected through buffer solution and flows across the surface of the biosensor, the binding between biomolecules causes the mass of the surface of the biosensor to increase, which results in the change of refractive index, and the change of the reaction between biomolecules is observed by monitoring the angle change of SPR. Constructing a visual target point with the ISO function by combining mass spectrometry and a computer fitting technology; after the target protein is determined, the target protein combined with ISO is interfered through the structural characteristics of the target protein, the phosphorylation changes of AKT and TSC 2S 939 sites and the changes of autophagy and mitochondrion autophagy are detected at the same time, and the protective effect of ISO on the activity of the podocyte is further researched;

(6) Cell activity assay:

the number of living cells was determined by MTT and apoptosis was analyzed by annexin V-PI double staining-flow cytometry and TUNEL staining.

Claims (1)

1. A traditional Chinese medicine monomer isoorientin for treating diabetic nephropathy is characterized in that the chemical structural formula of the isoorientin is as follows:

is a flavonoid compound containing luteolin structure, is present in medicinal or edible parts of herba Sonchi Oleracei, semen Fagopyri Esculenti, herba Passiflorae Caeruleae and semen Trigonellae, and has molecular formula of C ₂₁ H ₂₀ O ₁₁ Can be used for treating diabetic nephropathy.

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