The Effect and Mechanism of ICA Regulation of miR-122-5p which from Osteoblasts-Derived Exosomes on Osteogenesis and Migration of BMSCs


 BackgroundAvascular necrosis of femoral head（ANFH）is a common disease in orthopedics, which seriously affects the quality of life of patients. At present, there is no clear method to solve it in clinical practice. In order to cope with this worldwide problem, many scholars have devoted themselves to the research on the treatment of ANFH. Promoting the osteogenic differentiation and directed migration of stem cells is one of the main ideas for the treatment of ANFH. In recent years, more and more researches have begun to pay attention to Chinese medicine and Chinese medicine extracts. At present, in the field of bone regeneration, epimedium is the most in-depth study of Chinese herbal medicine, and Icariin is one of the main active ingredients of epimedium. MethodsDifferent concentrations of Icariin were applied to bone marrow mesenchymal stem cells (BMSCs), and the optimal concentration of Icariin to promote osteogenic differentiation and migration of BMSCs was observed. Then the exosomes from osteoblasts were combined with the optimal concentration of Icariin to BMSCs, observe the effect of promoting osteogenic differentiation and migration of BMSCs. Using high-throughput sequencing, analyze the composition of osteoblast-derived exosomes, predict the target miRNA, and then use miRNA minic and inhibitor to verify. ResultsThe optimal concentration of Icariin to promote osteogenic differentiation and migration of BMSCs is 1×10-7 M. Icariin combined with osteoblast-derived exosomes can effectively promote the osteogenic differentiation and migration of BMSCs. The top four miRNAs which content in osteoblast-derived exosomes are let-7a-5p, miR-100-5p, miR-21-5p, miR-122-5p, and analysis shows that miR-122-5p is related to osteogenic differentiation , The verification found that miR-122-5p can significantly promote the osteogenic differentiation and migration of BMSCs. ConclusionsIcariin can clearly promote the osteogenic differentiation and migration of BMSCs, and osteoblast-derived exosomes can also effectively promote the osteogenic differentiation and migration of BMSCs, the combination of Icariin and osteoblast-derived exosomes can enhance this effect. In this process, one of its targets is miR-122-5p.

Refer to John's method [16], take the two ends and stems of the limbs of a 6-month-old New Zealand white rabbit, separate the periosteum on the bone surface, cut it, centrifuge (600 g/min, 5min), add 0.2% type I collagenase and put it in a 37℃ thermostat for 3-4h. Take out the centrifuge tube and centrifuge (600g/min, 5min), discard the supernatant, add PBS containing double antibody and wash it again, centrifuge (600g/min, 5min) to remove the supernatant, repeat once. Finally, the precipitate obtained by removing the supernatant is mixed with 1640 medium by pipetting, and then aliquoted into T25 culture flasks, and cultured in a CO2 incubator (37℃, 20% O2 concentration, 5% CO2 concentration), when the cell fusion reaches 80% ~90%, pass it down again. Observe the cell morphology under a microscope, and identify the cells by Giemsa staining and Alizarin red staining.

