Combination of bone marrow mesenchymal stem cells and moxibustion restores cyclophosphamide-induced premature ovarian insufficiency by improving mitochondrial function and regulating mitophagy.

BACKGROUND: Premature ovarian insufficiency (POI) is a major cause of infertility. In this study, we aimed to investigate the effects of the combination of bone marrow mesenchymal stem cells (BMSCs) and moxibustion (BMSCs-MOX) on POI and evaluate the underlying mechanisms. METHODS: A POI rat model was established by injecting different doses of cyclophosphamide (Cy). The modeling of POI and the effects of the treatments were assessed by evaluating estrous cycle, serum hormone levels, ovarian weight, ovarian index, and ovarian histopathological analysis. The effects of moxibustion on BMSCs migration were evaluated by tracking DiR-labeled BMSCs and analyzing the expression of chemokines stromal cell-derived factor 1 (Sdf1) and chemokine receptor type 4 (Cxcr4). Mitochondrial function and mitophagy were assessed by measuring the levels of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), ATP, and the mitophagy markers (Drp1, Pink1, and Parkin). Furthermore, the mitophagy inhibitor Mdivi-1 and the mitophagy activator CCCP were used to confirm the role of mitophagy in Cy-induced ovarian injury and the underlying mechanism of combination therapy. RESULTS: A suitable rat model of POI was established using Cy injection. Compared to moxibustion or BMSCs transplantation alone, BMSCs-MOX showed improved outcomes, such as reduced estrous cycle disorders, improved ovarian weight and index, normalized serum hormone levels, increased ovarian reserve, and reduced follicle atresia. Moxibustion enhanced Sdf1 and Cxcr4 expression, promoting BMSCs migration. BMSCs-MOX reduced ROS levels; upregulated MMP and ATP levels in ovarian granulosa cells (GCs); and downregulated Drp1, Pink1, and Parkin expression in ovarian tissues. Mdivi-1 significantly mitigated mitochondrial dysfunction in ovarian GCs and improved ovarian function. CCCP inhibited the ability of BMSCs-MOX treatment to regulate mitophagy and ameliorate Cy-induced ovarian injury. CONCLUSIONS: Moxibustion enhanced the migration and homing of BMSCs following transplantation and improves their ability to repair ovarian damage. The combination of BMSCs and moxibustion effectively reduced the excessive activation of mitophagy, which helped prevent mitochondrial damage, ultimately improving ovarian function. These findings provide a novel approach for the treatment of pathological ovarian aging and offer new insights into enhancing the efficacy of stem cell therapy for POI patients.

Bioactive materials from berberine-treated human bone marrow mesenchymal stem cells promote alveolar bone regeneration by regulating macrophage polarization.

Alveolar bone regeneration has been strongly linked to macrophage polarization. M1 macrophages aggravate alveolar bone loss, whereas M2 macrophages reverse this process. Berberine (BBR), a natural alkaloid isolated and refined from Chinese medicinal plants, has shown therapeutic effects in treating metabolic disorders. In this study, we first discovered that culture supernatant (CS) collected from BBR-treated human bone marrow mesenchymal stem cells (HBMSCs) ameliorated periodontal alveolar bone loss. CS from the BBR-treated HBMSCs contained bioactive materials that suppressed the M1 polarization and induced the M2 polarization of macrophages in vivo and in vitro. To clarify the underlying mechanism, the bioactive materials were applied to different animal models. We discovered macrophage colony-stimulating factor (M-CSF), which regulates macrophage polarization and promotes bone formation, a key macromolecule in the CS. Injection of pure M-CSF attenuated experimental periodontal alveolar bone loss in rats. Colony-stimulating factor 1 receptor (CSF1R) inhibitor or anti-human M-CSF (M-CSF neutralizing antibody, Nab) abolished the therapeutic effects of the CS of BBR-treated HBMSCs. Moreover, AKT phosphorylation in macrophages was activated by the CS, and the AKT activator reversed the negative effect of the CSF1R inhibitor or Nab. These results suggest that the CS of BBR-treated HBMSCs modulates macrophage polarization via the M-CSF/AKT axis. Further studies also showed that CS of BBR-treated HBMSCs accelerated bone formation and M2 polarization in rat teeth extraction sockets. Overall, our findings established an essential role of BBR-treated HBMSCs CS and this might be the first report to show that the products of BBR-treated HBMSCs have active effects on alveolar bone regeneration.

Combination Therapy of Bone Marrow Mesenchymal Stem Cell Transplantation and Electroacupuncture for the Repair of Intrauterine Adhesions in Rats: Mechanisms and Functional Recovery.

