CN113069463A - Application of selenium-rich astragalus saponin in slowing down cartilage cell degeneration - Google Patents
Application of selenium-rich astragalus saponin in slowing down cartilage cell degeneration Download PDFInfo
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Abstract
The invention discloses application of selenium-rich astragaloside in slowing down cartilage cell degeneration, which takes degenerated cartilage cells as experimental objects and observes key genes of mTOR signal pathways of IL-1 beta-induced rat degeneration cartilage cells induced by the selenium-rich astragalosidePI3K、Akt、mTORAndBeclin1the possible target point of improving the cartilage cell degeneration is observed, the molecular mechanism of treating OA by using the selenium-rich astragaloside is analyzed, and the basis is provided for the feasibility of treating OA by using the selenium-rich astragaloside. The invention determines that the selenium-rich astragaloside can enhance the proliferative capacity of degenerative cartilage cells and weaken the degenerative cartilage cellsPI3K、AktAndmTORmRNA gene expression and cartilage extracellular matrix II improvementCollagen expression, protection of chondrocytes, enhancement of arthritic chondrocytesBeclin1mRNA gene expression, enhancing autophagy gene expression of degenerated chondrocytes.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to application of selenium-rich astragalus saponin in slowing down cartilage cell degeneration.
Background
Osteoarthritis (OA) is a degenerative disease of joint cartilage, and is characterized mainly by degeneration of joint cartilage, hyperosteogeny, etc. Related studies have found that chondrocyte degeneration plays a critical role in pathogenesis. In recent years, a plurality of researchers focus on researching the mechanism of traditional Chinese medicine intervention OA, and the model of IL-1 beta induced chondrocyte degeneration through traditional Chinese medicine intervention has the advantage of multi-target regulation, thereby providing a certain experimental basis for the prevention and treatment of OA. The astragaloside is used as an effective component of astragalus and has multiple physiological activities of resisting tumor, regulating immunity, influencing collagen synthesis and metabolism, protecting cells from ischemia and hypoxia injury and the like. Selenium is a necessary trace element for maintaining normal physiological activities of human bodies, and has the effects of oxidation resistance, aging resistance, cancer prevention, radiation protection and the like. Meanwhile, researches show that the selenium enrichment can improve the yield and the effective components of the astragalus.
Disclosure of Invention
The invention aims to take degenerated chondrocytes as an experimental object, and observe key genes of mTOR signal pathways of IL-1 beta-induced rat degenerated chondrocytes induced by selenium-rich astragalosidesPI3K、Akt、mTORAndBeclin1the expression of the selenium-rich astragalus saponin can be used for observing possible target spots of the selenium-rich astragalus saponin for improving the degeneration of the chondrocytes, analyzing the molecular mechanism of the selenium-rich astragalus saponin for treating OA, providing a new application of the selenium-rich astragalus saponin and providing basis for the feasibility of using the selenium-rich astragalus saponin for treating OA.
The invention relates to extracorporeal regressionThe cartilage cell change model is established by inducing with IL-1 beta (10 mug/mL), intervening with selenium-rich astragaloside (25-300 mmol/L), detecting the proliferation capacity of each group of chondrocytes by using a thiazole blue (MTT) method, observing the expression change of type II collagen of each group of chondrocytes by using an immunohistochemical method, and detecting each group of chondrocytes by using an RT-PCR methodPI3K、Akt、mTORAndBeclin1the results show that the proliferative capacity of degenerated chondrocytes of the selenium-rich astragaloside treatment group is enhanced in 24h, 48h and 72h, but the difference of 100mmol/L selenium-rich astragaloside in 48h is most obvious, and the expression of type II collagen of the selenium-rich astragaloside treatment group is combined with the expression of type II collagen of the selenium-rich astragalosideBeclin1The gene expression is also increased, and 100mmol/L selenium-rich astragalus saponinPI3K、AktAndmTORthe gene expression of (1) is reducedP<0.05）。
The invention discloses application of selenium-rich astragaloside to enhancement of the proliferative capacity of degenerated cartilage cells.
In a second aspect of the invention, selenium-enriched astragalosides are disclosed for attenuating degenerated chondrocytesPI3K、AktAndmTOR use of mRNA gene expression.
The third aspect of the invention discloses the application of selenium-rich astragalus saponin in improving the expression of cartilage extracellular matrix type II collagen.
In a fourth aspect of the invention, the application of selenium-rich astragaloside in cartilage cell protection is disclosed.
In the fifth aspect of the invention, selenium-rich astragaloside is disclosed for improving the content of arthritis chondrocytesBeclin1Use of mRNA gene expression.
The sixth aspect of the invention discloses application of selenium-rich astragaloside in enhancing autophagy gene expression of degenerated chondrocytes.
