Icariin upregulates methyltransferase-like 14-mediated prolyl 4-hydroxylase beta subunit m6A modification to promote osteogenic differentiation of bone marrow stem cells.

Prolyl 4-hydroxylase beta subunit (P4HB) plays a vital role in bone formation. This study intends to clarify the role of P4HB in the therapeutic effect of Icariin (ICA) on osteoporosis. Herein, in vivo and in vitro models were constructed by performing ovariectomy (OVX) in rats and inducing osteogenic differentiation in bone marrow stem cells (BMSCs), respectively. Hematoxylin and eosin staining and micro-computed tomography analysis were performed to evaluate osteoporosis in OVX rats. Alizarin Red staining, alkaline phosphatase staining, and the ALP activity test were employed to assess osteogenesis. m6A dot blotting and methylated RNA immunoprecipitation were used to determine m6A modification. We found that P4HB was downregulated in bone tissues of patients with osteoporosis and OVX rats. P4HB facilitated osteogenic differentiation of BMSCs. What's more, ICA upregulated P4HB expression, promoted osteogenic differentiation of BMSCs, and alleviated osteoporosis in OVX rats, which were reversed by knocking down P4HB. ICA enhanced the stability and m6A modification of P4HB. METTL14 mediated m6A modification of P4HB mRNA. In addition, METTL14 knockdown overturned the promotive effects of ICA on P4HB m6A level and BMSC osteogenic differentiation. To sum up, ICA elevated the METTL14-mediated m6A modification of P4HB to facilitate BMSC osteogenic differentiation.

Icariin triggers osteogenic differentiation of bone marrow stem cells by up-regulating miR-335-5p.

BACKGROUND: The physical health and quality of life of the elderly are severely affected by osteoporosis (OP). METHODS: We explored the regulatory mechanism of ICA in vivo and in vitro by constructing OP rats and inducing osteogenic differentiation of BMSCs. First, we determined the expression of miR-335-5p in bone tissues of OP patients, bone tissues of OP rats, and osteogenic BMSCs by RT-qPCR. Alizarin red staining was employed to detect the formation of calcium nodules in the cells. MTT was used to detect cell viability. Finally, we detected the bone tissue changes in OP rats by overexpression of miR-335-5p or oral ICA. RESULTS: miR-335-5p was lowly expressed in bone tissues of OP patients and OP rats. ICA treatment reversed the inhibitory effect of miR-335-5p inhibitor on BMSCs matrix mineralization. Moreover, PTEN was verified to be a downstream effector of miR-335-5p. During ICA induction, overexpression of PTEN reversed the promotive effect of miR-335-5p mimics on the osteogenic differentiation of BMSCs. In vivo experiments also found that overexpression of miR-335-5p or ICA treatment improved the pathogenesis of OP in rats. CONCLUSION: ICA improved OP by up-regulating miR-335-5p to inhibit PTEN, thereby providing a new strategy for the prevention and treatment of OP.

Anticancer Effects of Constituents of Herbs Targeting Osteosarcoma.

Osteosarcoma is a rare primary malignancy of bone that is prone to early metastasis. Resection surgery and chemotherapeutic regimens are current standard treatments for osteosarcoma. However, the long-term survival rate of patients with osteosarcoma is low due to a high risk of metastasis. Hence, a new approach is urgently needed to improve the treatment of osteosarcoma. Compared with chemotherapy, natural active constituents isolated from herbs exhibit less adverse effects and better anti-tumor effects. This study aimed to summarize the anticancer effects of constituents of herbs on the progression and metastasis of osteosarcoma cells. It showed that many constituents of herbs inhibited osteosarcoma by targeting proliferation, matrix metalloproteinases, integrin and cadherin, and angiogenesis. The findings might be beneficial for the development of new drugs and treatment strategies.

[EFFECT OF FETAL BOVINE SERUM ON OSTEOGENIC GROWTH PEPTIDE PROMOTING BONE MARROW MESENCHYMAL STEM CELLS PROLIFERATION AND DIFFERENTIATION].