Transplantation of bone marrow mesenchymal stem cells (BMSCs) has demonstrated promising clinical utility in the treatment of endometrial injury and the restoration of fertility. However, since the efficacy of BMSCs after transplantation is not stable, it is very important to find effective ways to enhance the utilisation of BMSCs. Electroacupuncture (EA) has some positive effects on the chemotaxis of stem cells and diseases related to uterine injury. In this study, we established the intrauterine adhesion (IUA) model of the Sprague-Dawley rat using lipopolysaccharide infection and mechanical scratching. Phosphate-buffered saline, BMSCs alone, and BMSCs combined with EA were randomly administered to the rats. Fluorescent cell labelling showed the migration of transplanted BMSCs. H&E staining, Masson staining, Western blot, immunohistochemistry, ELISA, and qRT-PCR were utilised to detect changes in endometrial morphology and expressions of endometrial receptivity-related factors, endometrial pro-inflammatory factors, and fibrosis factors. Finally, we conducted a fertility test to measure the recovery of uterine function. The results showed that EA promoted transplanted BMSCs to migrate into the injured uterus by activating the SDF-1/CXCR4 axis. Endometrial morphology showed the most significant improvement in the BMSC + EA group. The expressions of endometrial pro-inflammatory factors and fibrosis indexes in the BMSC + EA group were lower than those in the model and BMSC groups. Further studies revealed that the expression of endometrial receptivity-related factors and the number of embryos implanted on day 8 of gestation increased in the BMSC + EA group compared with the model group and the BMSC group.

[Exosome and prevention of Alzheimer's disease: based on theory of kidney storing essence].

The theory of kidney storing essence storage, an important part of the basic theory of traditional Chinese medicine(TCM), comes from the Chapter 9 Discussion on Six-Plus-Six System and the Manifestations of the Viscera in the Plain Questions, which says that "the kidney manages closure and is the root of storage and the house of Jing(Essence)". According to this theory, essence is the fundamental substance of human life activities and it is closely related to the growth and development of the human body. Alzheimer's disease(AD) is one of the common neurodegenerative diseases, with the main pathological features of Aß deposition and Tau phosphorylation, which activate neurotoxic reactions and eventually lead to neuronal dysfunction and cell death, severely impairing the patient's cognitive and memory functions. Although research results have been achieved in the TCM treatment of AD, the complex pathogenesis of AD makes it difficult to develop the drugs capable of curing AD. The stem cell therapy is an important method to promote self-repair and regeneration, and bone marrow mesenchymal stem cells(BMSCs) as adult stem cells have the ability of multi-directional differentiation. By reviewing the relevant literature, this paper discusses the association between BMSCs and the TCM theory of kidney storing essence, and expounds the material basis of this theory from the perspective of molecular biology. Studies have shown that TCM with the effect of tonifying the kidney in the treatment of AD are associated with BMSCs. Exosomes produced by such cells are one of the main substances affecting AD. Exosomes containing nucleic acids, proteins, and lipids can participate in intercellular communication, regulate cell function, and affect AD by reducing Aß deposition, inhibiting Tau protein phosphorylation and neuroinflammation, and promoting neuronal regeneration. Therefore, discussing the prevention and treatment of exosomes and AD based on the theory of kidney storing essence will provide a new research idea for the TCM treatment of AD.

Isoliquiritin treatment of osteoporosis by promoting osteogenic differentiation and autophagy of bone marrow mesenchymal stem cells.

Osteoporosis is a chronic progressive bone disease characterized by the decreased osteogenic ability of osteoblasts coupled with increased osteoclast activity. Natural products showing promising therapeutic potential for postmenopausal osteoporosis remain underexplored. In this study, we aimed to analyze the therapeutic effects of isoliquiritin (ISL) on osteoporosis in mice and its possible mechanism of action. An ovariectomy-induced osteoporosis mouse model and bone marrow mesenchymal stem cells (BMSCs) were used to analyze the effects of ISL on bone regeneration in vivo and in vitro, respectively. Mitogen-activated protein kinase (MAPK) and autophagy inhibitors were used, to investigate whether the MAPK signaling pathway and autophagy affect the osteogenic differentiation of BMSCs. ISL significantly improved bone formation and reduced bone resorption in mouse femurs without inducing any detectable toxicity in critical organs such as the liver, kidney, brain, heart, and spleen. In vitro experiments showed that ISL enhanced the proliferation and osteogenic differentiation of BMSCs and that its osteogenic effect was attenuated by p38/extracellular regulated protein kinase (ERK) and autophagy inhibitors. Further studies showed that the inhibition of phosphorylated p38/ERK blocked ISL autophagy in BMSCs. ISL promoted the osteogenic differentiation of BMSCs through the p38/ERK-autophagy pathway and was therapeutically effective in treating osteoporosis in ovariectomized mice without any observed toxicity to vital organs. These results strongly suggest the promising potential of ISL as a safe and efficacious candidate drug for the treatment of osteoporosis.

Synergistic effects of electroacupuncture and bone marrow mesenchymal stem cell transplantation on rat intrauterine adhesion： an observation. / 电针联合骨髓间å è´¨å¹²ç»†èƒžç§»æ¤æ²»ç–—å¤§é¼ å®«è ”ç²˜è¿žçš„ååŒæ•ˆåº”è§‚å¯Ÿ.

