CN117599105A - Traditional Chinese medicine compound composition with functions of delaying kidney aging and fibrosis as well as preparation method and application thereof - Google Patents
Traditional Chinese medicine compound composition with functions of delaying kidney aging and fibrosis as well as preparation method and application thereof Download PDFInfo
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- CN117599105A CN117599105A CN202311543505.8A CN202311543505A CN117599105A CN 117599105 A CN117599105 A CN 117599105A CN 202311543505 A CN202311543505 A CN 202311543505A CN 117599105 A CN117599105 A CN 117599105A
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Classifications
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K36/481—Astragalus (milkvetch)
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- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
Abstract
The invention discloses a Chinese medicinal compound composition for delaying kidney aging and fibrosis and a preparation method thereof, which is prepared from extracts prepared from 240-480 g of astragalus root, 240-480 g of flower of abelmoschus manihot, 240-480 g of centella asiatica, 120-240 g of root of red-rooted salvia and 40-80 g of liquorice and cordyceps mycelia. According to the invention, through years of clinical experience and combination of kidney aging pathogenesis, the screening formula is added with two medicines of centella asiatica and cordyceps sinensis mycelia on the basis of astragalus, okra, red sage root and liquorice, so that the effects of clearing and activating blood, tonifying qi and tonifying kidney can be obviously improved. The traditional Chinese medicine formula is prepared by harmonizing the traditional Chinese medicines, tonifying kidney qi, tonifying kidney essence, activating qi and blood, clearing damp turbidity and combining the treatment concept of protecting kidney and delaying aging, and becomes a common formula aiming at kidney function decline and early kidney injury clinically. Experimental results show that the traditional Chinese medicine formula can recover kidney functions, and kidney SA, HE, masson dyeing shows that the traditional Chinese medicine has the effects of resisting natural aging and prolonging life, and the symptoms of aging and fibrosis are relieved after the traditional Chinese medicine is treated.
Description
Technical Field
The invention relates to a traditional Chinese medicine composition, in particular to a traditional Chinese medicine compound composition for delaying kidney aging and fibrosis and a preparation method thereof, and belongs to the technical field of traditional Chinese medicines.
Background
The kidney is considered to store congenital and acquired essence in the theory of traditional Chinese medicine. The earliest is mentioned in the "Su-Liu Jie visceral theory": the kidney is the main sting and the essence is the part of the body. "Su Wen, jin Kui Zhen Lun" considers essence to be the root of life. Congenital essence is inherited from parents, acquired essence is obtained by digestion of the stomach, spleen qi transformation and condensation of cereal Shui Shiwu after ingestion into the body. The kidneys store essence to nourish the viscera and six fu organs, so the ability of kidneys to store essence and the running conditions of essence qi and water liquid, which are dominant by kidneys, tend to affect the health condition of the whole human body. Ancient doctors pointed out that kidney aging caused abnormal qi transformation, damp turbidity, stasis and toxin blocking triple energizer, and "kidney collaterals mass" formed, eventually a hundred diseases.
Kidneys are responsible for regulating the bodily fluid metabolism of the whole body, and are one of the most easily aged organs, due to their physiological functions. With the aging, the kidney structure and function are diseased, kidney fibrosis is generated, and the kidney function is further promoted to decline, and the research also shows that the kidney of the chronic kidney disease has similar characteristics as the aging kidney.
The institute of animal research at academy of China systematically studied senescence genes by whole genome gene editing technique, found that the difference in expression of KAT7 is most remarkable in the aging of primary mesenchymal stem cells (hMPCs) of patients with premature senility, and KAT7 can promote the expression of senescence marker genes and induce cellular senescence by selectively acetylating histone H3K 14. Animal model studies of natural aging have found that inactivating KAT7 can lead to a life span of over 130 weeks in 81% of mice, whereas only 27% of control mice can survive to 130 weeks. With the proposal of the role of KAT7 in aging, the research on the aging correlation of KAT7 with different organs is gradually emerging in recent years, which suggests the research value of KAT7 in different species, multiple organs and different aging-related diseases. This suggests that inhibition of KAT7 levels may be of significant research value in combating aging and extending longevity.
The traditional Chinese medicine has unique advantages in the aspects of resisting aging and prolonging the service life, so that the traditional Chinese medicine compound composition with obvious effects of delaying kidney aging and kidney fibrosis is preferred by combining clinical practice under the guidance of the theory of traditional Chinese medicine.
Disclosure of Invention
The invention aims to: the invention aims to solve the defects of the prior art, adopts the theory of protecting kidney and delaying aging, and adopts the traditional Chinese medicine compound composition which can effectively delay kidney aging and fibrosis through tonifying kidney qi, tonifying kidney essence, activating qi-blood, clearing damp turbidity and differentiating treatment. The invention also aims to provide a preparation method and application of the traditional Chinese medicine compound composition.
The technical scheme is as follows: in order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a Chinese medicinal compound composition for delaying kidney aging and fibrosis is prepared from the following raw materials:
astragalus root, flower of sunset abelmoschus, centella asiatica, root of red rooted saliva, licorice root and cordyceps sinensis hypha.
As a preferred scheme, the traditional Chinese medicine compound composition with the functions of delaying kidney aging and fibrosis is prepared from extracts of astragalus mongholicus, abelmoschus manihot, centella asiatica, salvia miltiorrhiza and liquorice and cordyceps sinensis mycelia.
