CN116966219A - Charcoal medicine with effect of inhibiting kidney injury caused by aristolochic acid - Google Patents
Charcoal medicine with effect of inhibiting kidney injury caused by aristolochic acid Download PDFInfo
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- CN116966219A CN116966219A CN202210413882.9A CN202210413882A CN116966219A CN 116966219 A CN116966219 A CN 116966219A CN 202210413882 A CN202210413882 A CN 202210413882A CN 116966219 A CN116966219 A CN 116966219A
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- charcoal
- astragalus
- aristolochic acid
- kidney injury
- inhibiting
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- BBFQZRXNYIEMAW-UHFFFAOYSA-N aristolochic acid I Chemical compound C1=C([N+]([O-])=O)C2=C(C(O)=O)C=C3OCOC3=C2C2=C1C(OC)=CC=C2 BBFQZRXNYIEMAW-UHFFFAOYSA-N 0.000 title claims abstract description 19
- 239000003814 drug Substances 0.000 title claims abstract description 18
- 206010061481 Renal injury Diseases 0.000 title claims abstract description 12
- 230000002401 inhibitory effect Effects 0.000 title claims abstract description 11
- 208000037806 kidney injury Diseases 0.000 title claims abstract description 10
- 239000003610 charcoal Substances 0.000 title claims description 22
- 230000000694 effects Effects 0.000 title description 14
- 241001061264 Astragalus Species 0.000 claims abstract description 29
- 235000006533 astragalus Nutrition 0.000 claims abstract description 29
- 210000004233 talus Anatomy 0.000 claims abstract description 29
- 230000006907 apoptotic process Effects 0.000 claims abstract description 5
- 230000036542 oxidative stress Effects 0.000 claims abstract description 4
- 206010061218 Inflammation Diseases 0.000 claims abstract 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 239000009636 Huang Qi Substances 0.000 claims description 5
- 230000003110 anti-inflammatory effect Effects 0.000 claims description 2
- 239000003963 antioxidant agent Substances 0.000 claims description 2
- 230000003078 antioxidant effect Effects 0.000 claims description 2
- 239000000843 powder Substances 0.000 claims description 2
- 230000035882 stress Effects 0.000 claims description 2
- 239000006187 pill Substances 0.000 claims 3
- 238000002360 preparation method Methods 0.000 claims 3
- 230000006909 anti-apoptosis Effects 0.000 claims 1
- 239000000839 emulsion Substances 0.000 claims 1
- 239000007902 hard capsule Substances 0.000 claims 1
- 230000008798 inflammatory stress Effects 0.000 claims 1
- 230000005764 inhibitory process Effects 0.000 claims 1
- 239000007924 injection Substances 0.000 claims 1
- 238000002347 injection Methods 0.000 claims 1
- 239000007788 liquid Substances 0.000 claims 1
- 239000002674 ointment Substances 0.000 claims 1
- 239000006072 paste Substances 0.000 claims 1
- 230000037361 pathway Effects 0.000 claims 1
- 239000000546 pharmaceutical excipient Substances 0.000 claims 1
- 239000007901 soft capsule Substances 0.000 claims 1
- 239000000829 suppository Substances 0.000 claims 1
- 239000003826 tablet Substances 0.000 claims 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 16
- 229910052799 carbon Inorganic materials 0.000 abstract description 15
- 229940079593 drug Drugs 0.000 abstract description 4
- 238000002474 experimental method Methods 0.000 abstract description 2
- 230000004054 inflammatory process Effects 0.000 abstract 1
- 241000699670 Mus sp. Species 0.000 description 26
- 210000005084 renal tissue Anatomy 0.000 description 17
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- 201000011040 acute kidney failure Diseases 0.000 description 7
- 108700000707 bcl-2-Associated X Proteins 0.000 description 7
- 102000055102 bcl-2-Associated X Human genes 0.000 description 7
- 230000001965 increasing effect Effects 0.000 description 7
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- 210000003734 kidney Anatomy 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 102100021569 Apoptosis regulator Bcl-2 Human genes 0.000 description 4
- 102100021943 C-C motif chemokine 2 Human genes 0.000 description 4
- 101710155857 C-C motif chemokine 2 Proteins 0.000 description 4
- 102100025064 Cellular tumor antigen p53 Human genes 0.000 description 4
- 101000971171 Homo sapiens Apoptosis regulator Bcl-2 Proteins 0.000 description 4
- 108090001005 Interleukin-6 Proteins 0.000 description 4