OBJECTIVES: To investigate the effects of electroacupuncture(EA) combined with bone marrow mesen-chymal stem cells(BMSCs) transplantation on the endometrium of rats with intrauterine adhesions(IUA), so as to explore the possible mechanisms underlying their combined therapeutic effects. METHODS: Forty adult female SD rats were randomly divided into control, model, cell, and combined groups. The IUA rat model was established using a dual injury method of mechanical scratching and lipopolysaccharide infection. After successful modeling, on days 1, 3, and 7, rats in the model group received tail vein injection of phosphate buffered solution, while rats in the cell group received tail vein injection of BMSCs suspension for BMSCs transplantation；and rats in the combined group received BMSCs transplantation combined with EA treatment (2 Hz/15 Hz, 1-2 mA), targeting the "Guanyuan"(CV4), bilateral "Zusanli"(ST36) and "Sanyinjiao"(SP6) for 20 min daily for 3 consecutive estrous cycles. After intervention, uterine tissue was collected from 5 rats in each group. Histological analysis was performed using hematoxylin and eosin staining to evaluate endometrial thickness and glandular number. Masson staining was used to assess endometrial fibrosis area. Immunohistochemistry was performed to detect the positive expressions of vascular endothelial growth factor(VEGF), proliferating cell nuclear antigen(PCNA), and estrogen receptor(ER). Western blot analysis was conducted to determine the protein expressions of homeobox A10(HoxA10) and leukemia inhibitory factor(LIF), both key regulators of endometrial receptivity. The remaining 5 rats in each group were co-housed with male rats, and the uterine function recovery was evaluated by assessing the number of embryo implantations. RESULTS: Compared with the control group, the model group showed thinning endometrium(P<0.001), decreased glandular number(P<0.001), increased endometrial fibrosis area(P<0.001), reduced positive expressions of VEGF, PCNA, ER, expressions of HoxA10 and LIF, and decreased embryo implantation number (P<0.001) on the injured side of the uterus. Compared with the model group, the combined group showed a reversal of the aforementioned indicators(P<0.001, P<0.01)；the cell group exhibited thicker endometrium(P<0.001) and reduced endometrial fibrosis area(P<0.001). Compared with the cell group, the combined group showed increased endometrial thickness(P<0.01), elevated glandular number(P<0.05), significantly decreased endometrial fibrosis area(P<0.05), enhanced positive expressions of VEGF, PCNA and ER, expressions of HoxA10 and LIF in the endometrium, and a significant increase in embryo implantation number (P<0.001, P<0.05, P<0.01) on the injured side of the uterus, indicating better results than the cell group. CONCLUSIONS: The combination of EA and BMSCs synergistically promotes the repair of damaged endometrium, improves endometrial morphology, reduces fibrosis levels, enhances vascular regeneration and matrix cell proliferation, improves endometrial receptivity, which ultimately facilitates embryo implantation.

Tauroursodeoxycholic Acid Inhibited Apoptosis and Oxidative Stress in H2O2-Induced BMSC Death via Modulating the Nrf-2 Signaling Pathway: the Therapeutic Implications in a Rat Model of Spinal Cord Injury.

Spinal cord injury (SCI) is a prevalent and significant injury to the central nervous system, resulting in severe consequences. This injury is characterized by motor, sensory, and excretory dysfunctions below the affected spinal segment. Transplantation of bone marrow mesenchymal stem cells (BMSCs) has emerged as a potential treatment for SCI. However, the low survival as well as the differentiation rates of BMSCs within the spinal cord microenvironment significantly limit their therapeutic efficiency. Tauroursodeoxycholic acid (TUDCA), an active ingredient found in bear bile, has demonstrated its neuroprotective, antioxidant, and antiapoptotic effects on SCI. Thus, the present study was aimed to study the possible benefits of combining TUDCA with BMSC transplantation using an animal model of SCI. The results showed that TUDCA significantly enhanced BMSC viability and reduced apoptosis (assessed by Annexin V-FITC, TUNEL, Bax, Bcl-2, and Caspase-3) as well as oxidative stress (assessed by ROS, GSH, SOD, and MDA) both in vitro and in vivo. Additionally, TUDCA accelerated tissue regeneration (assessed by HE, Nissl, MAP2, MBP, TUJ1, and GFAP) and improved functional recovery (assessed by BBB score) following BMSC transplantation in SCI. These effects were mediated via the Nrf-2 signaling pathway, as evidenced by the upregulation of Nrf-2, NQO-1, and HO-1 expression levels. Overall, these results indicate that TUDCA could serve as a valuable adjunct to BMSC transplantation therapy for SCI, potentially enhancing its therapeutic efficacy.

Zuo Gui Wan Promotes Osteogenesis via PI3K/AKT Signaling Pathway: Network Pharmacology Analysis and Experimental Validation.