As a further preferable scheme, the traditional Chinese medicine compound composition with the functions of delaying kidney aging and fibrosis is prepared from extracts of 240-480 g of astragalus mongholicus, 240-480 g of flos abelmoschus manihot, 240-480 g of centella asiatica, 120-240 g of radix salviae miltiorrhizae and 40-80 g of liquorice and cordyceps sinensis mycelia.
As a particularly preferred scheme, the traditional Chinese medicine compound composition with the functions of delaying kidney aging and fibrosis is prepared from extracts of 240g of astragalus mongholicus, 240g of flos abelmoschus manihot, 240g of centella asiatica, 120g of radix salviae miltiorrhizae and 40g of liquorice and cordyceps sinensis mycelia.
As a further preferable scheme, the traditional Chinese medicine compound composition with the functions of delaying kidney aging and fibrosis is prepared from the extract of the traditional Chinese medicine compound and cordyceps sinensis mycelia in a weight ratio of 1:0.88-1.
The invention relates to a traditional Chinese medicine compound composition for delaying kidney aging and fibrosis, which is prepared from the following extracts:
soaking radix astragali, flos Abelmoschi Manihot, herba Centellae, saviae Miltiorrhizae radix and Glycyrrhrizae radix in water, decocting, squeezing residues with gauze, collecting decoction, decocting residues with water, collecting decoction, filtering with gauze, rotary evaporating filtrate, concentrating, pre-freezing, and processing with vacuum freeze dryer to obtain lyophilized powder.
As a preferred scheme, the traditional Chinese medicine compound composition with the functions of delaying kidney aging and fibrosis is prepared from the following extracts:
soaking radix astragali, flos Abelmoschi Manihot, centella asiatica, radix salviae miltiorrhizae and liquorice in water with the weight of 5-15 times of the medicinal materials for 30-40 min, decocting and extracting for 30-60 min, squeezing the medicinal residues with gauze to collect decoction, decocting the medicinal residues with water with the volume of 5-15 times of the medicinal residues for 30-60 min, collecting the two decoction, filtering with gauze, rotationally evaporating and concentrating the filtrate, pre-freezing at-80 ℃ for 24h, and treating with a vacuum freeze dryer for 72h to obtain freeze-dried powder.
The Chinese medicinal compound composition and pharmaceutically acceptable carrier can be made into granule, tablet, capsule, pill, oral liquid, powder, mixture, and unguent.
When the invention is prepared into tablets, the traditional Chinese medicine extract is added with carrier lactose or corn starch, and a lubricant magnesium stearate is added when needed, and the mixture is uniformly mixed, and then the mixture is pressed into tablets.
When the invention is prepared into capsules, the traditional Chinese medicine extract is uniformly mixed with dextrin or starch, and the mixture is granulated and then encapsulated into capsules.
When the invention is prepared into granules, the traditional Chinese medicine extract is added with dextrin and the like to be uniformly mixed, granulated, dried and prepared into granules.
When the invention is prepared into mixture, the traditional Chinese medicine extract is dissolved in purified water to prepare the mixture.
When the invention is used for preparing the oral liquid, the traditional Chinese medicine extract is dissolved in purified water, steviosin or aspartame is added, and the pH is regulated to prepare the oral liquid.
The beneficial effects are that: compared with the prior art, the invention has the following advantages:
(1) According to the invention, through years of clinical experience and combination of kidney aging pathogenesis, the screening formula is added with two medicines of centella asiatica and cordyceps sinensis mycelia on the basis of astragalus, okra, red sage root and liquorice, so that the effects of clearing and activating blood, tonifying qi and tonifying kidney can be obviously improved. The traditional Chinese medicine formula is prepared by harmonizing the traditional Chinese medicines, tonifying kidney qi, tonifying kidney essence, activating qi and blood, clearing damp turbidity and combining the treatment concept of protecting kidney and delaying aging, and becomes a common formula aiming at kidney function decline and early kidney injury clinically. Experimental results show that the traditional Chinese medicine formula provided by the invention can recover kidney functions, and kidney SA, HE, masson staining shows that aging and fibrosis symptoms are relieved after the traditional Chinese medicine is used for treating. Has good effects of resisting natural aging and prolonging life.
(2) The traditional Chinese medicine compound composition with the functions of delaying kidney aging and resisting kidney fibrosis can be conveniently prepared into various dosage forms with pharmaceutical carriers, is convenient to clinically take, and has the advantages of reliable curative effect, safe use and no toxic or side effect after long-term taking.
Drawings
FIG. 1 shows that the traditional Chinese medicine adjusts KAT7 to reduce kidney aging and fibrosis (bar: 100 μm) in mice.
FIG. 2 shows that the traditional Chinese medicine regulates KAT7 to improve kidney aging and fibrosis related protein expression of mice. Filling: * Representative versus blank, # representative versus the previous group; * P, # P < 0.05, # P < 0.01, # P < 0.001, # P < 0.001.
FIG. 3 is a graph showing that serum-regulated KAT7 with the drug-containing traditional Chinese medicine reduces the aging and fibrosis of tubular epithelial cells (100X). Filling: * Representative versus blank, # representative versus the previous group; * P, # P < 0.05, # P < 0.01, # P < 0.001, # P < 0.001.