Extraction and identification of osteoblast-derived exosomes(OB-exo)
The P3 generation of osteoblasts are routinely cultured, and the status of the cells is observed with a microscope. If the cells are in good condition, subculture is carried out. The subcultured osteoblasts are then expanded and cultured. After the culture scale can meet the requirements for collecting the supernatant (the total volume of the supernatant is 240ml), stop expanding the culture and continue the culture for 24 hours. After the cell status is stable, discard the original medium and change to 1640 medium without FBS. After continuing the culture for 48 hours, place it in the ultraclean workbench to collect the cell supernatant and use it in the next centrifugation. ① Kit extraction method: After the osteoblasts are expanded and cultured, the supernatant is taken, centrifuged with a low-speed centrifuge (1200g/min, 15min), and the centrifuged supernatant is transferred to an ultrafiltration tube with a pore size of 100kD, under the condition of 4℃ to ultrafiltration, concentration and centrifugation (4000g/min, 40-60min). Then discard the liquid at the bottom of the tube, transfer the concentrated liquid in the upper tube to the EP tube, add onefifth volume of the exosome extraction reagent SBI, mix well, and incubate at 4°C overnight (not less than 16h ). On the 2nd day, the above-mentioned mixed solution was centrifuged again (4000g/min, 3-5min), and the supernatant was discarded after centrifugation. A white flake precipitate appeared at the bottom of the EP tube, and this white substance was exosomes. Place the EP tube containing exosomes in the centrifuge again to continue centrifugation (4000g/min, 3-5min), and suck up the remaining liquid. Use 20μl PBS to resuspend the exosomes at the bottom of the EP tube, mix by pipetting, and store at -80°C for later use. ②Ultracentrifugation extraction method: After the osteoblasts are expanded and cultured, the supernatant is taken, centrifuged with a lowspeed centrifuge (1200g/min, 15min) to remove cell debris, and the centrifuged supernatant is transferred to a high-speed centrifuge tube at 4°C high-speed centrifugation (4000g/min, 60min). Transfer the supernatant after centrifugation to an ultracentrifuge tube, ultracentrifuge at 4°C (4000g/min, 70min), discard the supernatant, repeat this ultracentrifugation operation once, when the second centrifugation is completed, the bottom of the centrifuge tube appears white flake precipitation, this white matter is exosomes. Gently discard the supernatant, pay attention to this step, be very careful, and move gently. Then use 200μl of PBS to resuspend the exosomes at the bottom of the centrifuge tube, pipette to mix, collect and transfer to a 1.6ml EP tube, store at -80°C for later use.

High-throughput sequencing and miRNAs analysis of OB-exo
When culturing osteoblasts, divide three independent culture flasks to extract OB-exo, and finally obtain three exosomes samples, perform high-throughput sequencing on the three samples, and finally take the average value , Get the sequencing result.

The effects of different concentrations of ICA on the proliferation, osteogenesis and migration of BMSCs
P3 generation BMSCs were inoculated into six-well plates, and the following tests were performed respectively: ①Divided ICA into 1×10 -9 M, 1×10 -8 M, 1×10 -7 M, 1×10 -6 M, 1×10 -5 M, 1×10 -4 M each group. CCK-8 kit detects the activity and proliferation ability of BMSCs at different concentrations of ICA; ②Different concentrations of ICA (0, 1×10 -6 M, 1×10 -7 M, 1×10 -8 M, 1×10 -9 M) and the mixture of culture medium was added to different wells after 2w culture and stained with Alizarin Red dye to observe the formation of mineralized nodules in each group; ③Different concentrations of ICA (0, 1×10 -6 M , 1×10 -7 M, 1×10 -8 M, 1×10 -9 M) and medium mixtures were added to different wells, and RNA of each group of cells was extracted on 3d and 7d for osteogenic differentiation and migration related gene mRNA detection; ④Add the mixture of ICA (0, 1×10 -6 M, 1×10 -7 M, 1×10 -8 M, 1×10 -9 M) and culture medium at different concentrations into different wells , Extract the protein of each group of cells on 3d and 7d, and perform Western blotting for osteogenic differentiation and migration related genes. Through these experiments we will determine the optimal concentration of ICA.

The effect of ICA at different times on the osteogenic differentiation and migration ability of BMSCs
P3 generation BMSCs were inoculated into six-well plates, and the following tests were performed: ①The mixture of 1×10 -7 M ICA and culture medium was added to different wells, and the cells of each group were extracted on 3d, 7d, and 14d. mRNA for osteogenic differentiation and migration related genes mRNA qPCR detection; ②Add 1×10 -7 M concentration of ICA and medium mixture to different wells, respectively on 1d, 2d, 3d, 4d, 5d, 6d, and 7d,then the mRNA of each group of cells was extracted, and the osteogenic differentiation and migration-related genes mRNA were detected by qPCR; ③The mixture of 1×10 -7 M ICA and culture medium was added to different wells, respectively, on 1d, 2d, 3d, 4d, 5d, 6d, and 7d, the cell proteins of each group were extracted, and Western blotting was performed to detect the proteins related to osteogenic differentiation and migration.