OBJECTIVE: Osteogenesis is vitally important for bone defect repair, and Zuo Gui Wan (ZGW) is a classic prescription in traditional Chinese medicine (TCM) for strengthening bones. However, the specific mechanism by which ZGW regulates osteogenesis is still unclear. The current study is based on a network pharmacology analysis to explore the potential mechanism of ZGW in promoting osteogenesis. METHODS: A network pharmacology analysis followed by experimental validation was applied to explore the potential mechanisms of ZGW in promoting the osteogenesis of bone marrow mesenchymal stem cells (BMSCs). RESULTS: In total, 487 no-repeat targets corresponding to the bioactive components of ZGW were screened, and 175 target genes in the intersection of ZGW and osteogenesis were obtained. And 28 core target genes were then obtained from a PPI network analysis. A GO functional enrichment analysis showed that the relevant biological processes mainly involve the cellular response to chemical stress, metal ions, and lipopolysaccharide. Additionally, KEGG pathway enrichment analysis revealed that multiple signaling pathways, including the phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) signaling pathway, were associated with ZGW-promoted osteogensis. Further experimental validation showed that ZGW could increase alkaline phosphatase (ALP) activity as well as the mRNA and protein levels of ALP, osteocalcin (OCN), and runt related transcription factor 2 (Runx 2). What's more, Western blot analysis results showed that ZGW significantly increased the protein levels of p-PI3K and p-AKT, and the increases of these protein levels significantly receded after the addition of the PI3K inhibitor LY294002. Finally, the upregulated osteogenic-related indicators were also suppressed by the addition of LY294002. CONCLUSION: ZGW promotes the osteogenesis of BMSCs via PI3K/AKT signaling pathway.

Exploring the mechanism of Astragali radix for promoting osteogenic differentiation based on network pharmacology, molecular docking, and experimental validation.

The present study used network pharmacology and molecular docking to predict the active ingredients and mechanisms of action of Astragalus radix (AR) to promote osteogenic differentiation of bone marrow mesenchymal stem cells (BM-MSCs), and cell experiments were conducted for verification. First, network pharmacology was used to predict the effective components, targets, and mechanisms of action of AR to promote osteogenic differentiation. The effective components and corresponding target proteins of AR, and the target proteins of osteogenic differentiation were collected through the database. The intersection targets of the two were used for the construction and analysis of a protein-protein interaction (PPI) network. Gene Oncology (GO) and Kyoto Encyclopedia of Genes, and Genomes (KEGG) enrichment analyses were conducted. Next, molecular docking technology was carried out to verify the interaction between the active ingredient and the target protein, and to select the appropriate effective active ingredient. Finally, the results of network pharmacology analysis were verified by in vitro experiments. A total of 95 potential targets were retrieved by searching the intersection of AR and osteogenic differentiation targets. PPI network analysis indicated that RAC-α-serine-threonine-protein kinase (Akt1) was considered to be the most reliable target for AR to regulate osteogenic differentiation. GO enrichment analysis included 21 biological processes, 21 cellular components and 100 molecular functions. KEGG enrichment analysis indicated that the class I phosphatidylinositol-3 kinase (PI3K)-serine-threonine kinase (Akt) signaling pathway may play an important role in promoting osteogenic differentiation. The results of molecular docking showed that quercetin's performance was improved compared with that of kaempferol. In vitro experiments showed that quercetin promoted the expression of osteogenic marker proteins (including collagen I, Runt-related transcription factor 2 and osteopontin) in BMSCs and activated the PI3K/Akt signaling pathway. AR acted on Akt1 targets through its main active component quercetin, and promoted the osteogenic differentiation of BM-MSCs by activating the PI3K/Akt signaling pathway.

[Electroacupuncture combined with bone marrow mesenchymal stem cell transplantation promotes repair of thin endometrium by regulating SDF-1/CXCR4 signaling].

OBJECTIVE: It is to explore, based on stromal cell derived factor 1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) signal axis, whether the electroacupuncture (EA) combined with bone marrow mesenchymal stem cells (BMSCs) transplantation can promote thin endometrium regeneration and improve endometrial receptivity, so as to further study its mechanisms underlying improvement of promoting BMSCs homing to repair thin endometrium. METHODS: Thirty matured female SD rats were randomly divided into normal control , model , BMSCs transplantation (BMSCs), BMSCs+AMD3100 (a specific antagonist of CXCR4, BMSCs+AMD3100), BMSCs+EA, and BMSCs+EA+AMD3100 groups, with 5 rats in each group. The thin endometrial model was established by intrauterine injection of 95% ethanol during the period of estrus. Rats of the model group received intravenous injection of PBS solution (tail vein) on day 1, 3 and 7 of modeling and intraperitoneal injection of normal saline once daily for 3 estrous cycles. Rats of the BMSCs group received intravenous injection of BMSCs suspension on day 1,3 and 7 of modeling, and those of the BMSCs+EA group received BMSCs transplantation and EA stimulation. EA (2 Hz/15 Hz, 1 mA) was applied to "Guanyuan" (CV4) and bilateral "Sanyinjiao"(SP9), "Zigong" (EX-CA1) for 15 min, once daily for 3 estrous cycles. Rats of the BMSCs+AMD3100 group received intravenous injection of BMSCs suspension (1×106/mL) and intraperitoneal injection of AMD3100 (5 mg/kg), and those of the BMSCs+EA+AMD3100 group received administration of BMSCs, AMD3100 and EA, with both groups being once daily for 3 estrous cycles. H.E. staining was used to observe histopathological changes of endometrium tissues, and immunohistochemistry was used to detect the expressions of cytokeratin (CK19) and vimentin in endometrium (for evaluating the damage and repair of endometrium). The expression levels of homeobox A10 (HOXA10), leukemia inhibitory factor (LIF), SDF-1 and CXCR4 proteins were detected by Western blot, and those of SDF-1 and CXCR4 mRNAs in the endometrium detected by real-time PCR. RESULTS: In comparison with the normal control group, the number of endometrial glands, the immunoactivity of CK19 and vimentin, the expression leve-ls of HOXA10, LIF and CXCR4 proteins and CXCR4 mRNA were significantly down-regulated (P<0.01), and the expression levels of SDF-1 protein and mRNA significantly up-regulated (P<0.05) in the model group. Compared with the model group, the number of endometrial glands, the immunoactivity of CK19 and vimentin, and the expression levels of HOXA10, LIF, CXCR4 proteins and CXCR4 mRNA in the BMSCs group, and the number of endometrial glands, the immunoactivity of CK19 and vimentin, the expression levels of HOXA10, LIF, CXCR4 proteins and CXCR4 mRNA, and SDF-1 protein and mRNA in the BMSCs+EA group were significantly up-regulated (P<0.05, P<0.01). Compared to the BMSCs group, the number of endometrial glands, and the expression levels of LIF, CXCR4 proteins and CXCR4 mRNA in the BMSCs+EA group were up-regulated (P<0.01, P<0.05)； the number of endometrial glands, the immunoactivity of CK19 and vimentin, the expression levels of HOXA10, LIF, CXCR4 proteins and CXCR4 mRNA in the BMSCs+AMD3100 group were down-regulated (P<0.01). Compared to the BMSCs+EA group, the number of endometrial glands, the immunoactivity of CK19 and vimentin, the expression levels of HOXA10, LIF, CXCR4 proteins and CXCR4 mRNA in the BMSCs+EA+AMD3100 group were down-regulated (P<0.01). Results of H.E. staining showed thin endometrium with absence of epithelial cells, and sparse glands and blood vessels, with smaller glandular cavity in the model group, which was relative milder in BMSCs and BMSCs+EA groups. CONCLUSION: EA can promote the transfer of transplanted BMSCs to the damaged site through SDF-1/CXCR4 signaling related stem cell homing, thereby promoting thin endometrial regeneration, repairing endometrial injury, and improving endometrial tolerance in rats with thin endometrium.