Detailed Description
The invention will be better understood from the following examples. However, it will be readily understood by those skilled in the art that the specific material ratios, process conditions and results thereof described in the examples are illustrative of the present invention and should not be construed as limiting the invention described in detail in the claims.
Example 1
1. A traditional Chinese medicine compound composition for delaying kidney aging and fibrosis is prepared from the following raw materials in parts by weight:
the weight ratio of the extract of the Chinese herbal compound to the cordyceps sinensis mycelium is 1:0.88.
The extract of the traditional Chinese medicine compound is prepared by the following method:
soaking 240g of astragalus membranaceus, 240g of flos abelmoschus manihot, 240g of centella asiatica, 120g of radix salviae miltiorrhizae and 40g of liquorice in water for 30min, supplementing 5L of pure water, boiling with strong fire, decocting with slow fire for 30min, squeezing the residues with gauze, collecting decoction, and decocting with 5L of pure water for 30min again; the two decoctions were collected and filtered using 8 layers of gauze. Concentrating by rotary evaporation to about 400mL, packaging into 50mL EP tube (15-20 mL per tube), pre-freezing at-80deg.C for 24 hr, and vacuum lyophilizing for 72 hr to obtain lyophilized powder.
Mixing the freeze-dried powder with Cordyceps sinensis mycelium powder according to the mass ratio of 1:0.88 to obtain compound freeze-dried mixed powder. When in use, the compound freeze-dried powder mixed powder is taken, a proper amount of ultrapure water is added, and 0.5 percent of sodium carboxymethyl cellulose is added for even stirring, and the compound freeze-dried powder mixed powder is prepared for use at present.
Example 2
1. A traditional Chinese medicine compound composition for delaying kidney aging and fibrosis is prepared from the following raw materials in parts by weight:
the weight ratio of the extract of the Chinese herbal compound to the cordyceps sinensis mycelia is 1:1.
The preparation method of the extract of the traditional Chinese medicine compound is the same as that of the example 1.
Example 3
1. A traditional Chinese medicine compound composition for delaying kidney aging and fibrosis is prepared from the following raw materials in parts by weight:
the weight ratio of the extract of the Chinese herbal compound to the cordyceps sinensis mycelium is 1:0.88.
The extract of the traditional Chinese medicine compound is prepared by the following method:
soaking 480g of astragalus membranaceus, 480g of flos abelmoschus manihot, 480g of centella asiatica, 240g of radix salviae miltiorrhizae and 80g of liquorice in water for 30min, supplementing to 10L of pure water, boiling with strong fire, decocting with slow fire for 40min, squeezing the residues with gauze, collecting decoction, and decocting with 10L of pure water for 30min again; the two decoctions were collected and filtered using 8 layers of gauze. Concentrating by rotary evaporation to about 800mL, packaging into 50mL EP tube (15-20 mL per tube), pre-freezing at-80deg.C for 24 hr, and vacuum lyophilizing for 72 hr to obtain lyophilized powder.
Mixing the freeze-dried powder with Cordyceps sinensis mycelium powder according to the mass ratio of 1:0.88 to obtain compound freeze-dried mixed powder.
When in use, the compound freeze-dried powder mixed powder is taken, a proper amount of ultrapure water is added, and 0.5 percent of sodium carboxymethyl cellulose is added for even stirring, and the compound freeze-dried powder mixed powder is prepared for use at present.
Example 4 efficacy test for delaying renal aging and fibrosis
1. Improving kidney aging of natural aging mice
1.1 Experimental materials
1.1.1 Experimental reagents
1.1.2 laboratory apparatus
| Main instrument | Model number | Manufacturer' s |
| Inverted fluorescent microscope | Axio Vert A1 | ZEISS |
| Multifunctional enzyme labeling instrument | PE | Perkin Elmer |
| Gel imaging system | LAS 4000 | FUJIFILM |
| Western blot gel electrophoresis equipment | MiNi ROTEAN | Bio-rad |
| Ultralow temperature refrigerator | UUS-410D-SS | ESCO |
| Water purifier | Milli-Q Integral 15 | MERCK |
| Super clean bench | ACB-4A1 | ESCO |
| CO2 cell incubator | HERAcell 150i | Thermo |
| Cell counter | C 10227 | Thermo |
。
1.1.3 laboratory animals
The experimental animals were selected from SPF grade C57BL/6 female mice. Of these, 8 mice of 2 months of age and 24 mice of 6 months of age were purchased from Jiangsu Hua Xinnuo biotechnology Co., ltd, and animal license number SCXK (Su) 2020-0009. The animals are raised in SPF environment of Nanjing university of Chinese medicine animal experiment center, and the raising environment is in 12 hours of light/dark cycle, the temperature is 22+/-3 ℃ and the humidity is 50+/-10%. Mice were given maintenance feed and sufficient clean drinking water. 30C 57BL/6 milk mice of 7-10 days old were purchased from Jiangsu Hua Xinnuo Biotech Co., ltd, animal license number SCXK 2020-0009. Ethical approval of animal experiment center of Nanjing university of traditional Chinese medicine, approval number: 202108A006.
