Studies have shown that ICA can promote the proliferation and osteogenic differentiation of BMSCs [19–30]. Cao et al. [31] demonstrated that intragastric ICA administration significantly accelerated the formation of calli and fracture healing in rats within 5 months of treatment. Fan et al. found that ICA could promote not only the proliferation of BMSCs in a dose-dependent manner in vitro but also their differentiation into osteoblasts at very low doses of 1×10− 9 M to 1×10− 6 M [32]. Interestingly, some researches have suggested that the proliferative and osteogenic effects of ICA could be achieved by inhibiting adipogenic differentiation [33, 35].
Many other studies have shown that the optimal concentration of ICA to improve the osteogenic differentiation of BMSCs is 1×10− 6 M [32, 34], but most researchers believe that the optimal concentration of ICA improving the osteogenic differentiation of BMSCs is 1×10− 7 M [19, 20, 35], which has been further confirmed in our study. In our experiments, we also observed that ICA at a high concentration (> 1×10− 5 M) had toxic effects on the cells. Most of the cells in the 1×10− 4 M ICA-treated group died, as shown by the CCK-8 assay, which is consistent with the literature.
In this study, we also found that the mRNA expression of osteogenic differentiation-related and migration-related genes peaked twice after 3 and 7 days, with expression higher on the 7th day. The Western blot results showed that from days 1–7, on the 4th and 7th days, the expression of osteogenic differentiation-related and migration-related proteins peaked twice, with protein expression higher on the 7th day. The results provide a very important reference for the use of ICA in clinical trials and even its medicinal application. In addition, osteogenic differentiation was positively related to the migration of BMSCs treated with ICA. These results indicate that an optimal window for the treatment of BMSCs with ICA exists and that the treatment effects are periodic, which may be related to cellular metabolism.
The Runx2 signalling axis and CXCR4 signalling axis may maintain cross-talk. Our previous work [36] revealed that there was a connection between osteogenic differentiation and migration mediated by CXCR4, but whether osteogenic differentiation and migration occur together has not been demonstrated. Furthermore, whether osteogenic differentiation initiates migration or migration promotes osteogenic differentiation remains to be determined.
By investigating whether ICA plays a role in the osteogenic differentiation and migration of BMSCs through the Runx2 signalling axis and CXCR4 signalling axis, we found that both si-Runx2 and AMD3100 effectively inhibited the effect of ICA. The results also verified that osteogenic differentiation and migration occur together and are co-regulated; when one is inhibited, the other is also suppressed. Thus, cross-talk between these two signalling axes, which promote and inhibit each other exists.
Exosomes, which have unique structural advantages, could be a good tool for transporting drugs to target cells to exert biological effects [37]. The outer layer of the phospholipid bilayer structure of exosomes can effectively and simultaneously protect the contents from various biological enzymes and maintain the activity of various biological molecules. Studies have reported [38–41] that electroporation, ultrafiltration, centrifugation, co-incubation and other methods can be used to load specific drugs into exosomes through their double-layer lipid structure, which allows the exosomes to carry the specific drug to the target cell, where it takes effect. Based on this special role of exosomes, the potential use of exosomes as pharmaceutical carriers has become an important application.
Our experimental suggested the presence of a concomitant relationship between osteogenic differentiation and migration. The results of qPCR and Western blot showed that OB-exos enhanced the osteogenic differentiation and migration of BMSCs, and the combination of ICA and OB-exos had a greater effect than treatment with ICA or OB-exos alone. These results showed that ICA and OB-exos have a synergistic effect.
How does the simultaneous application of ICA and OB-exos have a synergistic effect? We suggest that the mechanism is essentially based on three factors: ① Both ICA and OB-exos promoted the osteogenic differentiation and migration of BMSCs, but the effects were not simply additive. ② ICA efficiently entered BMSCs through the transport function of OB-exos to exert its biological effects, and the OB-exos also simultaneously exerted biological effects on the BMSCs. ③ ICA acted not only through the transport function of OB-exos but also by regulating and enhancing the expression of miRNAs in OB-exos. The hypotheses above need to be verified.
Because the composition of OB-exos is very complicated, we expected to find the target miRNA through comprehensive analysis of OB-exos. High-throughput sequencing allows researchers to obtain more accurate sequencing informatio. In our experiments, we found that miRNAs accounted for 17.47% of the total exosomal content, which indicated the presence of abundant miRNAs in the OB-exos, and the levels of individual mRNAs were also clearly different. A higher miRNA content indicates a closer relationship with osteogenic differentiation. However, some miRNAs may be extremely abundant in different exosomes, and further analysis is needed.
In the next experiment, we selected the top 20 most abundant miRNAs. First, we searched for target genes related to the regulation of bone metabolism in a predicted target gene library through software prediction to screen out miRNAs related to osteogenic differentiation. We identified miR-let-7a-5p, miR-100-5p, and miR-21-5p, none of which were “bone”-related genes in the top 3 gene banks, and a qualified gene in the candidate gene library for miR-122-5p, miR-122-5p, was ranked 4th. Our previous study have showed that miR-122-5p can clearly promote the proliferation of osteoblasts [42]. MiR-122-5p has been explored in many diseases, including acute kidney injury [43], cardiomyocyte injury [44], gastric cancer [45–48], kidney cancer [49], non-alcoholic steatohepatitis [50], breast cancer [51], and cervical cancer [52]. However, little information related to the regulation of osteogenic differentiation and migration in BMSCs.
In our study, the mRNA and protein expression of osteogenic differentiation-related and migration-related genes was found to be obviously increased when miR-122-5p mimic and miR-122-5p mimic + ICA were applied, while miR-122-5p inhibitor and miR-122-5p inhibitor + ICA significantly inhibited their expression. These results suggest that miR-122-5p may work by activating the interrelated genes of the Runx2 signalling axis and CXCR4 signalling axis. At the same time, the expression of these genes was greater in the presence of ICA. In summary, these results demonstrate that miR-122-5p plays a pivotal role in the effect of ICA.
We suggest that miR-122-5p is a target miRNA that regulates the differentiation and migration of BMSCs. The mechanism may be as follows: ① ICA promotes the osteogenic differentiation and migration of BMSCs through miR-122-5p; ② miR-122-5p is an effect medium of OB-exos. However, understanding how ICA and miR-122-5p target genes related to the Runx2 and CXCR4 signalling axes for activation requires further study.