Observe the influence of ICA on the expression of Runx2 signal axis and CXCR4 signal axis
P3 generation BMSCs were evenly inoculated into 4 wells in a six-well plate. When the cell fusion reached 60%-80%, the culture medium was discarded, the four groups of different treatments were added to the corresponding culture medium, and the four groups were cultured. The four groups of culture medium are blank group, ICA 1×10 -7 M group, ICA 1×10 -7 M +si-Runx2 group, and ICA 1×10 -7 M +AMD3100 group. Among them, the preparation process of si-Runx2 solution is as follows. Dilute the pre-prepared si-Runx2 solution and DMEM at a ratio of 1:50. At the same time, the transfection reagent Lipofectamin 2000 and DMEM are also diluted at a ratio of 1:50. The two solutions are diluted at the same time and incubated at room temperature for 5 minutes. After 5 minutes, the two solutions are mixed in equal proportions and placed at 37°C and incubated for 20 minutes. This is to prepare si-Runx2 solution to be added; The solution concentration of AMD3100 is 50μmol/L and added to the corresponding wells. After culturing for 3 days, the mRNA of each group of cells was extracted, and the mRNA of genes related to osteogenic differentiation and migration was detected.

The effect of ICA combined with OB-exo on the osteogenic differentiation and migration ability of BMSCs
Experimental grouping and culture conditions: The conclusion from the previous experiment is that the optimal concentration of ICA on BMSCs is 1×10 -7 M, and the ICA at this concentration is used for follow-up research. P3 generation BMSCs were inoculated into 4 wells of two six-well plates. When the cell fusion reached 60%-80%, the culture medium was discarded, and the four groups of different treatments were added to the corresponding culture medium. The culture medium was blank group, ICA group, OB-exo group, and ICA+OB-exo group. Among them, the amount of exosomes in the OB-exo group and the ICA+OB-exo group were both 20 μl. The PBS solution containing exosomes was thawed and mixed gently. On the 3rd day, one six-well plate was used to extract mRNA and one was used to extract protein. The mRNA and protein expression of genes related to bone differentiation and migration were detected by qPCR and Western blotting.
First, observe the effect of miR-122-5p minic and ICA on the osteogenic differentiation and migration ability of BMSCs. P3 generation BMSCs were inoculated into 4 wells of two six-well plates. When the cell fusion reached 60%-80%, the culture medium was discarded, and the four groups of different treatments were added to the corresponding culture medium. The culture media were miR-122-5p minic NC group, miR-122-5p minic group, miR-122-5p minic NC+ICA group, and miR-122-5p minic+ICA group. After culturing for 3 days, a six-well plate was used to extract mRNA and one was used to extract protein. The mRNA and protein expression of genes related to bone differentiation and migration were detected by qPCR and Western blotting.