1.1.4 Experimental reagent preparation
The traditional Chinese medicinal materials and decoction pieces in the experiment are provided by the department of preparation of middle hospitals in Jiangsu province. Preparing a traditional Chinese medicine compound sample: soaking 240g of astragalus membranaceus, 240g of flos abelmoschus manihot, 240g of centella asiatica, 120g of radix salviae miltiorrhizae and 40g of liquorice in water for 30min, supplementing 5L of pure water, boiling with strong fire, decocting with slow fire for 30min, squeezing the residues with gauze, collecting decoction, and adding 5L of pure water again for decoction; the two decoctions were collected and filtered using 8 layers of gauze. Concentrating by rotary evaporation to about 400mL, packaging into 50mL EP tubes (15-20 mL each tube), pre-freezing at-80deg.C for 24 hr, and vacuum freeze-drying for 72 hr to obtain 160g lyophilized powder with yield of 18.2%. Mixing the freeze-dried powder with Cordyceps sinensis mycelium powder according to the ratio of 1:0.88 to obtain compound freeze-dried mixed powder. When in use, the compound freeze-dried powder mixed powder is taken, a proper amount of ultrapure water is added, and 0.5 percent of sodium carboxymethyl cellulose is added for even stirring, and the compound freeze-dried powder mixed powder is prepared for use at present.
D-gal animals: 5-g D-gal is weighed, 100mL of sterile water is added, the mixture is uniformly mixed to obtain D-gal injection with the concentration of 500mg/mL, and the D-gal injection is stored at 4 ℃.
Electrophoresis buffer solution: tris 30.2g,Glycine 144g,SDS10g is weighed respectively, ddH2O is added, and the mixture is stirred uniformly, and the volume is fixed to 1000mL, so that electrophoresis 10X stock solution is obtained. 200mL of the stock solution was taken, and ddH2O was added to a volume of 2000mL to give 1 Xrunning buffer.
Transfer buffer: 50mL of 20 Xfast transfer membrane liquid is taken, 100mL of absolute ethyl alcohol is added, ddH2O is added to fix the volume to 1000mL, and 1 Xfast transfer membrane liquid is obtained.
TBS/TBST buffer: 80g of NaCl and 24.4g of Tris are weighed respectively, added with H2O and stirred uniformly, and the volume is fixed to 1000mL, thus obtaining 10 XTBS buffer solution. 200mL of 10 Xbuffer was added to H2O, 2mL of Tween 20 was added thereto and stirred, and the volume was fixed to 2000mL with ddH2O to obtain 1 XTBE buffer.
1.2 Experimental methods
1.2.1 animal intervention
After 6 month old mice were acclimatized for one week, sufficient maintenance feed and clean drinking water were given for continuous feeding to 16 months of age. The randomization was divided into 3 groups: the agent group, the Chinese herbal compound low-dose group (HY-L group) and the Chinese herbal compound high-dose group (HY-H group), 8 Chinese herbal compound low-dose groups (HY-H group). The administration is continued for 60 days after the respective gastric lavage physiological saline solution of 0.2mL/20g/d, the freeze-dried mixed powder of 2g/kg/d and the freeze-dried mixed powder of 4 g/kg/d. 6 mice of 2 months of age were kept to 4 months of age, and as a young control group, physiological saline was administered for 0.2mL/20g/d of gastric lavage treatment. Mice were sacrificed after each group of interventions and blood, urine, kidney tissue were collected for detection.
1.2.2 renal tissue protein expression
Detecting expression conditions of senescence-associated proteins P21, P16, laminB1 and gamma H2Ax by using a Western blot method; fibrosis related protein COL I, alpha-SMA expression; KAT7 and inflammatory minibody Guan Danbai NLRP3, IL-1 β expression.
The Western blot of kidney tissue was performed as follows: 50mg of kidney cortex was cut off using an ophthalmic scissors, kidney envelope was peeled off, PBS was washed, filter paper was sucked to remove excess water, the cut was put into a 2mL EP tube, 500. Mu.L of the mixed protein lysate (RIPA: protease inhibitor cocktail=100:1) was added, two 3mm steel balls were added, and homogenization was performed 2 times using a low temperature homogenizer at 30 power, 4℃for 3min. After the steel balls are taken out, the steel balls are subjected to ultrasonic treatment for 8s with 50% power and are cooled on ice for 8s, and ultrasonic treatment is repeated for 5 times. Centrifuging at 12000rpm and 4deg.C for 15min, collecting supernatant 5 μL for protein concentration measurement, adding 1/4 volume of 5×protein loading buffer solution into the rest supernatant, vortex mixing, denaturing at 99deg.C for 10min with metal bath, cooling, packaging, and storing at-80deg.C.
Gel with proper concentration is prepared by using a gel preparation kit, the gel is loaded according to the protein concentration measurement result, electrophoresis is carried out at a constant pressure of 160V, the rapid membrane transfer liquid is used for membrane transfer at 400mA current, and membrane transfer is carried out for 10-50min according to the molecular weight of target protein. The rapid blocking solution was blocked for 15min, washed 3min×3 times with TBST and the membranes were placed in the corresponding primary antibody for incubation overnight at 4 ℃. The next day the film was taken out and washed 5min×2 times +10min×2 times with TBST. The membrane was placed in the corresponding secondary antibodies, incubated at room temperature for 1 hour, washed 5min×2 times +10min×2 times with TBST, and exposed with ECL chemical developer. Each experiment was independently repeated 3 times, gray value calculation was performed using Image J, and statistics and mapping were performed using Graph Pad Prism.
