CN115814084A - Application of in-vivo serum exosome in anti-menopausal osteoporosis - Google Patents
Application of in-vivo serum exosome in anti-menopausal osteoporosis Download PDFInfo
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Abstract
The invention relates to the field of traditional Chinese medicines and biotechnology, and discloses application of in-vivo serum exosomes as a new target of an anti-postmenopausal osteoporosis drug, wherein OVX rats with ovaries removed on two sides and mesenchymal stem cells BMSCs are taken as research objects, a traditional Chinese medicine monomer icariin ICA is taken as a representative drug, an OVX osteoporosis rat model is established on one hand through experiments, and the pharmacodynamic effect of the ICA in preventing and treating osteoporosis is evaluated; on the other hand, rat serum exosomes are extracted, osteogenic differentiation conditions of ICA on BMSCs are researched based on BMSCs cells, and whether ICA plays an anti-osteoporosis role through the serum exosomes is verified, so that the serum exosomes are a new target of an anti-postmenopausal osteoporosis drug, and a new thought is provided for research on an action mechanism of traditional Chinese medicines for preventing and treating osteoporosis.
Description
Technical Field
The invention relates to the field of traditional Chinese medicines and biotechnology, in particular to application of in-vivo serum exosomes in resisting menopausal osteoporosis.
Background
Osteoporosis (OP) is a systemic metabolic bone disease characterized by a decrease in bone mass, a deterioration in bone microarchitecture leading to a decrease in bone strength, and an increase in the risk of fracture. With the accelerating aging of population, the incidence of OP is increased year by year, the number of sick people in China reaches 1.4 hundred million, and about 1.6 hundred million OP patients are estimated in China in 2050. OP can be classified into primary osteoporosis including postmenopausal osteoporosis (PMOP), senile Osteoporosis (SOP) and idiopathic osteoporosis, and secondary osteoporosis. Among them, PMOP is a clinically significant concern because of its steep decline in estrogen levels and its steep increase in OP prevalence, which is 51.6% in women over the age of 65.
OP can cause humpback, pain and dwarfing, and meanwhile, the incidence rate of fracture of patients is obviously increased, the life quality of OP patients is seriously harmed, the economic burden of society is increased, and the OP is a research focus in the medical field. Factors causing OP may be caused by changes in sex hormone levels, increase in age, nutritional status, lifestyle habits, etc., but the direct cause thereof is imbalance in bone metabolism, which is manifested by imbalance in osteoblast-mediated bone formation and osteoclast-mediated bone resorption dynamics in the bone marrow microenvironment. In the dynamic regulation process of the bone marrow microenvironment, osteoclasts are formed by induction and differentiation of monocytes/macrophages, and can form absorption cavities through recombination and polarization of cytoskeleton to secrete related lysosomes and cytokines to realize the bone absorption function. Osteoblasts are the main functional cells for Bone formation, and are differentiated from Bone marrow mesenchymal stem cells (BMSCs), and can synthesize, secrete and mineralize Bone matrix to form new Bone. Therefore, promoting new bone formation to replace lost bone tissue is critical to the treatment of OPs.
Clinically, the traditional Chinese medicine has obvious clinical effect of preventing and treating Osteoporosis (OP). In traditional Chinese medicine, OP belongs to the category of 'bone atrophy', and 'kidney governs bone and marrow generation' is an important guiding idea for preventing and treating OP, but the specific scientific connotation needs to be further clarified. The book Su Wen and the six sections Tibetan elephants treatise is characterized in that the kidney is the origin of the collection and the essence is filled in the bone, and the book Su Wen and the inverse tone treatise is characterized in that: "kidneys are born in bones and kidneys are not born, marrow is not full". Su Wen and Wei Lun (plain questions and atrophy): the kidney governs the bone marrow, indicating that the kidney is closely related to the bone. The book Su Wen Lun also clouds: the bone withering and marrow reduction caused by kidney essence deficiency indicates that the deficiency of the essence and marrow and the bone withering caused by kidney essence deficiency are the root causes of OP diseases, so that doctors of all ages and modern Chinese medicine apply kidney tonifying methods to prevent and treat OP and other bone metabolic diseases, obtain obvious curative effects, and have the effects of increasing the bone density of OP patients, relieving bone pain and improving clinical symptoms and physical signs. The 266 literatures for preventing and treating primary osteoporosis are also researched to analyze the medication rule, and the medication frequency of the traditional Chinese medicines for tonifying kidney, such as epimedium, prepared rehmannia root, teasel root, rhizoma drynariae and the like, is the highest, and the usage frequency ratio of the traditional Chinese medicines entering kidney meridian is the highest, so the traditional Chinese medicine for tonifying kidney is the main medicine for clinically preventing and treating OP in the traditional Chinese medicine. Modern pharmacology shows that a kidney-tonifying traditional Chinese medicine compound, a single kidney-tonifying traditional Chinese medicine and a kidney-tonifying traditional Chinese medicine monomer have wide and positive effects on prevention and treatment of OP, and play a role in resisting OP by promoting bone formation and bone repair of OP model animals, reducing the incidence rate of fracture, promoting homing of BMSCs, osteogenic differentiation of BMSCs and preosteoblasts, inhibiting generation of osteoclasts and the like, but specific pharmacological mechanisms of the compounds are still to be further clarified.