- 230000001640 apoptogenic effect Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 3
- 208000009304 Acute Kidney Injury Diseases 0.000 description 3
- 102000003777 Interleukin-1 beta Human genes 0.000 description 3
- 108090000193 Interleukin-1 beta Proteins 0.000 description 3
- 102000004889 Interleukin-6 Human genes 0.000 description 3
- 239000002033 PVDF binder Substances 0.000 description 3
- 102000013535 Proto-Oncogene Proteins c-bcl-2 Human genes 0.000 description 3
- 108010090931 Proto-Oncogene Proteins c-bcl-2 Proteins 0.000 description 3
- 208000033626 Renal failure acute Diseases 0.000 description 3
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 3
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 3
- 229940107666 astragalus root Drugs 0.000 description 3
- 230000002757 inflammatory effect Effects 0.000 description 3
- 231100000915 pathological change Toxicity 0.000 description 3
- 230000036285 pathological change Effects 0.000 description 3
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 2
- 101000721661 Homo sapiens Cellular tumor antigen p53 Proteins 0.000 description 2
- 206010030113 Oedema Diseases 0.000 description 2
- 239000006180 TBST buffer Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000001354 calcination Methods 0.000 description 2
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 2
- 210000004969 inflammatory cell Anatomy 0.000 description 2
- 230000003907 kidney function Effects 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 210000004926 tubular epithelial cell Anatomy 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- GOZMBJCYMQQACI-UHFFFAOYSA-N 6,7-dimethyl-3-[[methyl-[2-[methyl-[[1-[3-(trifluoromethyl)phenyl]indol-3-yl]methyl]amino]ethyl]amino]methyl]chromen-4-one;dihydrochloride Chemical compound Cl.Cl.C=1OC2=CC(C)=C(C)C=C2C(=O)C=1CN(C)CCN(C)CC(C1=CC=CC=C11)=CN1C1=CC=CC(C(F)(F)F)=C1 GOZMBJCYMQQACI-UHFFFAOYSA-N 0.000 description 1
- BBFQZRXNYIEMAW-UHFFFAOYSA-M Aristolochate I Natural products C1=C([N+]([O-])=O)C2=C(C([O-])=O)C=C3OCOC3=C2C2=C1C(OC)=CC=C2 BBFQZRXNYIEMAW-UHFFFAOYSA-M 0.000 description 1
- 241000726094 Aristolochia Species 0.000 description 1
- 206010003694 Atrophy Diseases 0.000 description 1
- 208000004884 Balkan Nephropathy Diseases 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 229940122907 Phosphatase inhibitor Drugs 0.000 description 1
- PNNCWTXUWKENPE-UHFFFAOYSA-N [N].NC(N)=O Chemical compound [N].NC(N)=O PNNCWTXUWKENPE-UHFFFAOYSA-N 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 230000037444 atrophy Effects 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000005252 bulbus oculi Anatomy 0.000 description 1
- 229940109239 creatinine Drugs 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 238000001879 gelation Methods 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 210000000738 kidney tubule Anatomy 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 231100000417 nephrotoxicity Toxicity 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000000242 pagocytic effect Effects 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 210000001539 phagocyte Anatomy 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000001603 reducing effect Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000002110 toxicologic effect Effects 0.000 description 1
- 231100000759 toxicological effect Toxicity 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
- A61K36/481—Astragalus (milkvetch)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
Landscapes
- Health & Medical Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Epidemiology (AREA)
- Microbiology (AREA)
- Medical Informatics (AREA)
- Botany (AREA)
- Alternative & Traditional Medicine (AREA)
- Mycology (AREA)
- Urology & Nephrology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The application belongs to the technical field of medicines, and particularly relates to an application of astragalus carbon in preparing a medicine for inhibiting kidney injury caused by aristolochic acid. Animal experiments show that the astragalus carbon can inhibit kidney injury caused by aristolochic acid through the ways of resisting oxidative stress, resisting inflammation and inhibiting apoptosis.
Description
Technical Field
The application relates to the field of medicines, in particular to a new application of astragalus carbon in the field of medicines.