1.2.3 renal function test
The mice were left on ice for 4h after blood was taken, centrifuged at 3000rpm for 15min at 4℃and serum was aspirated and stored in aliquots at-80 ℃. Urine from mice was collected using bladder compression and stored at-80 ℃. The level of mouse serum creatinine, serum urea nitrogen, serum uric acid, and urine protein was detected using a biochemical kit. Adding each working solution according to the instruction, respectively adding a sample, a standard substance and distilled water, incubating, detecting absorbance in an enzyme-labeled instrument, calculating the concentration of the target protein in the sample by using Office Excel according to an instruction algorithm, and carrying out statistics and mapping by using Graph Pad Prism.
1.2.4 renal SA-beta-gal staining
Frozen sections were made after OTC embedding of kidney tissue. Washing the slice with PBS for 5min×3 times, adding fixing solution, standing at room temperature for 20min, discarding the fixing solution, washing with PBS for 5min×3 times, and mixing according to component A: b: c: x=1: 1:93:5, preparing a dyeing working solution according to the proportion. 500. Mu.L of working solution was added dropwise to each slice, and the slices were placed in a moisture-retaining cassette and oven at 37℃overnight. The next day the working solution was discarded, 0.1% nuclear solid red dye solution was added for 20min, washed 5min x 3 times with PBS, and blue positive areas were observed under an optical microscope using a glycerol gelatin seal liquid seal.
1.2.5 renal histopathological staining
Paraffin sections were prepared and HE, masson, sirius red stained.
The sirius red dyeing method specifically comprises the following steps: the kidney tissue slice is baked for 40min in a 70 ℃ oven, and hydration treatment is carried out by sequentially using dimethylbenzene I, dimethylbenzene II and gradient alcohol. Soaking in saturated sirius red staining solution for 1h, washing with flowing water for 10s, soaking in 0.5% acetic acid solution for 20s, washing with flowing water, dehydrating with gradient ethanol and xylene, sealing with neutral resin, air drying, and observing and photographing with polarized light microscope.
1.2.6 immunofluorescent staining of kidney tissue
The kidney tissue slice is baked for 40min in a 70 ℃ oven, and hydration treatment is carried out by sequentially using dimethylbenzene I, dimethylbenzene II and gradient alcohol. Repairing with antigen retrieval liquid at 95deg.C for 10min, cooling, and washing with PBS for 5min×3 times. Adding 5% goat serum, sealing for 30min, removing serum, organizing pen ring tissue, and dripping 1: the Lamin B1 rabbit primary antibody diluted by 100 is subjected to moisture preservation and incubation for 8 hours at the temperature of 4 ℃ in a cassette. Taking out the product the next day, rewarming for 15min, shaking and washing for 5min×3 times by using Antibody wash buffer, and shaking and washing for 3min by using PBS. And then 1: the 100 diluted KAT7 murine anti-antibody was incubated for 8h and the shaking procedure was repeated. Dropwise adding 1:100 proportion of red fluorescent rabbit secondary antibody and green fluorescent mouse secondary antibody, and incubating for 1h at room temperature in dark. The same shaking operation as the primary antibody was performed. DAPI was added dropwise to dye nuclei for 15min, and the nuclei were washed 3min X3 times with PBS. And (3) using an anti-quenching sealing piece liquid sealing piece, observing red, green and blue fluorescence and shooting.
1.2.7 data processing
Protein gray value detection using Image J and Image processing using Image pro plus statisticsPathological staining, statistical data were analyzed using Prism GraphPad 8.0 and quantitative maps were made. The differences between groups are expressed by ANOVA single factor analysis of variance, the differences between two groups are expressed by LSD method, and the method is adoptedRepresenting the metrology data, P < 0.05 indicates that the difference is statistically significant.
2. Improving primary renal tubular epithelial cell aging of mice
2.1 Experimental materials
2.1.1 Experimental reagents and instruments
As in 1.1.1 and 1.1.2 above.
2.1.2 laboratory animals
SPF-grade male SD rats, 20, weighing about 250g, were purchased from Jiangsu Hua Xinnuo Biotech Inc., animal license number SCXK (Su) 2020-0009. The animals are raised in SPF environment of Nanjing university of Chinese medicine animal experiment center, and the raising environment is in 12 hours of light/dark cycle, the temperature is 22+/-3 ℃ and the humidity is 50+/-10%. Rats were given maintenance feed and sufficient clean drinking water.
2.1.3 Experimental reagent preparation
The tested traditional Chinese medicine compound is 1.1.3 as above, when in use, the compound freeze-dried powder mixed powder is taken, a proper amount of ultrapure water is added, and 0.5 percent of sodium carboxymethyl cellulose is added for even stirring, and the traditional Chinese medicine compound is prepared for use.
D-gal cell medication: 4.504g D-gal is weighed, 50mL of sterile water is added, the mixture is uniformly mixed to obtain a D-gal stock solution with the concentration of 500mM, the stock solution is filtered by a 0.22 mu m filter head and then is split into 1.5mLEP pipes, and the split pipes are sealed and then stored at the temperature of minus 20 ℃.