Myricitrin promotes osteogenesis and prevents ovariectomy bone mass loss via the PI3K/AKT signalling pathway.

This study aimed to explore the effect of myricitrin on osteoblast differentiation in mice immortalised bone marrow mesenchymal stem cells (imBMSCs). Additionally, ovariectomy (OVX) mice were employed to examine the effect of myricitrin on bone trabecular loss in vivo. The effect of myricitrin on the proliferation of imBMSCs was evaluated using a cell counting kit-8 assay. Alizarin red staining, alkaline phosphatase staining were performed to elucidate osteogenesis. Furthermore, qRT-PCR and western blot determined the expression of osteo-specific genes and proteins. To screen for candidate targets, mRNA transcriptome genes were sequenced using bioinformatics analyses. Western blot and molecular docking analysis were used to examine target signalling markers. Moreover, rescue experiments were used to confirm the effect of myricitrin on the osteogenic differentiation of imBMSCs. OVX mice were also used to estimate the delay capability of myricitrin on bone trabecular loss in vivo using western blot, micro-CT, tartaric acid phosphatase (Trap) staining, haematoxylin and eosin staining, Masson staining and immunochemistry. In vitro, myricitrin significantly enhanced osteo-specific genes and protein expression and calcium deposition. Moreover, mRNA transcriptome gene sequencing and molecular docking analysis revealed that this enhancement was accompanied by an upregulation of the PI3K/AKT signalling pathway. Furthermore, copanlisib, a PI3K inhibitor, partially reversed the osteogenesis promotion induced by myricitrin. In vivo, western blot, micro-CT, hematoxylin and eosin staining, Masson staining, Trap staining and immunochemistry revealed that bone trabecular loss rate was significantly alleviated in the myricitrin low- and high-dose groups, with an increased expression of osteopontin, osteoprotegerin, p-PI3K and p-AKT compared to the OVX group. Myricitrin enhances imBMSC osteoblast differentiation and attenuate bone mass loss partly through the upregulation of the PI3K/AKT signalling pathway. Thus, myricitrin has therapeutic potential as an antiosteoporosis drug.

Herpetin Promotes Bone Marrow Mesenchymal Stem Cells to Alleviate Carbon Tetrachloride-Induced Acute Liver Injury in Mice.

Herpetin, an active compound derived from the seeds of Herpetospermum caudigerum Wall., is a traditional Tibetan herbal medicine that is used for the treatment of hepatobiliary diseases. The aim of this study was to evaluate the stimulant effect of herpetin on bone marrow mesenchymal stem cells (BMSCs) to improve acute liver injury (ALI). In vitro results showed that herpetin treatment enhanced expression of the liver-specific proteins alpha-fetoprotein, albumin, and cytokeratin 18; increased cytochrome P450 family 3 subfamily a member 4 activity; and increased the glycogen-storage capacity of BMSCs. Mice with ALI induced by carbon tetrachloride (CCl4) were treated with a combination of BMSCs by tail-vein injection and herpetin by intraperitoneal injection. Hematoxylin and eosin staining and serum biochemical index detection showed that the liver function of ALI mice improved after administration of herpetin combined with BMSCs. Western blotting results suggested that the stromal cell-derived factor-1/C-X-C motif chemokine receptor 4 axis and the Wnt/ß-catenin pathway in the liver tissue were activated after treatment with herpetin and BMSCs. Therefore, herpetin is a promising BMSC induction agent, and coadministration of herpetin and BMSCs may affect the treatment of ALI.