Background
Aristolochic acid is widely present in plants of the genus aristolochia and has pharmacological actions of enhancing phagocytic function of phagocytes and enhancing immunity of the organism. However, in recent years, the report of aristolochic acid nephropathy caused by traditional Chinese medicine is increasing, so that the medical world at home and abroad is more concerned about the toxicological effects of traditional Chinese medicine and Chinese patent medicine containing aristolochic acid. How to reasonably reduce the toxic and side effects of the traditional Chinese medicine containing aristolochic acid becomes a hot spot of clinical research. The research at present shows that after the traditional Chinese medicine is matched with the traditional Chinese medicine containing aristolochic acid, the traditional Chinese medicine with the kidney attenuation effect comprises four types of tonifying, blood activating, heat clearing and purgation.
Finding a safe and inexpensive strategy for inhibiting aristolochic acid-induced kidney injury is an urgent need in the current clinic. Literature describes that Huang Qi carbon is used together with other drugs to tonify kidney and stop bleeding. But can inhibit renal toxicity of aristolochic acid? There have been no reports.
Disclosure of Invention
In view of the above, the present application aims to: provides a safe and low-cost traditional Chinese medicine charcoal medicine for inhibiting kidney injury caused by aristolochic acid, so as to be matched or singly used in clinic.
Specifically, the application is realized in such a way that the application of astragalus carbon in preparing a medicament for inhibiting the kidney injury caused by aristolochic acid comprises the following steps:
1) Adding water or 0-100% ethanol into radix astragali charcoal, extracting, and concentrating the extractive solution;
2) The extractive solution is administered via gastrointestinal tract, and can inhibit renal injury caused by aristolochic acid by antioxidant stress, antiinflammatory and apoptosis inhibiting means.
The application provides a new application of astragalus carbon.
Drawings
Fig. 1: effect of astragalus charcoal on BUN (fig. 1A) and CRE (fig. 1B) in AAI-induced AKI mouse serum.
Fig. 2: effect of astragalus charcoal on AAI-induced AKI mouse kidney tissue MCP-1 (fig. 2A), TNF- α (fig. 2B), IL-6 (fig. 2C) and IL-1β (fig. 2D).
Fig. 3: effect of astragalus charcoal on AAI-induced SOD (fig. 3A), MDA (fig. 3B), and GSH (fig. 3C) in kidney tissue of AKI mice.
Fig. 4: renal histopathological changes (HE staining, ×200 and×400 fields) in mice of each group, fig. 4A and 4F. Normal group; FIGS. 4B and 4G. Model sets; FIGS. 4C and 4H. Astragalus charcoal high dose group; FIGS. 4D and 4I. Dose group in astragalus charcoal; fig. 4E and 4J. Astragalus charcoal low dose group.
Fig. 5: number of apoptosis in kidney tissue of mice in each group.
Fig. 6: effect of astragalus charcoal on p53 (fig. 6D), bax (fig. 6E), bcl-2 (fig. 6F) and Bax/Bcl-2 (fig. 6G) protein expression levels in AAI-induced AKI mouse kidney tissue (fig. 6A).
Detailed Description
The present application is further illustrated below with reference to specific examples, which are to be construed as merely illustrative of the application and not limiting of its scope, as various equivalent modifications to the application will fall within the scope of the application as defined in the appended claims after reading the application.
Example 1: a method for preparing astragalus carbon, comprising the following steps:
placing the astragalus medicinal material into a sealed crucible, and calcining for 1h by a muffle furnace (the calcining temperature is 200-400 ℃). And taking out the carbonized astragalus after the muffle furnace is naturally cooled to room temperature.
Example 2: a method for preparing astragalus carbon, comprising the following steps:
placing radix astragali in a pot, uncovering, and parching at 50-900deg.C until the inside and outside are black, to obtain radix astragali charcoal. Grinding radix astragali charcoal into powder. Then, the astragalus carbon powder was extracted with water/ethanol (0-100%) of different concentrations as a solvent, and the extract was concentrated into a thick paste, so that the following experiment was performed.