Electrophoresis buffer solution: tris 30.2g,Glycine 144g,SDS10g is weighed respectively, ddH2O is added, and the mixture is stirred uniformly, and the volume is fixed to 1000mL, so that electrophoresis 10X stock solution is obtained. 200mL of the stock solution was taken, and ddH2O was added to a volume of 2000mL to give 1 Xrunning buffer.
Transfer buffer: 50mL of 20 Xfast transfer membrane liquid is taken, 100mL of absolute ethyl alcohol is added, ddH2O is added to fix the volume to 1000mL, and 1 Xfast transfer membrane liquid is obtained.
TBS/TBST buffer: 80g of NaCl and 24.4g of Tris are weighed respectively, added with H2O and stirred uniformly, and the volume is fixed to 1000mL, thus obtaining 10 XTBS buffer solution. 200mL of 10 Xbuffer was added to H2O, 2mL of Tween 20 was added thereto and stirred, and the volume was fixed to 2000mL with ddH2O to obtain 1 XTBE buffer.
2.2 Experimental methods
2.2.1 preparation of medicated serum
After 3 days of adaptive feeding, rats were randomized into 2 groups, 5 of which were given physiological saline 2mL/250g/d for lavage. 15 Chinese medicinal materials are administrated to the stomach of the freeze-dried mixed powder solution of the Chinese medicinal materials for each time in the morning and evening every day, and the dosage is 1.1g/kg. Serum was collected 4 days after gavage by anesthesia. The specific operation is as follows: after the injection of pentobarbital sodium for full anesthesia, the abdominal cavity is opened to push away viscera, the connective tissue is passively separated by using a cotton ball, the abdominal aorta is exposed, the blood is collected by using a disposable blood taking needle and a vacuum blood taking tube, and the rat is sacrificed after the collection is finished. The blood collection tube was left to stand on ice for 2 hours and centrifuged at 3000r for 15min at 4 ℃. The supernatant was pipetted into a 50mLEP tube and placed in a 56℃water bath for endotoxin inactivation for 30min. Transferring into an ultra clean bench, filtering with 0.22 μm filter head, packaging into 1.5mL tube, and storing at-80deg.C.
2.2.2 primary cell extraction and intervention
The primary cell extraction and culture method is as follows: the milk rats are killed, fully sterilized by soaking in 75% alcohol, double kidneys are rapidly taken out from the back, the envelopes and medulla are cleaned and removed in PBS, and the milk rats are moved into Hanks buffer solution to be sheared into the size of about 1mm 3. Transfer into a 15mL tube, supplement 5mL Hanks buffer, centrifuge at 1500rpm for 5min. The supernatant was discarded, 3mL of 0.1% collagenase IV was added, and after resuspension was performed by pipetting, the mixture was transferred to a 60mm dish and digested at 37℃for different times (15, 30, 45, 60 min). Digestion was stopped by adding 3mL DMEM complete medium containing 10% fbs after the end of the timer. The tissue fluid was transferred to a 15mL centrifuge tube and centrifuged at 1000rpm for 3min, and the supernatant was removed. The remaining tissue pellet was added to 5mL Hanks solution and centrifuged at 500rpm for 3min, and this step of centrifugation was repeated three times in total. 1mL of DMEM complete medium (hereinafter referred to as primary cell culture medium) containing 10% FBS, 1% ITS, 1% P/S was added, and the resuspended cells were blown. The suspension was added dropwise to a 100 mesh cell sieve, gently ground 5 times using a 5mL syringe stopper, rinsed with 1mL primary cell complete medium, and gently ground again. The filtrate was collected and added dropwise to a 200 mesh cell sieve, and the milling operation was repeated. The screened cell suspension was collected and inoculated into T25 flasks at a ratio of one T25 flask per 4 kidneys. Fresh primary cell culture medium was replaced 24-36 hours after inoculation for observation, and passaging or intervention was performed about 48 hours later. Primary cells in good condition were digested into cell suspensions, and cell counts were performed. Inoculated into a 6-well plate at a density of 4×105 cells/well, inoculated into a 12-well plate at a density of 105 cells/well, inoculated into a 96-well plate at a density of 104 cells/well, and cultured by adding a primary cell culture medium. When the fusion rate reaches 70% -80%, 10% blank serum, 10% blank serum+150 mM D-gal,5% medicated serum+D-gal, 10% medicated serum+D-gal, 15% medicated serum+D-gal are added respectively, and the mixture is intervened for 24 hours.
2.2.3 cell Activity assay
24 hours after the 96-well plate described above was tampered with. After sucking out the old culture medium, preparing 10% concentration CCK-8 working solution corresponding to the culture medium, adding 100 μl of working solution into each hole, placing in an incubator, incubating, and detecting absorbance of each hole at 450nm with a multifunctional enzyme-labeled instrument every 15min until absorbance of the blank control group is about 1.