Regulation of bone and fat balance by Fructus Ligustri Lucidi in ovariectomized mice.

CONTEXT: Fructus Ligustri Lucidi (FLL), a commonly used herb of traditional Chinese medicine (TCM), is the fruit of Ligustrum lucidum Ait. (Oleaceae). The ethanol extract of FLL is a potential candidate for preventing and treating postmenopausal osteoporosis (PMOP) by nourishing the liver and kidneys. OBJECTIVE: This study determines whether an ethanol extract of FLL has anti-osteoporotic effects in ovariectomized (OVX) mice and explores the underlying mechanism. MATERIALS AND METHODS: The OVX model of eight-week-old C57BL/6J female mice was taken, and ovariectomy was used as PMOP. Mice were divided into five groups: sham-operated group (n = 10), OVX group (n = 10), OVX + E2 group (n = 10; 0.039 mg/kg), OVX + FLL group (n = 10; 2 g/kg) and OVX + FLL group (n = 10; 4 g/kg). Mice were treated by gavage with FLL or CMCNa once daily for 8 weeks. We harvested uteri, femur, and tibias from mice; bone mineral density (BMD) and bone microstructure were obtained by X-ray absorptiometry and micro-CT. Furthermore, the effect of FLL on the balance of osteoblast and adipocyte differentiation was investigated using bone marrow mesenchymal stem cells (BMMSCs). RESULTS: The results indicated that FLL did not affect OVX-induced estradiol reduction. Compared with OVX mice, FLL significantly increased BMD (63.54 vs. 61.96), Conn. D (86.46 vs. 57.00), and left tibial strength (13.91 vs. 11.27), decreased Tb. Sp (0.38 vs. 0.44) and body fat content (4.19% vs. 11.24%). FLL decreased osteoclast activity and enhanced RUNX2 expression; inhibited perilipin peroxisome proliferator-activated receptor gamma (PPARγ) expression and adipocyte differentiation from BMMSCs. CONCLUSIONS: FLL prevented additional bone loss and improved bone microstructure in OVX mice by modulating bone and fat balance, suggesting that FLL might be a therapeutic agent for PMOP.

Extracellular Vesicles Derived from Bone Mesenchymal Stem Cells Carrying circ_0000075 Relieves Cerebral Ischemic Injury by Competitively Inhibiting miR-218-5p and Up-regulating E3 Ubiquitin Ligase SMURF2.

Extracellular vesicle (EV)-encapsulated circRNAs have the potential role in affecting brain disorders. However, the role of circ_0000075 in cerebral ischemic injury remains unclear. Here, we tried to investigate the mechanism of bone marrow mesenchymal stem cell (BMSC)-derived EVs carrying circ_0000075 in the control of cerebral ischemic injury. Initially, a mouse model with cerebral ischemic injury was induced by middle cerebral artery occlusion (MCAO), followed by the determination of circ_0000075 expression. Then, neurons were isolated and subjected to oxygen-glucose deprivation/reperfusion. BMSCs were isolated for extraction of EVs. The correlation among circ_0000075, microRNA (miR)-218-5p, and Smad ubiquitination regulatory factor 2 (SMURF2) was detected with their roles in cerebral ischemic injury analyzed in vivo and in vitro. circ_0000075 was down-regulated in MCAO mice and engineered RVG-EVs were internalized by neurons to up-regulate circ_0000075 expression. Treatment of RVG-circ_0000075-EVs reduced brain tissue damage, increased neuronal count, and significantly curtailed apoptosis rate, suppressing cerebral ischemic injury in vitro and in vivo. miR-218-5p was targeted by circ_0000075 in neurons, which promoted SMURF2 expression. A negative correlation between SMURF2 and transcriptional regulator Yin Yang 1 (YY1) was identified. In vitro experiments further proved that circ_ 00,000 75 could down-regulate the expression of YY1 through SMURF2, and finally relieving cerebral ischemic injury. Collectively, engineered EVs delivered circ_0000075 into brain tissues and increased circ_0000075 expression, which down-regulated miR-218-5p and up-regulated SMURF2, thus alleviating cerebral ischemic injury.