The seventh aspect of the invention discloses that the molar concentration of the selenium-rich astragalus saponin is 100mmol/L, and the action time is 48 h.
Compared with the prior art, the invention has the beneficial effects that:
the invention determines that the selenium-rich astragaloside can enhance the proliferative capacity of degenerative cartilage cells.
The invention determines that the selenium-rich astragaloside can weaken degenerated cartilage cellsPI3K、AktAndmTOR mRNA gene expression.
The invention determines that the selenium-rich astragaloside can improve the expression of cartilage extracellular matrix II type collagen.
The invention determines that the selenium-rich astragaloside can protect chondrocytes.
The invention determines that the selenium-rich astragaloside can improve the content of arthritis in chondrocytesBeclin1mRNA gene expression.
The invention determines that the selenium-enriched astragaloside can enhance autophagy gene expression of degenerated cartilage cells.
The invention determines that the effect is best when the molar concentration of the selenium-rich astragalus saponin is 100mmol/L and the action time is 48 hours.
Drawings
FIG. 1 shows the effect of different concentrations of selenium-enriched astragaloside on the proliferative capacity of degenerated chondrocytes at different time points;
FIG. 2 is the protein expression of type II collagen in each group of chondrocytes;
fig. 3 shows the morphological changes of chondrocytes in each group.
Detailed Description
The invention is further described with reference to the following figures and detailed description.
Experimental materials:
1. cell:
normal rat chondrocytes, purchased from Qishi Biotech, Inc., Jiangsu.
2. Drugs and reagents:
selenium-rich astragalus saponin powder, the specification is as follows: each package 20g, batch number: 160405, available from Shanghai Yongsi Biotechnology, Inc. 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyltetrazolium bromide (MTT) reagent, batch No.: 303H0521, purchased from solibao bio-production; high-sugar cell culture medium (DMEM) batch No.: AAJ205976, available from Hyclone, usa; CLARK serum, lot No.: JC50166, available from Hyclone, USA; interleukin-1 beta (IL-1 beta), batch No.: 100991-1D0616, available from PEPROTECH, USA; the reverse transcription kit and the fluorescence quantitative SYBR Premix EX Taq II kit are purchased from Shanghai Bao biology company.
Establishment and administration of a degenerative chondrocyte model:
normal rat chondrocytes were divided into 7 groups after 3 passages in an incubator at 37 ℃: a normal control group, a model group, a 25mmol/L selenium-rich astragaloside treatment group, a 50mmol/L selenium-rich astragaloside treatment group, a 100mmol/L selenium-rich astragaloside treatment group, a 200mmol/L selenium-rich astragaloside treatment group and a 300mmol/L selenium-rich astragaloside treatment group. Adding DEME culture medium containing 10% calf serum into normal control group, adding culture solution containing IL-1 beta 10 μ g/mL into model group, and adding culture solution containing IL-1 beta 10 μ g/mL and selenium-rich astragaloside 25, 50, 100, 200, 300mmol/L into selenium-rich astragaloside 25, 50, 100, 200, 300 mmol/L.
The detection method comprises the following steps:
1. MTT method for detecting proliferation capacity of each group of chondrocytes
The MTT method is adopted to observe the influence of the selenium-rich astragalus saponin with different concentrations on the proliferative capacity of the degenerated cartilage cells in 24h, 48h and 72 h. And (3) continuously culturing 10 mu L of MTT added into each hole of the cells after 20h, 44h and 68h for 4h, removing the supernatant, adding 150 mu L of DMSO, shaking for 10min, and measuring the absorbance value (A value) of each group at 490nm by using an enzyme-labeling instrument.
2. Observing the morphological change of each group of chondrocytes under an inverted microscope
The chondrocytes of a normal control group, a model group and a 100mmol/L selenium-rich astragalus saponin treatment group which are cultured for 48 hours in a cell culture bottle are observed under an inverted microscope for morphological change and photographed.
3. Immunohistochemical method for detecting protein expression of cartilage cell type II collagen
Preparing cell climbing tablets, and performing immunohistochemical staining according to a method in a literature, wherein the method comprises the following specific steps: antigen retrieval: the citrate buffer was heated by microwave for 10min, cooled and washed with PBS. Sealing with sealing liquid: blocking with 5% fetal calf serum for 1 h. ③ incubating the antibody: adding rabbit anti-mouse type II collagen primary antibody (diluted concentration of type II collagen is 1: 300) dropwise at 4 deg.C overnight, washing with PBS, adding goat anti-rabbit secondary antibody working solution dropwise, and incubating at 37 deg.C for 30 min. And fourthly, color development and observation: DAB color development and washing; counterstaining, dehydrating, transparentizing, sealing, reading by two pathology teachers in the pathology teaching and research laboratory of the university of traditional Chinese medicine in Gansu, and taking pictures. Staining with either a yellow or tan coloration of the chondrocyte cytoplasm is positive. The pictures were analyzed using Image-Pro Plus 6.0 professional Image analysis software to analyze 3 fields (x 200) of view at the same site for each tissue section, and the gray scale value (IOD) within the same area was calculated.