OBJECTIVE: To explore the effect of fetal bovine serum (FBS) of different concentrations in the culture medium on osteogenic growth peptide (OGP) promoting bone marrow mesenchymal stem cells (BMSCs) proliferation and differentiation. METHODS: BMSCs were separated from limb bones of 8 Sprague Dawley rats (5 weeks old) and purified by adherence method, and BMSCs at passage 3 were divided into 4 groups according to OGP concentration: OGP 1 x 10(-10) mol/L group, OGP 1 x 10(-9) mol/L group, OGP 1 x 10(-8) mol/L group, and control group without OGP; and 0, 2%, 5%, 8%, and 10% FBS concentration gradient was used in each group. The cell proliferation rate was detected by MTT method at 1, 3, 5, 7, 9, and 12 days after culture, and the activity of intracellular alkaline phosphatase (ALP) was determined by the method of p-nitrophenyl phosphate disodium at 9 days after culture. RESULTS: BMSCs showed adherent growth, rapid proliferation, long fiber vortex, and typical morphology. MTT analysis showed that cells could not sustain proliferation when FBS concentration was less than 5% in each group; when FBS concentration was above 8%, cells proliferated continually. Proliferation promoting effect of OGP 1 x 10(-8) mol/L and 1 x 10(-9) mol/L groups was significantly higher than that of the control group in all serum concentrations (P < 0.05); when FBS concentration was lower than 10%, the proliferation promoting effect of OGP 1 x 10(-8) mol/L group was significantly higher than that of the other 2 OGP groups (P < 0.05), but when FBS concentration was 10%, OGP 1 x 10(-8) mol/L group had no advantage of promoting proliferation. ALP test results showed that as the FBS concentration increased, ALP activity of all groups also significantly increased (P < 0.05). Under the condition of 5% FBS and 8% FBS, the ALP activity of each OGP group was significantly greater than that of the control group, and it was the highest in OGP 1 x 10(-8) mol/L group (P < 0.05). Under the condition of 10% FBS, the ALP activity of each OGP group was still greater than that of the control group (P < 0.05), but no significant difference was found between the OGP 1 x 10(-8) mol/L group and OGP 1 x 10(-9) mol/L group (P > 0.05). CONCLUSION: The concentration of 8% FBS is the best concentration of serum for OGP promoting the proliferation and differentiation of BMSCs, and the most suitable concentration of promoting the proliferation and differentiation of BMSCs is OGP 1 x 10(-8) mol/L.

Potential role of mesenchymal stem cells in alleviating intestinal ischemia/reperfusion impairment.

BACKGROUND: Transplantation of bone marrow mesenchymal stem cells (MSCs) provides a promising therapeutic efficiency for a variety of disorders caused by ischemia or reperfusion impairment. We have previously demonstrated the efficacy of MSCs in mitigating intestinal ischemia/reperfusion (I/R) injuries in rats, but the mechanism by which MSCs engraft ameliorates I/R injuries has largely been unknown. The present study aimed at investigating probable mechanisms by which MSCs exert their function. METHODS: Male donor derived rat MSCs were implanted into intestine of female recipient rat by direct submucosal injection after superior mesenteric artery clamping and unclamping. The homed MSCs were detected by Y chromosome in situ hybridization probe, and the tumor necrosis factor-α (TNF-α) content in intestinal mucosa was determined by ELISA. Expression of proliferative cell nuclear antigen (PCNA) in bowel mucosa was assayed by real-time PCR and intestinal mucosa expression of phosphorylation extracellular signal-regulated kinase (pERK1/2) and nuclear factor-κB (NF-κB) were evaluated by western blot. RESULTS: Four and seven days after MSCs transplantation, the TNF-α content of bowel mucosa in MSCs group was significantly lower than that in saline group. The PCNA in bowel mucosa showed higher expression in MSCs treated group than the saline group, both at 4 and 7 days after cell transplantation. The expression of intestinal mucosal pERK1/2 in MSCs treated group was markedly higher than that in saline group, and the expression of NF-κB in MSCs treated group was noticeably decreased than that in saline group at 4 and 7 days post MSCs transplantation. CONCLUSION: The present investigation provides novel evidence that MSCs have the potential to reduce intestinal I/R injuries probably due to their ability to accelerate cell proliferation and decrease the inflammatory response within intestinal mucosa after ischemia and reperfusion.