Then, observe the effect of miR-122-5p inhibitor and ICA on the osteogenic differentiation and migration ability of BMSCs. P3 generation BMSCs were inoculated into 4 wells of two six-well plates. When the cell fusion reached 60%-80%, the culture medium was discarded, and the four groups of different treatments were added to the corresponding culture medium. The culture medium is the miR-122-5p inhibitor NC group, miR-122-5p inhibitor group, miR-122-5p inhibitor NC+ICA group, and miR-122-5p inhibitor+ICA group. After culturing for 3 days, a six-well plate was used to extract mRNA and one was used to extract protein. The mRNA and protein expression of genes related to bone differentiation and migration were detected by qPCR and Western blotting. The primer sequence of each gene is shown in Table 1. Table 1 The primer sequence of each gene

Statistical analysis
For the data, use SPSS 21.0 statistical software to perform statistical analysis. Measurement data are expressed as mean ± standard deviation(X -±S). Count data is subjected to chi-square test. Oneway analysis of variance is used between multiple samples. The t test was used for comparison between groups. p<0.05 means the difference is statistically significant. GraphPad Prism 6 software was used to process the data to generate histograms. Figure 1 shows that under different magnification microscopes, BMSCs are spindle-shaped and grow in a whirlpool shape. Flow cytometry identified the three cell phenotypes of CD29, CD44, and CD45. CD29 and CD44 were positive, and CD45 was negative. Figure 2 shows that the OB cells are flat and spread, with various shapes, such as fusiform, polygonal, triangular, etc., with protrusions of different lengths and nuclei. At the same time, the cytoplasm is abundant, with 2 nuclei occasionally, and a spiral growth trend. The OB cells stained by Giemsa are fusiform or triangular, with rich cytoplasm and stained purple blue, while the nucleus is stained dark blue. The nucleus is located in the center of the cell and is round, there are 2 nucleoli in some cells, which are more obvious. After OB cultured for 2 weeks, a large number of scattered mineralized nodules were seen, and the mineralized nodules were stained orange-red after staining with Alizarin Red. Figure 3 shows that under different magnifications of the transmission electron microscope field of view, the OB-exo show a clear vesicle-like structure with a particle size between 50nm-200nm. Compared with the control group, the expression levels of Tsg101, Hsp70, and CD63 three marker proteins of the exosomes obtained by ultracentrifugation and the exosomes obtained by the kit were significantly higher, indicating that the two methods obtained were exosomes. At the same time, the amount of exosomes obtained by the kit is greater than that obtained by ultracentrifugation.  After 2w culture, Alizarin Red staining found that the cells in the ICA concentration of 1×10 -7 M group had the most abundant mineralized nodules and the best mineralization effect. Figure 6 is to observe the effect of different concentrations of ICA on the bone formation and migration of BMSCs. It can be seen in the figure that at 3d and 7d, ICA with a concentration of 1× 10 -7 M co-cultured with BMSCs has the highest mRNA expression of osteogenic differentiation and migration related genes, and the difference is statistically significant (P<0.05), indicating that the concentration of ICA on BMSCs to promote their osteogenic differentiation and migration is 1×10 -7 M . Figure 7 is to observe the change trend of osteogenic differentiation and migration ability of BMSCs by ICA on 1-7 days. Compared with the blank group, the mRNA expression of osteogenic differentiation-related genes (Runx2, BMP-2, Osterix, OPN, OCN) and migration-related genes (SDF-1α, CXCR4) in the ICA group were higher at 3d, 7d, and 14d, and the difference was statistically significant (P<0.05). The expression of osteogenic differentiation-related genes and migration-related genes mRNA reached the first peak on the 3rd day and reached the second peak on the 7th day. Among them, the expression was higher on the 7th day, the difference was statistically significant (P<0.05). The expression of osteogenic differentiation-related and migration-related proteins reached the first peak on the 4th day, and reached the second peak on the 7th day, the difference between the two peaks was statistically significant (P<0.05). Figure 8 is to observe the signal pathway that ICA affects the osteogenic differentiation and migration ability of BMSCs. It can be seen in the figure that the difference between the ICA group and the ICA+si-Runx2 group, and the ICA+AMD3100 group is statistically significant (P<0.05). It shows that both si-Runx2 and AMD3100 can effectively inhibit the promotion of ICA on the osteogenic differentiation and migration ability of BMSCs. Figure 9 shows that the ICA group, OB-exo group, and ICA+OB-exo group respectively compared with the blank group, the osteogenic differentiation and migration-related gene mRNA expression was significantly increased, while the ICA+OB-exo group was higher than the ICA group and OBexo group, and the difference was statistically significant (P<0.05). It shows that ICA combined with OB-exo can significantly promote the expression of mRNAs related to osteogenic differentiation and migration of BMSCs. Compared with the blank group, ICA group, OB-exo group, and ICA+OB-exo group had significantly higher expression of osteogenic differentiation and migration-related protein. At the same time, ICA+OB-exo was also higher than ICA and OB-exo protein expression, the difference was statistically significant (P<0.05). It shows that ICA combined with OB-exo can significantly promote the expression of genes and proteins related to osteogenic differentiation and migration of BMSCs. Figure 10 and Figure 11 observe the effect of ICA combined with miR-122-5p on the osteogenic differentiation and migration ability of BMSCs. Figure 10 observes the effect of miR-122-5p minic on the osteogenic differentiation and migration ability of BMSCs, and Figure 11 observes the effect of miR-122-5p inhibitor on the osteogenic differentiation and migration ability of BMSCs. It can be seen in the figure that the combined application of miR-122-5p minic and ICA can significantly enhance the mRNA and protein expression of osteogenic differentiation and migration in BMSCs, comparison between groups, the difference was statistically significant (P<0.05). While miR-122-5p inhibitor combined with ICA, the expression of osteogenic differentiation and migration in BMSCs decreased, comparison between groups, the difference was statistically significant (P<0.05). Experimental results confirmed that the expression of miR-122-5p can significantly affect the osteogenic differentiation and migration ability of BMSCs, and the combination with ICA can synergistically enhance the osteogenic differentiation and migration ability of BMSCs.