Taohong Siwu Decoction enhances human bone marrow mesenchymal stem cells proliferation, migration and osteogenic differentiation via VEGF-FAK signaling in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Taohong Siwu Decoction (THSWD) is a conventional traditional Chinese prescription aiming at promoting blood circulation and alleviating blood stasis. It is widely prescribed in instances of ischemic strokes, cardiovascular diseases, osteoporosis and bone fracture. However, its molecular functions in bone formation remain uncharacterized. AIM OF STUDY: This study aims to explore the potential effects of THSWD treatment on human bone marrow mesenchymal stem cells (BMSCs) proliferation, osteogenic differentiation, and migration. MATERIALS AND METHODS: BMSCs undergo osteogenic, adipogenic, and chondrogenic differentiation to determine cell stemness. BMSCs were treated with low dose (200 µg/ml), medium dose (400 µg/ml) and high dose (600 µg/ml) THSWD. The cell viability was determined by CCK-8 assays, the osteogenic differentiation ability was determined by alizarin red staining and ALP staining, and cell migration was determined by wound healing and transwell assays. The effect of THSWD on the vascular endothelial growth factor (VEGF)/focal adhesion kinase (FAK) pathway was determined by immunoblotting. RESULTS: THSWD time-dependently and dose-dependently promoted BMSC viability. Moreover, THSWD also promoted BMSC osteogenic differentiation and migration. As opposed to THSWD, VEGF receptor inhibitor Bevacizumab suppressed BMSC osteogenic differentiation and migration. In BMSCs that have been co-treated with THSWD and Bevacizumab, THSWD effects on BMSC functions were partially eliminated by Bevacizumab. Moreover, THSWD treatment boosted VEGF content in the supernatant and was conducive to the phosphorylation of FAK and Src, whereas Bevacizumab exerted opposite effects; similarly, Bevacizumab partially abolished THSWD effects on VEGF and FAK (Tyr397) and Src (Tyr418) phosphorylation. CONCLUSION: THSWD enhances the capacities of BMSCs to proliferate, differentiate, and migrate, possibly through VEGF and the FAK-Src, thereby improving fracture healing.

Alpinetin alleviates osteoporosis by promoting osteogenic differentiation in BMSCs by triggering autophagy via PKA/mTOR/ULK1 signaling.

Osteoporosis, a systemic bone disease that is characterized by a reduction in bone mass and destruction of bone microstructure, is becoming a serious problem worldwide. Bone marrow mesenchymal stem cells (BMSCs) can differentiate into bone-forming osteoblasts, and play an important role in maintaining homeostasis of bone metabolism, thus being a potential therapeutic target for osteoporosis. Although the phytochemical alpinetin (APT) has been reported to possess a variety of pharmacological activities, it is still unclear whether APT can influence the osteogenic differentiation of on BMSCs and if it can improve osteoporosis. In this study, we found that APT treatment was able to enhance osteogenic differentiation levels of human BMSCs in vitro and mouse ones in vivo as revealed by multiple osteogenic markers including increased alkaline phosphatase activity and osteocalcin expression. Mechanistically, the protein kinase A (PKA)/mTOR/ULK1 signaling was involved in the action of APT to enhance the osteogenic differentiation of BMSCs. In addition, oral administration of APT significantly mitigated the bone loss in a dexamethasone-induced mouse model of osteoporosis through strengthening PKA signaling and autophagy. Altogether, these data demonstrate that APT promotes osteogenic differentiation in BMSCs by augmenting the PKA/mTOR/ULK1 autophagy signaling, highlighting its potential therapeutic application for treating osteoporotic diseases.

Chinese Medicine Regulates Signaling Pathways Related to Proliferation and Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells： A Review / 中国实验方剂学杂志

Bone marrow mesenchymal stem cells （BMSCs） are derived from stem cells isolated from bone marrow and have the potential for multidirectional differentiation and self-renewal. Under certain conditions， BMSCs can be induced to differentiate into osteoblast （OB）， chondrocyte， adipocyte， fibroblast， etc. BMSCs play an important role in maintaining the stability of bone structure and balancing bone metabolism. Promoting the proliferation of BMSCs and inducing their differentiation into OB of great significance for the clinical prevention and treatment of osteoporosis， bone defects， fracture healing， and other diseases. Because the proliferation and osteogenic differentiation of BMSCs are complex processes controlled by multiple genes and regulated by multiple signal transduction pathways， traditional Chinese medicine （TCM） happens to have the advantages of multi-bioactive component， multi-target， and multi-pathway synergism， which can affect the proliferation and differentiation of BMSCs through multiple channels and induce the proliferation of BMSCs. The transcription and expression of genes related to osteogenesis can be enhanced to promote the differentiation of BMSCs into OB， so as to achieve the purpose of preventing and treating osteoporosis， bone defects， and other bone diseases. Based on the literature on the intervention of TCM monomers and compounds in the proliferation and osteogenic differentiation of BMSCs， this study reviewed TCM monomers and compounds in promoting the proliferation and osteogenic differentiation of BMSCs by regulating secreted glycoprotein （Wnt）， neurogenic locus notch homolog protein （Notch）， mitogen-activated protein kinase （MAPK）， phosphatidylinositol-3 kinase （PI3K） /protein kinase B （Akt）， bone morphogenetic protein （BMP）/Smad， Janus kinase （JAK）/signal transducer and activator of transcription protein （STAT）， osteoprotegerin （OPG）/receptor activator of nuclear factor-kappa B （RANK）/RANK ligand （RANKL）， and other signaling pathways to provide new ideas for the research and clinical application of Chinese medicine in the prevention and treatment of orthopedic diseases.

Fabrication and in vitro evaluation of 3D printed porous silicate substituted calcium phosphate scaffolds for bone tissue engineering.