Clinically, the traditional Chinese medicine Epimedium (Epimedium brevicornum Maxim.) for tonifying kidney is pungent, sweet and warm in nature, has the effects of tonifying kidney yang, strengthening muscles and bones and dispelling wind-damp, and the total flavonoid components of the traditional Chinese medicine are developed into various new traditional Chinese medicine preparations for treating osteoporosis, cardiovascular diseases and the like. Icariin (ICA) is the main component of epimedium total flavone, is an 8-isopentenyl flavonoid glycoside compound (CAS number: 489-32-7) in the form of light yellow needle crystal, and has wide bioactivity on skeletal system, nervous system and cardiovascular and cerebrovascular system. In the research of bone metabolism, ICA has obvious effect of preventing and treating PMOP patients and OVX osteoporosis rats. A randomized, double-blind placebo-controlled clinical trial with a 24-month period showed that ICA was effective in preventing PMOP, significantly alleviated clinical symptoms in patients, had relatively low side effects, and was a potential therapeutic agent for PMOP; the effectiveness of ICA in treating postmenopausal osteoporosis of rats is evaluated by systematic evaluation, meta-analysis and other methods, and the inventor finds that ICA can inhibit bone resorption by reducing TRAP5b level, promote bone formation by increasing content levels of Ca, OPG, E2 and the like in serum, and further inhibit the decrease of femoral and lumbar vertebrae density of ovariectomized rats to play an anti-OP role. Research shows that ICA can improve the mean width of femoral trabecula and the thickness of bone cortex of OVX rats in OP model animals, obviously increase the number of osteoblasts and increase the mRNA expression ratio of OPG/RANKL so as to reduce the production of osteoclasts; in the research of BMSCs, ICA can mediate and promote the expression of estrogen receptor Er alpha by activating Wnt/beta-catenin, notch, MAPK, OPG/RANK/RANKL, BMP-2/Smad-4 and other signal channels, improve the protein expression levels of bone morphogenetic protein-2 (BMP-2), RUNX2, ALP, collagen I (COL 1) and osteopontin, thereby inducing the osteogenic differentiation of BMSCs, and meanwhile ICA can also inhibit the adipogenic differentiation of BMSCs by reducing the formation of lipid, reducing the formation of adipogenic transcription factors C/EBP and PPAR. In the research of osteoblasts, ICA can improve the activity of osteoblasts, and obviously improve the osteogenic differentiation capacity of cells by increasing the autophagy of osteoblasts and the expression level of mRNA of Bmp-2 and Runx2 genes. However, the mechanism research of the action of ICA in the paracrine pathway and the like in vivo for preventing OP has not been related, and the relevant action target is still to be further explored.