2.2.4SA-beta-gal staining
After the above 12-well plate was interfered for 24 hours, the culture solution was discarded, and washed 1 time with PBS, 500. Mu.L of the staining fixative was added to each well, the wells were fixed at room temperature for 20 minutes, and the PBS was added to shake wash 5min X3 times at 60rpm of the shaking table. According to A: b: c: x=1: 1:93:5, preparing a dyeing working solution according to the proportion. Adding 500 mu L of dyeing working solution into each hole, dripping a proper amount of PBS (phosphate buffered saline) into gaps among the holes for moisturizing, sealing a film sealing plate, wrapping an aluminum foil, placing the aluminum foil in a constant temperature box at 37 ℃ for overnight in a dark place, discarding the working solution the next day, changing the working solution into PBS, and observing and shooting by using an optical microscope.
2.2.5 Primary tubular epithelial cell protein expression
Detecting expression conditions of senescence-associated proteins P21, P16, laminB1 and gamma H2Ax by using a Western blot method; fibrosis related protein COL I, alpha-SMA expression; KAT7 and inflammatory minibody Guan Danbai NLRP3, IL-1 β expression.
The Western blot specific procedure for primary tubular epithelial cells was as follows: old medium in 6 well plates was discarded, washed 2 times with PBS, 150 μl of the mixed protein lysate (RIPA: protease inhibitor cocktail = 100:1) was added, lysed on ice for 15min with shaker 60rpm, scraped off cells with a cell scraper, gently blown with a pipette, briefly vortexed, transferred into a 1.5mL EP tube, sonicated for 8s at 50% power + cooled on ice for 8s, and repeated 5 times. Centrifuging at 12000rpm and 4deg.C for 15min, collecting supernatant 5 μl for protein concentration measurement, adding 1/4 volume of protein loading buffer into the rest supernatant, mixing, denaturing at 99deg.C for 10min with metal bath, cooling, packaging, and storing at-80deg.C.
Gel with proper concentration is prepared by using a gel preparation kit, the gel is loaded according to the protein concentration measurement result, electrophoresis is carried out at a constant pressure of 160V, the rapid membrane transfer liquid is used for membrane transfer at 400mA current, and membrane transfer is carried out for 10-50min according to the molecular weight of target protein. The rapid blocking solution was blocked for 15min, washed 3min×3 times with TBST and the membranes were placed in the corresponding primary antibody for incubation overnight at 4 ℃. The next day the film was taken out and washed 5min×2 times +10min×2 times with TBST. The membrane was placed in the corresponding secondary antibodies, incubated at room temperature for 1 hour, washed 5min×2 times +10min×2 times with TBST, and exposed with ECL chemical developer. Each experiment was independently repeated 3 times, gray value calculation was performed using Image J, and statistics and mapping were performed using Graph Pad Prism.
2.2.6 data analysis
Protein grey values were detected using Image J and the images were processed, pathological staining was counted using Image pro plus, statistical data was analyzed using Prism GraphPad 8.0 and a quantitative graph was made. The differences between groups are expressed by ANOVA single factor analysis of variance, the differences between two groups are expressed by LSD method, and the method is adoptedRepresenting the metrology data, P < 0.05 indicates that the difference is statistically significant.
3. Experimental results
3.1 Chinese medicinal composition for improving kidney aging and fibrosis of mice with natural aging in vivo
As shown in the experimental results of fig. 1 and 2. HE staining showed that kidney medium and tubular and glomerular morphology was intact in 4 month old mice; the pathological changes of inflammatory cell infiltration, tubular epithelial cavitation, glomerular atrophy and the like of the mice at 18 months of age are obviously reduced after intervention of the traditional Chinese medicine compound composition with different doses.
Renal function testing showed: the serum creatinine, uric acid, urea nitrogen and urine protein levels are obviously increased at 18 months of age, and are reduced to different degrees after intervention of the Chinese herbal compound composition.
Masson staining showed that collagen deposition was evident in 18 month old mice kidneys, and the group of traditional Chinese medicine compound compositions exhibited significant improvement. The sirius red staining polarized light observation shows that partial type III collagen and a small amount of type I collagen are present in the kidneys of young mice, and the type I collagen and the type III collagen are deposited in a large amount in the kidneys of 18 months old mice, so that the type IV collagen is increased, and the traditional Chinese medicine compound has obvious downregulation effect on the type I collagen. Western blot shows that the kidney COL I and alpha-SMA levels of young mice are lower, the levels of the mice in 18 months are obviously increased, and the expression level of the mice is down-regulated after the intervention of the Chinese herbal compound composition. The protein expression difference has statistical significance (P < 0.05, P < 0.01).
Renal SA- β -gal shows: the kidneys of 18-month-old mice are stained in a large number of blue positive colors, and the positive areas of aging staining are obviously reduced through the intervention of the Chinese herbal compound composition. Western blot shows: the young group had higher levels of kidney LaminB1, while P21, P16, γh2ax were lower. The decrease in Lamin B1 was evident in 18 months of age, while P21, P16, γh2ax. The expression of the aging marker protein is reduced after the traditional Chinese medicine compound composition is dried, and the difference has statistical significance (P is less than 0.05 and P is less than 0.01).
Meanwhile, western blot results show that KAT7, NLRP3 and IL-1 beta are improved in the 18 month old group, the protein expression level is reduced after the traditional Chinese medicine compound composition is dried, and the difference has statistical significance (P is less than 0.05).