Silicate-substituted calcium phosphate (Si-CaP) ceramics, alternative materials for autogenous bone grafting, exhibit excellent osteoinductivity, osteoconductivity, biocompatibility, and biodegradability; thus, they have been widely used for treating bone defects. However, the limited control over the spatial structure and weak mechanical properties of conventional Si-CaP ceramics hinder their wide application. Here, we used digital light processing (DLP) printing technology to fabricate a novel porous 3D printed Si-CaP scaffold to enhance the scaffold properties. Scanning electron microscopy, compression tests, and computational fluid dynamics simulations of the 3D printed Si-CaP scaffolds revealed a uniform spatial structure, appropriate mechanical properties, and effective interior permeability. Furthermore, compared to Si-CaP groups, 3D printed Si-CaP groups exhibited sustained release of silicon (Si), calcium (Ca), and phosphorus (P) ions. Furthermore, 3D printed Si-CaP groups had more comprehensive and persistent osteogenic effects due to increased osteogenic factor expression and calcium deposition. Our results show that the 3D printed Si-CaP scaffold successfully improved bone marrow mesenchymal stem cells (BMSCs) adhesion, proliferation, and osteogenic differentiation and possessed a distinct apatite mineralization ability. Overall, with the help of DLP printing technology, Si-CaP ceramic materials facilitate the fabrication of ideal bone tissue engineering scaffolds with essential elements, providing a promising approach for bone regeneration.

Physical and Chemical Properties, Biosafety Evaluation, and Effects of Nano Natural Deer Bone Meal on Bone Marrow Mesenchymal Stem Cells.

At present, bone-based products are abundant, and the main sources are bovine bone and pig bone, but there are few studies on the development of deer bone as a bone repair material. Deer bone has important osteogenic effects in the theory of traditional Chinese medicine. It is rich in protein, ossein, and a variety of trace elements, with the effect of strengthening tendons and bones. Nanomaterials and their application in the repair of bone defects have become a research hotspot in bone tissue engineering. In this study, nano-deer bone meal (nBM), nano-calcined deer bone meal, and nano-demineralized bone matrix were successfully prepared. It was found that the Ca/P ratio in deer bone was significantly higher than that in cow bone and human bone tissue, and deer bone contained beneficial trace elements, such as potassium, iron, selenium, and zinc, which were not found in cow bone. The three kinds of deer bone powders prepared in this study had good biocompatibility and met the implantation standards of medical biomaterials. Cell function studies showed that compared with other bone powders, due to the presence of organic active ingredients and inorganic calcium and phosphate salts, nBM had excellent performance in the proliferation, adhesion, migration, and differentiation of bone marrow mesenchymal stem cells. These findings indicate that nBM can be used as a potential osteoinductive active nanomaterial to enhance bone tissue engineering scaffolds with certain application prospects.

Application of vancomycin-impregnated calcium sulfate hemihydrate/nanohydroxyapatite/carboxymethyl chitosan injectable hydrogels combined with BMSC sheets for the treatment of infected bone defects in a rabbit model.

BACKGROUND: The choice of bone substitutes for the treatment of infected bone defects (IBDs) has attracted the attention of surgeons for years. However, single-stage bioabsorbable materials that are used as carriers for antibiotic release, as well as scaffolds for BMSC sheets, need further exploration. Our study was designed to investigate the effect of vancomycin-loaded calcium sulfate hemihydrate/nanohydroxyapatite/carboxymethyl chitosan (CSH/n-HA/CMCS) hydrogels combined with BMSC sheets as bone substitutes for the treatment of IBDs. METHODS: BMSCs were harvested and cultured into cell sheets. After the successful establishment of an animal model with chronic osteomyelitis, 48 New Zealand white rabbits were randomly divided into 4 groups. Animals in Group A were treated with thorough debridement as a control. Group B was treated with BMSC sheets. CSH/n-HA/CMCS hydrogels were implanted in the treatment of Group C, and Group D was treated with CSH/n-HA/CMCS+BMSC sheets. Gross observation and micro-CT 3D reconstruction were performed to assess the osteogenic and infection elimination abilities of the treatment materials. Histological staining (haematoxylin and eosin and Van Gieson) was used to observe inflammatory cell infiltration and the formation of collagen fibres at 4, 8, and 12 weeks after implantation. RESULTS: The bone defects of the control group were not repaired at 12 weeks, as chronic osteomyelitis was still observed. HE staining showed a large amount of inflammatory cell infiltration around the tissue, and VG staining showed no new collagen fibres formation. In the BMSC sheet group, although new bone formation was observed by gross observation and micro-CT scanning, infection was not effectively controlled due to unfilled cavities. Some neutrophils and only a small amount of collagen fibres could be observed. Both the hydrogel and hydrogel/BMSCs groups achieved satisfactory repair effects and infection control. Micro-CT 3D reconstruction at 4 weeks showed that the hydrogel/BMSC sheet group had higher reconstruction efficiency and better bone modelling with normal morphology. HE staining showed little aggregation of inflammatory cells, and VG staining showed a large number of new collagen fibres. CONCLUSIONS: Our preliminary results suggested that compared to a single material, the novel antibiotic-impregnated hydrogels acted as superior scaffolds for BMSC sheets and excellent antibiotic vectors against infection, which provided a basis for applying tissue engineering technology to the treatment of chronic osteomyelitis.