In recent years, exosomes have attracted attention as a class of intercellular signaling substances. Exosomes are bilayer membrane vesicles of about 30-200nm in diameter, and can serve as important mediators for intercellular signaling. The research finds that various active substances such as nucleic acid, protein and the like encapsulated in exosome can be effectively delivered to target cells and perform functional regulation on the target cells. The current research proves that exosomes in the bone marrow microenvironment play an important role in bone remodeling, and the exosomes can regulate the proliferation, osteogenic differentiation and other functions of bone-related cells through key factors thereof, mediate balanced bidirectional signal transmission between osteoclasts and osteoblasts, reverse bone loss caused by pathological destructive bone diseases and promote bone regeneration. The existing research shows that compared with normal people, the serum-derived exosome of OP patients or osteopenia patients can obviously enhance osteoclast-mediated bone resorption; serum exosomes produced by patients with reduced bone mass and normal old people can compensate and increase the osteoblastic differentiation level and promote osteoblastic bone formation, but serum exosomes of OP patients inhibit osteoblastic mediated bone formation, and the relevant mechanism of the serum exosomes is probably closely related to the down regulation of integrins beta 1, beta 3 and CD34 protein in the exosomes and the interference of bone formation. Meanwhile, the expression of miRNA-21 in the exosomes derived from the MSCs in vivo of the OP patient is higher than that of healthy adults, and the expression of SMAD7 combined with the miRNA-21 is lower, so that the change can obviously reduce the expression of MSC osteogenesis related genes Alpl and Runx2, and obviously inhibit the MSEs-mediated osteogenesis. The miR-31 level is obviously increased in the blood plasma of the elderly and osteoporosis patients, and further research finds that miR-31 secreted by exosomes derived from aging endothelial cells is taken by BMSCs, so that osteogenic differentiation of mesenchymal stem cells is inhibited, and the development of osteoporosis is mediated. The study carries out detection and screening on miRNA in serum exosomes of 20 PMOP and 20 healthy women, and miR-140-3p and miR-23b-3p are found to be potential biomarkers of PMOP and osteoporotic fracture. Further research shows that the exosome has obvious regulation and control effect on bone cells such as BMSCs and the like, and a feasible research direction is provided for clarifying the action mechanism of preventing OP by using the medicine. Research shows that the MSC-derived exosomes have treatment effect on various injury or disease indications including bone defects and osteoporosis, wherein BMSCs have the potential of differentiating into osteoblasts, and the BMSCs-derived exosomes (BMSCs-Exos) can effectively improve OP symptoms, promote proliferation, osteogenic differentiation and bone regeneration of the BMSCs and the osteoblasts, and are the main research content of the exosomes in bone metabolism at present. In addition, BMSCs-Exos can slow down the bone loss effect of mice with Ovariectomized Ovaries (OVX) and delayed fracture healing, improve the content of serum Osteocalcin (OCN) of the OVX mice, and improve the bone loss repair capacity, and the related action mechanism is closely related to the reduction of the expression of apoptosis-related proapoptotic protein cytochrome C and apoptosis protease caspase3 in osteoblasts through MAPK, apoptosis and other signal paths, and the promotion and differentiation of bone cell proliferation.
Therefore, paracrine pathways such as exosomes have important regulation effect on bone metabolism, but the mechanism of traditional Chinese medicine and monomer intervention OP is studied from the aspect of exosomes and is still in the preliminary stage. In the existing research on OP-related exosome mechanisms, exosomes derived from bone cells such as BMSCs are mostly used as entry points, and further research on in-vivo serum exosomes from osteoporosis animal models is still needed. The clinical application of traditional Chinese medicine emphasizes the holistic concept, and the research on the action mechanism of traditional Chinese medicine is carried out based on the basic change of substances in vivo after the medicine is taken, so that the traditional Chinese medicine is more pertinent and in line with the research on clinical traditional Chinese medicine. At present, the pharmacodynamic action of the traditional Chinese medicine monomer ICA for preventing and treating OP is reported, but whether the monomer ICA mediates in vivo exosome to play the anti-OP action is not reported, and whether in vivo serum exosome is an action target of the traditional Chinese medicine for preventing and treating PMOP is worthy of further exploration.
The method adopts Ovariectomized (OVX) rats to simulate postmenopausal osteoporosis, takes OVX rats and BMSCs cells as research objects, takes a traditional Chinese medicine monomer ICA as a representative medicine, finds that in vivo serum exosomes can be used as a new target point for preventing and treating postmenopausal osteoporosis, and provides a new idea for researching an action mechanism of the traditional Chinese medicine for preventing and treating OP.
Disclosure of Invention
In order to solve the technical problems, the invention provides application of in-vivo serum exosomes in anti-menopausal osteoporosis, the in-vivo serum exosomes are new targets of anti-postmenopausal osteoporosis drugs, and the anti-postmenopausal osteoporosis drugs are Icariins (ICAs).