4. RT-PCR method for detecting chondrocytesPI3K、Akt、mTORAndBeclin1expression of the gene
Collecting chondrocytes of a normal control group, a model group and a 100mmol/L selenium-rich astragaloside treatment group which are cultured in a 6-well plate, extracting total RNA of cells by a TRizol method, and measuring the content and the purity of the RNA by an ultra-micro ultraviolet spectrophotometer. Primer 3-phosphoinositide kinase (phosphoinositide 3-kinase,PI3K) Protein kinase B (protein kinase B,Akt）、mTOR、Beclin1、GAPDHdesigned and synthesized by TaKaRa Biotechnology engineering (Dalian) Co., Ltd. The gene sequences are shown in Table 1. First strand cDNA was synthesized and PCR was performed according to the procedures of Promega kit, and the reaction conditions were as follows: pre-denaturation at 95 deg.C for 2min, denaturation at 95 deg.C for 15s, annealing at 58 deg.C for 45s, extension at 60 deg.C for 1 min. For a total of 45 cycles. Each sample was replicated 3 times and the data was 2-ΔΔCtAfter the treatment, the relative expression amount analysis was performed.
TABLE 1PI3K、Akt、mTORAndBeclin1primer sequences
Tab 1Primer sequences of PI3K、Akt、mTORand Beclin1
5. Statistical treatment
Software analysis was performed using SPSS 19.0 statistics. The metrology data is expressed, using One-Way-Anova,P<0.05 had significant differences.
The experimental results are as follows:
1. selenium-rich astragalosides for enhancing degenerative cartilage cell proliferation capacity
Compared with the normal control group, the proliferation capacity of the chondrocytes in the model group is obviously weakened at 24h, 48h and 72h (P<0.05); compared with the model group, the proliferation capacity of chondrocytes of each intervention group of the selenium-rich astragalosides is enhanced, and the proliferation promoting effect of the experiment group of intervention of 100mmol/L selenium-rich astragalosides for 48 hours is most obvious (P<0.05), and the degenerated chondrocytes treated for 48 hours by 100mmol/L selenium-enriched astragaloside are used as a selenium-enriched astragaloside treatment group in subsequent experiments. The results are shown in FIG. 1.
2. Method for weakening degenerated cartilage cells by using selenium-enriched astragalosidesPI3K、AktAndmTOR mRNA gene expression but increased in arthritic chondrocytesBeclin1mRNA gene expression, by attenuation or inhibitionPI3K、AktAndmTOR expression of mRNA gene to activate its downstream regulatory geneBeclin1Expression of mRNA genes, i.e. enhancing autophagy gene expression of degenerated chondrocytes:
in the model group, compared with the normal control groupPI3K、AktAndmTORthe expression of the gene is increased,Beclin1reduction of gene expression: (P<0.05); comparing with model group, 100mmol/L selenium-rich astragaloside treatment groupPI3K、AktAndmTORthe expression of the gene is reduced, and,Beclin1increased gene expression (P<0.05). The results are shown in Table 2.
TABLE 2 groups of chondrocytesPI3K、Akt、mTORAndBeclin1the effects of mRNA expression (,n=6）
Tab 2 Effects of PI3K, Akt, mTOR and Beclin1 mRNA expression on the chondrocytes in each group（，n=6）
note: compared with the normal control group,△ P<0.05; in comparison with the set of models,* P<0.05
MTT result display, modelThe proliferation capacity of the chondrocytes of the type group is obviously weakened in 24h, 48h and 72h, the proliferation capacity of the chondrocytes of each intervention group of selenium-enriched astragalosides is enhanced, but the proliferation promoting effect of the experiment group of intervention of 100mmol/L selenium-enriched astragalosides for 48h is most obvious. Therefore, the experiment observes that the content of 100mmol/L selenium-rich astragaloside after being interfered by degenerated chondrocytes for 48 hoursPI3K、Akt、mTORAndBeclin1 mRNA expression changes, and the result shows that the IL-1 beta induces the rat chondrocyte model group established 48h laterPI3K、AktAndmTORthe expression of the gene(s) of (2) is enhanced,Beclin1the gene expression of (A) is weakened, and after the selenium-rich astragaloside intervenes for 48 hours, the cartilage cells are degeneratedPI3K、Akt、mTORThe expression of the gene(s) of (a),Beclin1the gene expression of (3) is enhanced. The result indicates that the selenium-enriched astragaloside can enhance and restore the expression level of autophagy-related genes, thereby down-regulating the apoptosis level of articular chondrocytes.