Example 3: treatment effect of astragalus carbon on aristolochic acid-induced acute kidney injury mice
3.1 Grouping, modeling and administration
40C 57BL/6J mice were randomly divided into a control group, a model group, and astragalus carbon high, medium and low dose administration groups, 5 groups, and 8 groups each. Astragalus root charcoal administration group mice are according to 0.1 ml.10g -1 Is administered by intragastric administration, while the control group and model group mice are administered physiological saline by intragastric administration for 7 days. 2h after 5 th day of administration, mice of model group and astragalus carbon administration group were intraperitoneally injected with aristolochic acid I (AAI, 10mg.kg) -1 ) And establishing an acute kidney injury model for 3 days continuously. After the last administration, all mice were fasted for 12h and were free to drink water.
3.2 Renal function testing
Mice pick eyeballs, take blood and stand for 4 hours, and then are 2500 r.min -1 Centrifuging for 10min, and collecting upper serum.Creatinine (CRE) and urea nitrogen (BUN) levels were then measured by a fully automated biochemical analyzer and the renal function differences were compared for each group.
After three consecutive days of intraperitoneal injection of AAI for model group mice, BUN and CRE in serum of mice are obviously increased (P is less than 0.01) compared with the control group; after the astragalus carbon administration, the serum BUN and CRE levels of mice in each administration group were significantly reduced (P < 0.01) compared with the model group. The above results demonstrate that astragalus charcoal is effective in alleviating acute kidney injury in AAI-induced mice (fig. 1).
3.3 Inflammatory factor index detection
Immediately after the mice were sacrificed, the kidneys on both sides were removed and the kidney membranes were peeled off. The right kidney of the mice was washed in 0.9% physiological saline, and the water was sucked dry with filter paper. A10% tissue homogenate was prepared at a ratio of kidney tissue weight (g) to phosphate buffer volume (mL) =1:9. Setting 4000 r.min in a centrifuge -1 Centrifuging for 10min, collecting supernatant, and measuring the content of inflammatory factors such as MCP-1, IL-1 beta, IL-6, TNF-alpha, etc. according to the specification of ELISA kit.
The inflammatory factor index detection result is shown in figure 2. Compared with the normal group, the levels of MCP-1, TNF-alpha, IL-6 and IL-1 beta in the lung tissue homogenate of the mice in the model group are all obviously increased (P is less than 0.01). Compared with the model group, after the low, medium and high dosage groups of astragalus root charcoal are used, the levels of MCP-1, TNF-alpha, IL-6 and IL-1 beta in the kidney tissue homogenate of the mice are obviously reduced (P is less than 0.01).
3.4 Oxidative stress index detection
The 10% supernatant of kidney tissue homogenate from mice was used to detect and calculate MDA and GSH contents and SOD enzyme activities in kidney tissue according to the operation method of each test box.
The detection result of the oxidative stress index shows that the MDA content in the kidney tissue of the model group mice is obviously increased (P is less than 0.01) compared with the control group, and the SOD activity and GSH activity are obviously reduced (P is less than 0.01). The high, medium and low doses of astragalus root charcoal have remarkable reducing effect on the MDA content increase of the kidney tissue of the mice and remarkable increasing effect on SOD activity and GSH activity (P is less than 0.01) (figure 3).
3.5 Renal histopathological examination
The left kidney of the mouse is fixed in 4% paraformaldehyde solution, eluted by ethanol gradient with different concentrations, embedded in paraffin, cut into slices with the thickness of 4 mu m, HE stained, and observed under an optical microscope for pathological changes of kidney tissues of the mouse.
Morphological observations of HE staining of kidney tissue are shown in fig. 4. The kidney tissue of the control mice did not show pathological changes. The kidney tissue of the mice in the model group is seriously damaged, tubular expansion or atrophy occurs, part of tubular epithelial cells fall off, the mice are denatured and necrotized, and inflammatory cells infiltrate into renal interstitial spaces and cause congestion and edema. The kidney tubule expansion and interstitial expansion edema of mice in the astragalus carbon high-dose group are obviously reduced, inflammatory cell infiltration is obviously reduced, and the pathological changes of other dose groups are reduced to a certain extent, so that the dose dependency is presented (figure 4).
3.6 TUNEL method for detecting apoptosis
Paraffin sections of kidney tissue were taken, dewaxed with xylene, dehydrated with ethanol gradient, rinsed with distilled water, and rehydrated. The sections were capped with proteinase K added working solution and incubated in TUNEL reaction mixtures at 37℃for 2 hours. Nuclei were counterstained with DAPI. Images were taken with a fluorescence microscope and photographed. The number of apoptotic cells stained for the selected region TUNEL was counted at random 5 fields per slide.