In order to solve the technical problems, the invention also provides a method for verifying in vivo serum exosomes as a new target for preventing and treating postmenopausal osteoporosis, which is characterized by comprising the following steps:
(1) Grouping SD rats, setting three groups of a Sham operation group (a Sham group), a bilateral ovariectomy group (an OVX group) and an icariin administration group (an ICA group), and evaluating the drug effect of ICA on preventing and treating postmenopausal osteoporosis;
(2) Extracting rat serum exosomes of each group, and performing particle size, surface marker and morphological identification;
(3) Extracting BMSCs (rat bone marrow mesenchymal stem cells), and identifying BMSCs surface markers;
(4) Setting a blank group, a Sham-operated rat serum exosome dry preparation group (Sham-Exos group), a bilateral ovariectomized rat serum exosome dry preparation group (OVX-Exos group) and an icariin-administered rat serum exosome dry preparation group (ICA-Exos group) by taking BMSCs as a research platform, and determining the influence of serum exosomes with different concentrations on the proliferation of the BMSCs;
(5) The osteogenic differentiation of BMSCs was examined after the BMSCs were dried in each group of serum exosomes.
Preferably, the evaluation of the pharmacodynamic action of ICA in preventing and treating osteoporosis in the step 1 comprises the analysis of the weight and uterine changes of rats in each group, the detection of bone microstructure by Micro-CT and HE staining, and the detection of the expression levels of osteogenic differentiation related factors ALP, BMP-2, COL1 and RUNX2 proteins in bone tissues by Western-blot.
Preferably, the step 2 of identifying the rat serum exosomes of each group comprises detecting the particle size of the serum exosomes by a laser nanometer particle sizer, detecting the form of the serum exosomes by a transmission electron microscope, and detecting the surface markers of the serum exosomes by Western-blot.
Preferably, step 5 is to detect osteogenic differentiation conditions of the BMSCs, including alizarin red staining to detect mineralized nodules of the BMSCs, an ALP kit to detect ALP activity in the BMSCs, and Q-PCR and Western-blot to detect the expression levels of osteogenic differentiation related factors ALP, BMP-2, COL1 and RUNX2 proteins and encoding genes Alpl, bmp-2, col1al and Runx2 of the BMSCs.
Compared with the prior art, the invention has the beneficial effects that: at present, the research on the mechanism of exosome for preventing OP of traditional Chinese medicine is in the initial stage, and the exosome from cell source is obtained by interfering cells with traditional Chinese medicine extracts or traditional Chinese medicine monomers, so as to research the mechanism of bone-promoting action of the exosome on bone-related cells. However, the clinical application of TCM emphasizes the holistic concept, and the research on the action mechanism of TCM based on the change of the substance basis in vivo after administration will be more appropriate and consistent with the clinical study of TCM. The invention takes a traditional Chinese medicine monomer ICA as a representative medicine, finds that in vivo serum exosomes can be used as a new target point for preventing and treating postmenopausal osteoporosis, and provides a new idea for researching an action mechanism of the traditional Chinese medicine for preventing and treating OP.
Drawings
FIG. 1 is a schematic design diagram of the technical scheme of the invention;
FIG. 2 is a graph showing the changes in body weight (A), body weight gain (B), uterine shape (C) and uterine coefficient (D) of each group of animals after 12 weeks of administration of the present invention;
FIG. 3 shows the change of bone relationship between femur (A) and lumbar vertebra (B) tissues of rats in each group;
FIG. 4 shows the change of bone microstructure of femur (A) and lumbar (B) tissues of rats in each group according to the present invention;
FIG. 5 shows the protein level changes of factors involved in bone differentiation in femoral tissues of rats in each group according to the present invention;
FIG. 6 shows the results of the identification of the particle size (A), surface marker (B) and shape (C) of exosomes of the present invention;
FIG. 7A is a graph showing the identification of the surface marker CD44 (+) of BMSCs according to the present invention;
FIG. 7B shows the identification of the surface marker CD29 (+) of BMSCs according to the present invention;
FIG. 7C shows the identification of the surface marker CD11b (-) of BMSCs according to the present invention;
FIG. 7D shows the identification of the surface marker CD45 (-) of BMSCs according to the present invention;
FIG. 8 shows the change of cell proliferation activity of BMSCs under the intervention of different concentrations of various groups of serum exosomes of the present invention;
FIG. 9A is the mRNA change of the gene encoding the osteogenic differentiation associated factor for BMSCs 24 hours after intervention of various serum exosomes at different concentrations;
FIG. 9B shows the mRNA changes of the gene encoding the osteogenic differentiation associated factor for BMSCs 48 hours after intervention of various serum exosomes at different concentrations;
FIG. 10 is a graph showing changes in mineralized nodules in BMSCs after 28 days of drying for each group of serum exosomes of the present invention;
FIG. 11 is a graph showing ALP activity changes in BMSCs in the presence of various sets of serum exosomes of the present invention;
FIG. 12 is a graph showing changes in protein levels of osteogenic differentiation-associated factors of BMSCs in the presence of a serum exosome of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to the accompanying drawings and examples, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not meant to limit the present invention.