3. Selenium-rich astragalus saponin for improving cartilage extracellular matrix II type collagen expression
The type II collagen expression positive staining is tan or tan, and is mainly located in cell cytoplasm. The chondrocytes of the normal control group are evenly stained; the staining of the degenerated chondrocytes of the model group becomes obviously lighter, and the color of cytoplasmic granules becomes lighter; the 100mmol/L selenium-rich astragaloside treatment group degenerated chondrocyte staining is relatively uniform and obviously enhanced. Decreased IOD of type II collagen expression in the model group compared to the normal control group: (P<0.05); compared with the model group, the IOD value of 100mmol/L selenium-rich astragalus saponin treatment group II collagen expression is obviously increased (P<0.05). The results are shown in FIG. 2 and Table 3.
Table 3 effect of selenium-rich astragalosides on type ii collagen expression IOD values in various groups of chondrocytes (,n=6）
Tab 3 Effects of seleno enriched Astragalus Saponin on the IOD Level expression of collagen type II in each degenerated chondrocytes group（，n=6）
note: compared with the normal control group,△ P<0.05; in comparison with the set of models,* P<0.05
type ii collagen is a major component constituting the structural framework of articular cartilage and is also a major component of the extracellular matrix of cartilage. The type II collagen can maintain the normal physicochemical property and mechanical property of the articular cartilage. The normal body articular chondrocyte collagen II metabolism is in dynamic balance. OA may occur due to a cause of less than complete degradation of body type II collagen synthesis, which ultimately leads to loss of cartilage matrix and destruction of cartilage function. The research finds that the protein expression of mouse type II collagen of the OA model is obviously reduced. The experimental result also proves that the protein expression of the II type collagen of the degenerated chondrocyte is obviously weakened, and the protein expression of the II type collagen is obviously enhanced after the selenium-rich astragaloside is given, which shows that the selenium-rich astragaloside plays a role in treatment by improving the protein expression of the cartilage extracellular matrix II type collagen.
4. Selenium-rich astragalus saponin protecting cartilage cell
The cell morphology of the chondrocytes of the normal control group is spindle-shaped in the growth process, the cell morphology of the chondrocytes of the model group is changed in a long spindle-shaped or irregular shape in the growth process, and the cell morphology of the chondrocytes of the 100mmol/L selenium-rich astragaloside treatment group is spindle-shaped or long spindle-shaped in the growth process. The results are shown in FIG. 3.
The intervention effect of the selenium-rich astragaloside on degenerated chondrocytes can be preliminarily determined through the microscopic observation of the chondrocyte morphology. The experimental result proves that the microscopic morphology of the degenerated chondrocytes of the 100mmol/L selenium-rich astragaloside treatment group has a tendency of gradually recovering the normal chondrocyte morphology, which indicates that the selenium-rich astragaloside can protect the chondrocytes.
Claims (7)
1. The application of the selenium-rich astragalus saponin in slowing down the degeneration of chondrocytes is characterized in that: the selenium-rich astragaloside is used for enhancing the proliferative capacity of degenerated cartilage cells.
2. The selenium enriched astragaloside of claim 1Use for slowing down the degeneration of chondrocytes, characterized in that: the selenium-rich astragaloside has effect in weakening degenerative chondrocytePI3K、AktAndmTORuse of mRNA gene expression.
3. The use of the selenium enriched astragaloside of claim 1 for slowing cartilage cell degeneration, wherein: the selenium-rich astragaloside is used for improving the expression of cartilage extracellular matrix II type collagen.
4. The use of the selenium enriched astragaloside of claim 1 for slowing cartilage cell degeneration, wherein: the selenium-rich astragaloside is used for protecting chondrocytes.
5. The use of the selenium enriched astragaloside of claim 1 for slowing cartilage cell degeneration, wherein: the selenium-rich astragaloside can improve the content of arthritis cartilage cellsBeclin1 Use of mRNA gene expression.
6. The use of the selenium enriched astragaloside of claim 5 for slowing cartilage cell degeneration, wherein: the selenium-enriched astragaloside is applied to enhancing autophagy gene expression of degenerated cartilage cells.
7. The use of the selenium enriched astragaloside of any of claims 1-5 for slowing chondrocyte degeneration, wherein: the molar concentration of the selenium-rich astragalus saponin is 100mmol/L, and the action time is 48 h.
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