Nuclei were blue under ultraviolet excitation after DAPI counterstaining, and positive apoptotic nuclei were green. The number of tubular epithelial cells in the model group apoptotic was significantly increased compared to the blank group. The number of apoptotic positive cells was significantly reduced (P < 0.01) for each dose of astragalus charcoal compared to the model group (FIG. 5).
3.7 Western blot method for detecting cell p53, bax and Bcl-2 protein expression
The tissue is lysed in RIPA lysis buffer containing phosphatase inhibitor and PMSF, and total protein is extracted. Protein concentration was determined using BCA protein concentration determination kit. SDS-PAGE gel is prepared, and after gelation, the gel is loaded, electrophoresed, PVDF membrane transferred and BSA sealing membrane are carried out. The corresponding primary antibody was diluted with blocking solution and PVDF membranes were immersed in the primary antibody incubation solution overnight at 4 ℃. After TBST was used to wash PVDF membrane well, secondary antibody was incubated at 37℃for 120min, TBST was washed 3 times, ECL developed, and the grey scale value of the bands was analyzed using imageJ software.
Western blotting results show that compared with a blank group, the expression level of P53 and Bax proteins in kidney tissue cells of a model group mouse is obviously improved (P is less than 0.01), the content of Bcl-2 proteins is obviously reduced (P is less than 0.01), and the content of Bax/Bcl-2 is obviously increased (P is less than 0.01). Compared with a model group, after astragalus carbon pre-administration, the high, medium and low dosage administration groups can obviously improve the Bcl-2 protein content (P is less than 0.01) in kidney tissue cells of mice, reduce the P53 protein expression level (P is less than 0.01), and have no obvious difference (P is more than 0.05) in the low dosage administration groups; the high and medium doses of astragalus carbon reduce the Bax protein content (P < 0.05, P < 0.01); the high, medium and low dose groups of astragalus charcoal significantly reduced Bax/Bcl-2 (FIG. 6).
3.8 Statistical treatment
Data results were counted and analyzed using SPASS25.0 software, data results were expressed as x+ -s, and differences between groups were statistically analyzed by one-way analysis of variance (ANOVA), with P < 0.05 being considered statistically significant.
Claims (4)
1. Application of radix astragali charcoal in preparing medicine for inhibiting renal injury caused by aristolochic acid is provided.
2. The use according to claim 1, wherein the action mechanism of the astragalus charcoal for inhibiting kidney injury caused by aristolochic acid is: through antioxidant stress, anti-inflammatory and apoptosis inhibition pathways.
3. The use according to claim 2, wherein the kidney injury inhibiting effect is aristolochic acid-induced, other causes-induced, kidney injury characterized by inflammation or oxidative stress or apoptosis.
4. The use according to claim 1, wherein the astragalus charcoal and pharmaceutically acceptable excipients are prepared into a clinical preparation; the clinical preparations include, but are not limited to, injections, oral liquid preparations, hard capsules, soft capsules, tablets, suppositories, water pills, honeyed pills, concentrated pills, ointments, powders, emulsions, pastes, and smears.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210413882.9A CN116966219A (en) | 2022-04-21 | 2022-04-21 | Charcoal medicine with effect of inhibiting kidney injury caused by aristolochic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210413882.9A CN116966219A (en) | 2022-04-21 | 2022-04-21 | Charcoal medicine with effect of inhibiting kidney injury caused by aristolochic acid |
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| Publication Number | Publication Date |
|---|---|
| CN116966219A true CN116966219A (en) | 2023-10-31 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210413882.9A Pending CN116966219A (en) | 2022-04-21 | 2022-04-21 | Charcoal medicine with effect of inhibiting kidney injury caused by aristolochic acid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116966219A (en) |
-
2022
- 2022-04-21 CN CN202210413882.9A patent/CN116966219A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| 赵怡蕊 等: "国医大师张大宁教授巧用炭剂治肾病", 世界中医药, vol. 10, no. 12, 15 December 2015 (2015-12-15), pages 1096 - 1097 * |
| 赵怡蕊 等: "张大宁教授应用"升清降浊"法治疗肾脏病的"理"与"效"", 世界中医药, vol. 8, no. 9, 14 November 2013 (2013-11-14), pages 1008 * |
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