Example (b): method for verifying serum exosome as new target for preventing and treating postmenopausal osteoporosis
Fig. 1 is a practical schematic diagram of the technical scheme of the invention, and the specific experimental process is as follows:
1. animal experiment grouping: 30 SD female rats were randomly divided into a Sham group (Sham group, n = 10), a bilateral ovariectomized group (OVX group, n = 10), and an icariin-administered group (ICA group, n = 10), wherein the OVX group and the ICA group underwent bilateral ovariectomy. The specific method for bilateral ovariectomy of the OVX group and the ICA group comprises the following steps: rats were fasted for 12 hours before surgery without water deprivation, weighed, and anesthetized by intraperitoneal injection of 2% sodium pentobarbital (0.2 mL/100 g); after the rats are deeply anesthetized, the abdomen is opened along the leucorrhea line, the muscle layer and the connective tissue of the rats are separated layer by layer, the ovaries are found through turning, the oviduct tissue is ligated through an operation line, the ovaries on the two sides are cut off through scissors and are sutured layer by layer, the sutured port is cleaned through overturning by 0.5% medical iodophor disinfectant, water is fed at proper time, and the rats are placed in the cage box after waking up. Sham groups removed equal volumes of adipose tissue near the ovaries. After 1 week of operation, the ICA group is administered by gastric gavage with ICA (125 mg/Kg. D) for 12 weeks, and the other groups are administered with physiological saline of the same amount; wherein the evaluation of the drug effect of ICA in preventing and treating postmenopausal osteoporosis comprises the following steps:
(1) The rats in each group were analyzed for changes in body weight and uterus. As shown in FIG. 2, the weight of the rats in the OVX group was significantly increased (P < 0.05), and the uterine atrophy, uterine weight and uterine coefficient were significantly decreased (P < 0.05) compared to the Sham group; compared with the OVX group, the rats in the ICA group have obviously reduced body weight (P < 0.05) and obviously improved uterine atrophy (P < 0.05).
(2) micro-CT (Suzhou Haisifeld information technology Co., ltd., china) observes changes in bone phase relationship of rat bone tissues of each group. As shown in figure 3, compared to Sham group rats, OVX group rats had sparse, fractured, incomplete femoral and lumbar trabeculae; the density of the femur bone is obviously reduced (P is less than 0.01), the number of the bone body integral, the number of the trabeculae and the thickness of the trabeculae are all obviously reduced (P is less than 0.01), and the gap between the trabeculae is obviously increased (P is less than 0.05); the volume fraction of lumbar vertebrae and the number of trabeculae are both obviously reduced (P is less than 0.05), the trabecular gap is obviously increased (P is less than 0.05), the trabecular thickness is reduced to a certain extent, but no obvious change exists; compared with the Sham rats, the micro-structures of the thighbone and the lumbar of the rats in the ICA group are improved, the bone density of the thighbone and the number of the trabeculae are obviously increased (P is less than 0.05), the gap between the trabeculae is obviously reduced (P is less than 0.05), the thickness of the trabeculae is not obviously changed, and the volume of the trabeculae is increased to a certain degree but has no obvious difference; the bone number integral of the lumbar vertebra is obviously increased (P is less than 0.05), the thickness of the trabecular bone is not obviously changed, the number of the trabecular bone is increased to a certain degree, and the gap between the trabecular bone is reduced to a certain degree but has no significant difference.
(3) HE staining was performed to observe changes in bone microstructure in rat bone tissue of each group. As shown in FIG. 4, compared with Sham rats, the fat vacuoles of femur and lumbar vertebrae of OVX rats were significantly increased and enlarged, and the trabecular bone was significantly fractured; compared with Sham rats, ICA rats have obviously reduced and smaller fat vacuoles of thighbone and lumbar vertebra, and the fracture of trabecula is repaired.
