The Potential of MSC-Based Cell-Free Therapy in Wound Healing-A Thorough Literature Review.

A wound is an interruption of the normal anatomic structure and function of the skin, which is critical in protecting against foreign pathogens, regulating body temperature and water balance. Wound healing is a complex process involving various phases, including coagulation, inflammation, angiogenesis, re-epithelialization, and re-modeling. Factors such as infection, ischemia, and chronic diseases such as diabetes can compromise wound healing, leading to chronic and refractory ulcers. Mesenchymal stem cells (MSCs) have been used to treat various wound models due to their paracrine activity (secretome) and extracellular vehicles (exosomes) that contain several molecules, including long non-coding RNAs (lncRNAs), micro-RNAs (miRNAs), proteins, and lipids. Studies have shown that MSCs-based cell-free therapy using secretome and exosomes has great potential in regenerative medicine compared to MSCs, as there are fewer safety concerns. This review provides an overview of the pathophysiology of cutaneous wounds and the potential of MSCs-based cell-free therapy in each phase of wound healing. It also discusses clinical studies of MSCs-based cell-free therapies.

The Application of Adipose Tissue-Derived Mesenchymal Stem Cells (ADMSCs) and a Twin-Herb Formula to the Rodent Wound Healing Model: Use Alone or Together?

Our previous study reported that mesenchymal stem cells (MSCs) accelerated the wound healing process through anti-inflammatory, anti-apoptotic, and pro-angiogenetic effects in a rodent skin excision model. NF3 is a twin-herb formula, which presents similar effects in promoting wound healing. Research focusing on the interaction of MSCs and Chinese medicine is limited. In this study, we applied MSCs and the twin-herb formula to the wound healing model and investigated their interactions. Wound healing was improved in all treatment groups (MSCs only, NF3 only, and MSCs + NF3). The combined therapy further enhanced the effect: more GFP-labelled ADMSCs, collagen I and collagen III expression, Sox9 positive cells, and CD31 positive cells, along with less ED-1 positive cells, were detected; the expressions of proinflammatory cytokine IL-6 and TNF-α were downregulated; and the expression of anti-inflammatory cytokine IL-10 was upregulated. In vitro, NF3 promoted the cell viability and proliferation ability of MSCs, and a higher concentration of protein was detected in the NF3-treated supernatant. A proteomic analysis showed there were 15 and 22 proteins in the supernatants of normal ADMSCs and NF3-treated ADMSCs, respectively. After PCR validation, the expressions of 11 related genes were upregulated. The results of a western blot suggested that the TGFß/Smad and Wnt pathways were related to the therapeutic effects of the combined treatment. Our study suggests for the first time that NF3 enhanced the therapeutic effect of MSCs in the wound healing model and the TGFß/Smad and Wnt pathways were related to the procedure.

Deteriorating effect on bone metabolism and microstructure by passive cigarette smoking through dual actions on osteoblast and osteoclast.

There is no clear evidence to show the direct causal relationship between passive cigarette smoking and osteoporosis. Furthermore, the underlying mechanism is unknown. The objective of this study is to demonstrate the effects of long-term passive cigarette smoking on bone metabolism and microstructure by a mouse model and cell culture systems. BALB/c mice were exposed to 2 or 4 % cigarette smoke for 14 weeks. The bone turnover biochemical markers in urine and serum and also the bone micro-architecture by micro-CT were compared with the control group exposed to normal ambient air. In the cell culture experiments, mouse MC3T3-E1 and RAW264.7 cell lines to be employed as osteoblast and osteoclast, respectively, were treated with the sera obtained from 4 % smoking or control mice. Their actions on cell viability, differentiation, and function on these bone cells were assessed. The urinary mineral and deoxypyridinoline (DPD) levels, and also the serum alkaline phosphatase activity, were significantly higher in the 4 % smoking group when compared with the control group, indicating an elevated bone metabolism after cigarette smoking. In addition, femoral osteopenic condition was observed in the 4 % smoking group, as shown by the decrease of relative bone volume and trabecular thickness. In isolated cell studies, osteoblast differentiation and bone formation were inhibited while osteoclast differentiation was increased. The current mouse smoking model and the isolated cell studies demonstrate that passive cigarette smoke could induce osteopenia by exerting a direct detrimental effect on bone cells differentiation and further on bone remodeling process.

The effects of an antiosteoporosis herbal formula containing epimedii herba, ligustri lucidi fructus and psoraleae fructus on density and structure of rat long bones under tail-suspension, and its mechanisms of action.

An innovative anti-osteoporosis herbal formula containing epimedii herba, ligustri lucidi fructus and psoraleae fructus (ELP) has been previously shown its bone protecting effects in ovariectomized osteoporotic rats and also in post-menopausal osteopenic women. This study aimed to investigate the efficacy of ELP against bone loss during physical inactivity or weightlessness. A hindlimb unloading tail-suspended rat model was used for studying the effects of ELP on bone mineral density (BMD) and bone micro-architecture. For in vitro mechanistic studies, rat mesenchymal stem cells (MSCs) and mouse macrophage cells (RAW264.7) were used for studying the effects of ELP on osteogenic/adipogenic differentiations and osteoclastogenesis, respectively. Our data illustrated that ELP had a significant preventive effect against bone loss induced by tail-suspension (TS) at day 28 (p < 0.01) as indicated in the reduction in BMD loss and the preservation of bone micro-architecture. ELP could significantly promote the osteogenesis and suppress the adipogenesis (p < 0.05) in MSCs. Besides, significant inhibition of osteoclast formation (p < 0.01) was found in ELP-treated RAW264.7 cells upon receptor activator of nuclear factor kappa-B ligand induction. Our study presents the first scientific evidence that ELP had a significant preventive effect against bone loss induced by TS through the actions of enhancing osteogenesis, suppressing adipogenesis and osteoclastogenesis.