Discussion
A large number of studies have found [17][18][19][20][21][22][23][24][25][26][27][28][29][30] that ICA can promote the proliferation and osteogenic differentiation of BMSCs. Cao et al. [31] showed that within 5 months of treatment, intragastric administration of ICA significantly accelerated the formation of callus and fracture healing in rats. Zhang et al. found that ICA inhibited the differentiation of BMSCs into adipocytes and promoted their differentiation into osteoblasts [32]. Fan et al. found that ICA not only promotes the proliferation of BMSCs in vitro in a dose-dependent manner, but also promotes their differentiation into osteoblasts at very low doses (1×10 -9 M to 1×10 -6 M). Of course, a higher concentration of 1×10 -5 M is toxic and inhibits its differentiation towards osteogenic [33]. Huang et al. [34] showed that ICA can effectively prevent femoral head necrosis, improve the proliferation of BMSCs induced by prednisolone, enhance osteogenic differentiation and inhibit adipogenic differentiation. In addition, low concentrations of ICA (1×10 -9 M to 1×10 -5 M) significantly enhanced the proliferation ability of BMSCs, especially at the concentration of 1×10 -6 M [33]. The above studies show that ICA can significantly promote the osteogenic differentiation of BMSCs. Some studies have shown that the optimal concentration of ICA on the osteogenic differentiation of BMSCs is 1×10 -6 M [33,34], and most researchers believe that the optimal concentration of ICA on the osteogenic differentiation of BMSCs is 1×10 -7 M [17,18,35], this concentration has also been further confirmed in our study.
In the experiment, we also observed that high-concentration ICA (>1×10 -5 M) has a toxic effect on cells. In the CCK-8 test, most of the cells in the 1×10 -4 M group have died, which is basically consistent with the results reported in the literature. ICA acts on BMSCs, not only can promote their differentiation toward osteogenic direction, but also enhance their migration ability, which is not clearly proposed in the previous literature. Our research team has verified in the previous study that the Runx2 signal axis and the CXCR4 signal axis can cross-talk, suggesting that there may be a connection between osteogenic differentiation and migration [36]. But it is the first time to clarify that ICA can clearly promote the osteogenic differentiation and migration of BMSCs at the same time. At the same time, this study found that the optimal concentration of ICA to promote osteogenic differentiation and migration of BMSCs is 1×10 -7 M. In the research, we also found that over time, ICA with a concentration of 1×10 -7 M promotes the osteogenic differentiation and migration of BMSCs, which has certain regularity. In 1-7d, the 3rd and 7th days are the two peak periods of osteogenic differentiation and migration-related gene mRNA expression. Among the two, the 7th day's expression is higher; and Western blotting test found that in 1-7 days, the 4th and 7th days are the two peak periods of osteogenic differentiation and migration protein expression, of which the 7th day's expression is higher. The results of this study showed that ICA has the best time to act on BMSCs and has a certain periodicity, reaching another peak at an interval of about 3 days, which may be related to the metabolic cycle of cells. At the same time, it was found that the first peak of mRNA for detecting bone formation and migration occurred on the 3rd day and the first peak of protein occurred on the 4th day, the results of mRNA and protein expression are inconsistent, but the second peak of mRNA and protein both occurred on the 7th day. According to our analysis, in the early stage of cell culture, the expression of mRNA occurs before the protein, and the protein immediately follows. However, when the 7th day is reached, the role of ICA in promoting the osteogenic differentiation and migration of BMSCs becomes stronger and stronger, so that the peaks of mRNA and protein appeared in the same period. This research result provides a very important reference for the future of ICA to clinical trials and even clinical application of medication. In addition, it was also found in this study that ICA is positively related to the occurrence of bone formation and migration of BMSCs. Our previous study [36] have revealed that there will be a crosstalk between osteogenic differentiation and migration, and there is a crossover point between the two, namely CXCR4, but it has not been demonstrated that they are a co-occurring relationship. Then, whether osteogenic differentiation initiates migration, or migration promotes osteogenic differentiation, remains to be further studied.