(4) And detecting the protein change condition of osteogenic differentiation related factors of the bone tissues of the rats in each group by using Western-blot. As shown in FIG. 5, compared to Sham group rats, the levels of ALP, BMP-2, COL1 and RUNX2 proteins were significantly reduced relative to the expression levels in the femurs of OVX group rats (P < 0.05); compared with the OVX group rats, the relative expression levels of ALP, BMP-2, COL1 and RUNX2 proteins of the femurs of the ICA group rats are obviously increased (P < 0.05).
From the above examples, it can be seen that bilateral Ovariectomy (OVX) can cause weight gain, uterine atrophy, bone mass loss and destruction of bone microstructure in rats, and that addition of ICA drugs to bilateral Ovariectomy (OVX) can significantly reverse or improve the relevant symptoms and promote bone formation and repair in bilateral ovariectomized rats, thus showing that ICA has a good anti-osteoporosis efficacy.
Extracting rat serum exosomes of each group by an ExoQuick kit (System Biosciences, USA), and identifying the Particle size, surface markers and morphology by adopting a scanning electron microscope (Thermo Fisher Scientific, USA), a laser nanometer Particle sizer (Particle Meter, germany) and a surface antigen detection method of Western-blot; as shown in fig. 6, the serum exosomes extracted from the ExoQuick kit are in a double-layer membrane structure, and the shape is "hemispherical"; the particle size range is 30-120nm, and the average particle size is about 100nm; the expression of the exosome marker proteins CD9, CD63 and Tsg101 were positive.
3. BMSCs were isolated by whole bone marrow adherent culture and surface markers of BMSCs were identified by flow cytometry (Becton, dickinson and Company, USA). As shown in fig. 7A, 7B, 7C, and 7D, the surface markers CD29 and CD44 of BMSCs were positively expressed and CD45 and CD11B were negatively expressed.
4. Separating the exosome-free fetal calf serum by an ultra-high speed centrifugation method, and preparing an exosome-free alpha-MEM culture medium containing 10% fetal calf serum.
5. BMSCs are taken as a research platform and cultured in an exosome-free alpha-MEM culture medium containing 10% fetal bovine serum, a blank group, a Sham-operated rat serum exosome dry pre-group (Sham-Exos group), a bilateral ovariectomized rat serum exosome dry pre-group (OVX-Exos group) and an ICA administration rat serum exosome dry pre-group (ICA-Exos group) are arranged, and the serum exosomes are verified to be a new target point for preventing and treating postmenopausal osteoporosis as follows:
(1) The CCK8 method detects the change of the cell proliferation activity of BMSCs under the intervention of different concentrations of various groups of serum exosomes. As shown in FIG. 8, when the serum exosome concentration is 25. Mu.g/mL, 50. Mu.g/mL, 100. Mu.g/mL, 200. Mu.g/mL, the BMSCs cell proliferation of the Sham-Exos group, the OVX-Exos group and the ICA-Exos group are different, wherein the serum exosomes at the concentration of 50. Mu.g/mL and 100. Mu.g/mL can maintain or promote the cell proliferation activity of the BMSCs in each group.
(2) And (3) detecting the change condition of BMSCs osteogenic differentiation index mRNA after different concentrations of the serum exosomes of each group are dried by a Q-PCR method. As shown in fig. 9A and 9B, the relative expression levels of mRNA of Alpl, bmp-2, col1al, and Runx2 genes in OVX-Exos group BMSCs were significantly decreased (P < 0.05) compared to Sham-Exos group after intervention at a serum exosome concentration of 100 μ g/mL for 24 hours or 48 hours; compared with OVX-Exos group, the relative expression levels of mRNA of Alpl, bmp-2, col1al and Runx2 genes in BMSCs of ICA-Exos group were significantly increased (P < 0.05).
(3) Alizarin red staining detects mineralized nodules in BMSCs. As shown in fig. 10, after intervention at a serum exosome concentration of 100 μ g/mL for 28 days, mineralized nodule volume and number of BMSCs were significantly reduced in the OVX-Exos group compared to the Sham-Exos group; compared with the OVX-Exos group, the BMSCs mineralized nodules in the ICA-Exos group had significantly increased volume and number.