Immunofluorescence showed that in the kidneys of 18 month old natural aging mice, the green fluorescence representing KAT7 expression appeared to be opposite to the red fluorescence representing lamin B1 expression. Meanwhile, the expression of kidney Lamin B1 is increased while KAT7 is reduced by the intervention of traditional Chinese medicine.
3.2 in vitro improvement of Primary tubular epithelial cell aging and fibrosis by serum containing drug of Chinese herbal Compound composition
As shown by the experimental results in fig. 3. The cell activity detection results show that: the addition of drug-containing serum improved the activity of mPTCs to a different extent than the model group with the addition of the blank serum and D-gal, with 10% of the drug-containing serum improving most significantly and a 10% reduction in effect at 15% concentration.
SA-beta-gal detection results show that blue-green positive staining of mPTCs cells cultured by using blank serum is increased after D-gal intervention, and positive staining is reduced to different degrees by intervention of drug-containing serum with different concentrations, wherein 5% and 10% of positive staining are obviously reduced.
Western blot results show that: the intervention of the serum containing the traditional Chinese medicine compound composition can up-regulate the expression level of the Lamin B1 protein in D-gal induced aging mPCs and down-regulate the levels of P21, P16 and gamma H2Ax. And down-regulation of COL I, alpha-SMA expression levels was observed, the differences between these groups of changes were statistically significant (P < 0.05 or P < 0.01).
The phenomenon in inflammatory body regulation is similar to in vivo experiments. Western blot results show that KAT7, NLRP3 and IL-1 beta are reduced in expression after intervention of compound drug-containing serum, and experimental results inside and outside the compound drug-containing serum indicate that the traditional Chinese medicine compound can inhibit activation of inflammatory corpuscles, so that the aging renal function and fibrosis condition are improved.
The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the present invention and to implement it, but not limit the scope of the present invention, and all equivalent changes or modifications made according to the spirit of the present invention should be included in the scope of the present invention.
Claims (10)
1. A traditional Chinese medicine compound composition for delaying kidney aging and fibrosis is characterized by being prepared from the following raw materials:
astragalus root, flower of sunset abelmoschus, centella asiatica, root of red rooted saliva, licorice root and cordyceps sinensis hypha.
2. The compound Chinese medicinal composition for delaying kidney aging and fibrosis according to claim 1, wherein the compound Chinese medicinal composition is prepared from extracts of astragalus mongholicus, abelmoschus manihot, centella asiatica, salvia miltiorrhiza and liquorice and cordyceps sinensis mycelia.
3. The compound Chinese medicine composition for delaying kidney aging and fibrosis according to claim 2, wherein the compound Chinese medicine composition is prepared from extracts of 240-480 g of astragalus mongholicus, 240-480 g of flos abelmoschus manihot, 240-480 g of centella asiatica, 120-240 g of radix salviae miltiorrhizae and 40-80 g of liquorice and cordyceps sinensis mycelia.
4. The compound Chinese medicinal composition for delaying kidney aging and fibrosis according to claim 3, wherein the compound Chinese medicinal composition is prepared from extracts of 240g of astragalus mongholicus, 240g of flos abelmoschus manihot, 240g of centella asiatica, 120g of radix salviae miltiorrhizae and 40g of liquorice and cordyceps sinensis mycelia.
5. The compound Chinese medicinal composition for delaying kidney aging and fibrosis according to claim 3 or 4, wherein the weight ratio of the extract to the cordyceps sinensis mycelia is 1:0.88-1.
6. The compound traditional Chinese medicine composition for delaying kidney aging and fibrosis according to any one of claims 2 to 5, wherein the extract is prepared by the following method:
soaking radix astragali, flos Abelmoschi Manihot, herba Centellae, saviae Miltiorrhizae radix and Glycyrrhrizae radix in water, decocting, squeezing residues with gauze, collecting decoction, decocting residues with water again, collecting decoction, filtering with gauze, rotary evaporating filtrate, concentrating, pre-freezing, and processing with vacuum freeze dryer to obtain lyophilized powder.
7. The compound traditional Chinese medicine composition for delaying kidney aging and fibrosis according to claim 6, wherein the extract is prepared by the following method:
soaking radix astragali, flos Abelmoschi Manihot, centella asiatica, radix salviae miltiorrhizae and liquorice in water with the weight of 5-15 times of the medicinal materials for 30-40 min, decocting and extracting for 30-60 min, squeezing the medicinal residues with gauze to collect decoction, decocting the medicinal residues with water with the volume of 5-15 times of the medicinal residues for 30-60 min, collecting the two decoction, filtering with gauze, rotationally evaporating and concentrating the filtrate, pre-freezing at-80 ℃ for 24h, and treating with a vacuum freeze dryer for 72h to obtain freeze-dried powder.
8. A traditional Chinese medicine preparation for delaying kidney aging and fibrosis, which is characterized in that the traditional Chinese medicine compound composition of any one of claims 1-7 and a pharmaceutically acceptable carrier are prepared into granules, tablets, capsules, pills, oral liquid, powder, mixture and ointment.
9. The use of a compound Chinese medicinal composition according to any one of claims 1 to 7 for the preparation of a medicament for the prevention and treatment of renal aging or renal fibrosis.
10. The use of a compound Chinese medicinal composition according to any one of claims 1 to 7 for the preparation of a medicament for the prevention and treatment of renal failure.
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