In addition, in experiments to explore whether ICA plays a role through the Runx2 signal axis and CXCR4 signal axis, we found that both si-Runx2 and AMD3100 can effectively inhibit ICA's role in promoting osteogenic differentiation and migration. This result, from the opposite side, also verified that osteogenic differentiation and migration occur concurrently, inhibiting one of them, and the other effect is also inhibited. There is a cross-talk between these two signal axes, which can promote and inhibit each other.
Exosomes, due to their unique structural advantages, have the potential to become a good tool for transporting drugs to target cells to play a biological role. The outer layer of its phospholipid bilayer structure can effectively protect the contents from various biological enzymes in the environment, and at the same time, it can maintain the activity of various biological molecules. After reaching the target cell, its content communicates with the cell's content, thereby exerting a biological effect. Studies have found [37][38][39][40][41] that the use of electroporation, ultrafiltration, centrifugation, coincubation and other methods can load specific drugs into the exosomes through the double-layer lipid structure of the exosomes, allowing the exosomes to carry the specific drug reaches the target cell and takes effect. Based on this, the pharmaceutical carrier has become an important direction of application of exosomes, which has broad application prospects.
The experimental results suggest that there is a concomitant relationship between osteogenic differentiation and migration. At the same time as osteogenic differentiation, they can reach a distant place through migration, and then continue to demonstrate the role of osteogenic. In this process, both ICA and OB-exo can promote the osteogenic differentiation and migration of BMSCs, and putting them together can enhance this effect. The results of detection of mRNA and protein related to osteogenic differentiation and migration by qPCR and Western blotting found that compared with the ICA group or the OB-exo group, the combined effect of ICA and OB-exo on BMSCs is better than the other two groups. It shows that ICA and OB-exo have a synergistic enhancement effect in effectively promoting the osteogenic differentiation and migration of BMSCs. In addition, the experimental results also show that, compared with the control group, the ICA group and the OBexo group had significant differences in the expression of osteogenesis and migration mRNA and protein, indicating that both ICA and OB-exo can promote the osteogenic differentiation and migration of BMSCs. So, when ICA and OB-exo are used at the same time, in what way do they play a synergistic effect? Is it a simple superposition or a more specific synergy mechanism? We think that the mechanism by which ICA and OB-exo can play a synergistic effect is roughly based on three factors: ① Both ICA and OB-exo have their own effects on BMSCs, promoting their osteogenic differentiation and migration, and finally their effects are superimposed, but from the experimental results, the result of the ICA+OB-exo group is not a simple addition of the results of the ICA group and the OB-exo group; ②ICA can enter the BMSCs more efficiently through the transport function of OB-exo, and then play a biological role. At the same time, OB-exo also exerts a biological effect on BMSCs; ③ICA not only uses the transport function of OB-exo, but also regulates certain components in OB-exo such as miRNAs, promotes its expression, and then released into BMSCs to promote the expression of osteogenic differentiation and migration. The above are just our conjectures and still need to be verified.