(4) ALP kit (Nanjing, china, institute of bioengineering) for detecting ALP enzyme activity of BMSCs. As shown in FIG. 11, ALP enzyme activity was significantly decreased in OVX-Exos BMSCs (P < 0.05) compared to the Sham-Exos group 7 days after the intervention at a serum exosome concentration of 100 μ g/mL; compared with OVX-Exos group, ALP activity was significantly increased in BMSCs of ICA-Exos group (P < 0.01).
(5) Western-blot was used to determine the relative expression level of the proteins of osteogenic differentiation-associated factors in BMSCs. As shown in FIG. 12, after intervention at a serum exosome concentration of 100. Mu.g/mL for 3 days, the relative expression levels of ALP, BMP-2, COL1 and RUNX2 were significantly reduced (P < 0.05) for BMSCs in the OVX-Exos group compared to the Sham-Exos group; compared with the OVX-Exos group, the relative expression levels of ALP, BMP-2, COL1 and RUNX2 proteins of BMSCs in the ICA-Exos group are remarkably increased (P < 0.05).
From the above examples, it can be seen that bilateral ovariectomized rat serum exosomes have osteogenic differentiation inhibitory effect on BMSCs, whereas ICA-administered rat serum exosomes have osteogenic differentiation inhibitory effect on BMSCs by reversing bilateral ovariectomized rat serum exosomes. From this, it is known that ICA can promote osteogenic differentiation of BMSCs by serum exosome, thereby exerting an anti-postmenopausal osteoporosis effect.
Therefore, the in vivo serum exosome can be proved to be a new target for preventing and treating postmenopausal osteoporosis, and a new thought is provided for the mechanism research of traditional Chinese medicines.
The above is an embodiment of the present invention. The embodiments and specific parameters in the embodiments are only for the purpose of clearly illustrating the verification process of the invention and are not intended to limit the scope of the invention, which is defined by the claims, and all equivalent structural changes made by using the contents of the specification and the drawings of the present invention should be covered by the scope of the present invention.
Claims (5)
1. The application of in-vivo serum exosomes in anti-menopausal osteoporosis is characterized in that the in-vivo serum exosomes are new targets of anti-postmenopausal osteoporosis drugs, and the anti-postmenopausal osteoporosis drugs are Icariins (ICAs).
2. A method for verifying in vivo serum exosomes as a new target for preventing and treating postmenopausal osteoporosis is characterized by comprising the following steps:
(1) Grouping SD rats, setting three groups of a Sham operation group (a Sham group), a bilateral ovariectomy group (an OVX group) and an icariin administration group (an ICA group), and evaluating the drug effect of ICA on preventing and treating postmenopausal osteoporosis;
(2) Extracting rat serum exosomes of each group, and performing particle size, surface marker and morphological identification;
(3) Extracting rat bone marrow mesenchymal stem cells BMSCs and identifying BMSCs surface markers;
(4) Setting a blank group, a Sham-operated rat serum exosome dry preparation group (Sham-Exos group), a bilateral ovariectomized rat serum exosome dry preparation group (OVX-Exos group) and an icariin-administered rat serum exosome dry preparation group (ICA-Exos group) by taking BMSCs as a research platform, and determining the influence of serum exosomes with different concentrations on the proliferation of the BMSCs;
(5) And (3) detecting the osteogenic differentiation condition of the BMSCs after the BMSCs are dried and pre-treated by the serum exosomes of each group.
3. The method of claim 3, wherein the step 1 of evaluating the pharmacodynamic action of ICA in preventing and treating osteoporosis comprises analyzing the change of body weight and uterus of rats in each group, detecting bone microstructure by Micro-CT and HE staining, and detecting the expression levels of ALP, BMP-2, COL1 and RUNX2 proteins, which are factors related to osteogenic differentiation of bone tissues, by Western-blot.
4. The method according to claim 3, wherein the step 2 of identifying the serum exosomes of each group of rats comprises detecting the particle size of the serum exosomes by a laser nanometer particle sizer, detecting the morphology of the serum exosomes by a transmission electron microscope, and detecting surface markers of the serum exosomes by Western-blot.
5. The method of claim 3, wherein the step 5 of detecting osteogenic differentiation of BMSCs comprises alizarin red staining to detect mineralized nodules of BMSCs, ALP kit to detect ALP activity in BMSCs, Q-PCR and Western-blot to detect the expression levels of the osteogenic differentiation related factors ALP, BMP2, COL1 and RUNX2 proteins and their coding genes Alpl, bmp-2, col1al and Runx2 of BMSCs.
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