The chemical composition of ICA is very clear, but the composition of OB-exo is more complicated, we hope to find the target miRNA that plays a role through a comprehensive analysis of the composition of OB-exo, so as to complete further verification work. High-throughput sequencing detection is an important method of modern scientific research, which allows people to obtain more accurate sequencing information. In the experiment, we sequenced OB-exo and analyzed the composition of it and found that miRNAs accounted for 17.47% of the total exosomes, indicating that miRNAs are abundant in OB-exo. Among them, the content of each miRNA is also very different. The existence of this difference implies that each miRNA has a different degree of activity in osteoblast metabolism. The high content of miRNA indicates that it is highly related to osteogenic differentiation,however, there may also be some miRNAs in various exosomes with high content, so further analysis is needed. Based on this, we select the top 20 miRNAs with content to enter the next step of analysis.
In order to screen out miRNAs related to osteogenic differentiation, we first searched for target genes related to the regulation of bone metabolism in the predicted target gene library, through software prediction, we found that miR-let-7a-5p, miR-100-5p, and miR-21-5p, none of the top 3 candidate gene banks have the above-mentioned "bone" related genes, but a qualified gene was found in the candidate gene library of miR-122-5p, and miR-122-5p was ranked 4th. In addition, our previous research found that miR-122-5p can clearly promote the proliferation of osteoblasts [42]. Combined with the results of this previous research, we predict that miR-122-5p is one of the target miRNAs for osteogenic differentiation.
MiR-122-5p has been used as a target for the study of many diseases, including acute kidney injury [43], cardiomyocyte injury [44], gastric cancer [45][46][47][48], kidney cancer [49], non-alcoholic Steatohepatitis [50], breast cancer [51], cervical cancer [52], etc. However, there are few studies related to the regulation of osteogenic differentiation and migration of BMSCs. A study by our team suggests that [42] miR-122-5p is involved in the regulation of osteoblast proliferation, but it does not involve the promotion of osteogenic differentiation and migration of stem cells.
In the experiment, we found that miR-122-5p minic and miR-122-5p minic+ICA can effectively promote the expression of osteogenesis and migration-related mRNAs and proteins, while miR-122-5p inhibitor and miR-122-5p inhibitor+ICA can effectively inhibit the expression of mRNAs and proteins related to osteogenic differentiation and migration. This shows that miR-122-5p can target and activate related genes on the Runx2 signal axis and CXCR4 signal axis, and promote the expression of mRNAs and proteins related to osteogenic differentiation and migration. At the same time, in the presence of ICA, miR-122-5p target activation of Runx2 signal axis and CXCR4 signal axis related genes expression is more obvious, the expression of osteogenic and migration-related proteins is also more obvious, which in turn also shows that ICA promotes the osteogenic differentiation and migration of BMSCs, and miR -122-5p is playing a promoting role.
Based on the experimental results, we further analyzed that miR-122-5p in OB-exo is one of the target miRNAs that regulates the differentiation and migration of BMSCs. We can infer that:①ICA regulation of miR-122-5p promotes the osteogenic differentiation and migration of BMSCs;②miR-122-5p is the target of OB-exo to promote the osteogenic differentiation and migration of BMSCs; ③miR-122 -5p is also a target for ICA and OB-exo to promote osteogenic differentiation and migration of BMSCs. Based on this, we believe that after ICA-carrying exosomes (or ICA and exosomes are in a coexisting state) enter BMSCs, both ICA and miR-122-5p to activate the Runx2 signal axis and the CXCR4 signal axis related genes (such as Runx2, BMP-2, Osterix, SDF-1α, CXCR4) to promote their expression, and then promote the expression of related proteins. Regarding the status of ICA and miR-122-5p in exosomes, and how they target and activate related genes on the Runx2 signal axis and CXCR4 signal axis, further studies are needed.

Conclusions
Icariin can clearly promote the osteogenic differentiation and migration of BMSCs, and osteoblastderived exosomes can also effectively promote the osteogenic differentiation and migration of BMSCs, the combination of Icariin and osteoblast-derived exosomes can enhance this effect. In this process, one of its targets is miR-122-5p.