Traditional Chinese Medicine for Topical Treatment of Skeletal Muscle Injury.

Muscle injuries are common musculoskeletal problems, but the pharmaceutical agent for muscle repair and healing is insufficient. Traditional Chinese Medicine (TCM) frequently uses topical treatments to treat muscle injuries, although scientific evidence supporting their efficacy is scarce. In this study, an in vitro assay was used to test the cytotoxicity of a topical TCM formula containing Carthami Flos, Dipsaci Radix, and Rhei Rhizoma (CDR). Then, a muscle contusion rat model was developed to investigate the in vivo effect and basic mechanisms underlying CDR on muscle regeneration. The in vitro assay illustrated that CDR was non-cytotoxic to immortalized rat myoblast culture and increased cell viability. Histological results demonstrated that the CDR treatment facilitated muscle repair by increasing the number of new muscle fibers and promoting muscle integrity. The CDR treatment also upregulated the expression of Pax7, MyoD and myogenin, as evidenced by an immunohistochemical study. A gene expression analysis indicated that the CDR treatment accelerated the regeneration and remodeling phases during muscle repair. This study demonstrated that topical CDR treatment was effective at facilitating muscle injury repair.

Rat Plantar Fascia Stem/Progenitor Cells Showed Lower Expression of Ligament Markers and Higher Pro-Inflammatory Cytokines after Intensive Mechanical Loading or Interleukin-1ß Treatment In Vitro.

The pathogenesis of plantar fasciitis is unclear, which hampers the development of an effective treatment. The altered fate of plantar fascia stem/progenitor cells (PFSCs) under overuse-induced inflammation might contribute to the pathogenesis. This study aimed to isolate rat PFSCs and compared their stem cell-related properties with bone marrow stromal cells (BMSCs). The effects of inflammation and intensive mechanical loading on PFSCs' functions were also examined. We showed that plantar fascia-derived cells (PFCs) expressed common MSC surface markers and embryonic stemness markers. They expressed lower Nanog but higher Oct4 and Sox2, proliferated faster and formed more colonies compared to BMSCs. Although PFCs showed higher chondrogenic differentiation potential, they showed low osteogenic and adipogenic differentiation potential upon induction compared to BMSCs. The expression of ligament markers was higher in PFCs than in BMSCs. The isolated PFCs were hence PFSCs. Both IL-1ß and intensive mechanical loading suppressed the mRNA expression of ligament markers but increased the expression of inflammatory cytokines and matrix-degrading enzymes in PFSCs. In summary, rat PFSCs were successfully isolated. They had poor multi-lineage differentiation potential compared to BMSCs. Inflammation after overuse altered the fate and inflammatory status of PFSCs, which might lead to poor ligament differentiation of PFSCs and extracellular matrix degeneration. Rat PFSCs can be used as an in vitro model for studying the effects of intensive mechanical loading-induced inflammation on matrix degeneration and erroneous stem/progenitor cell differentiation in plantar fasciitis.

In Vivo Study on the Pharmacological Interactions between a Chinese Herbal Formula ELP and Antiresorptive Drugs to Counteract Osteoporosis.

Antiresorptive drugs, alendronate and raloxifene, are effective in lowering bone mineral density (BMD) loss in postmenopausal women. However, long-term treatment may be associated with serious side effects. Our research group has recently discovered that a Chinese herbal formula, ELP, could significantly reduce BMD loss in animal and human studies. Therefore, the present study aimed to investigate the potential synergistic bone-protective effects of different herb-drug combinations using ovariectomized rats. To assess the efficacy of different combinations, the total BMD was monitored biweekly in the 8-week course of daily oral treatment. Bone microarchitecture, bone strength, and deoxypyridinoline level were also determined after 8 weeks. From our results, coadministration of ELP and raloxifene increased the total tibial BMD by 5.26% (2.5 mg/kg/day of raloxifene; P = 0.014) and 5.94% (0.25 mg/kg/day of raloxifene; P = 0.026) when compared with the respective dosage groups with raloxifene alone. Similar synergistic effects were also observed in BMD increase at distal femur (0.25 mg/kg/day; P = 0.001) and reduction in urinary deoxypyridinoline crosslink excretion (2.5 and 0.25 mg/kg/day; both P = 0.02). However, such interactions could not be observed in all alendronate-treated groups. Our data provide first evidence that ELP could synergistically enhance the therapeutic effects of raloxifene, so that the clinical dosage of raloxifene could be reduced.

Green tea extract synergistically enhances the effectiveness of an antiresorptive drug on management of osteoporosis induced by ovariectomy in a rat model.

Antiresorptive drugs are effective for reducing bone loss in postmenopausal women, but their long-term application may be associated with adverse effects. The present study aimed to investigate the potential in vivo synergistic effects of green tea extract (GTE) and alendronate or raloxifene on the management of osteoporosis. Ovariectomized rats were fed orally with GTE, alendronate and raloxifene at different concentrations and various combinations for 4 weeks. Bone mineral density (BMD) at the lumbar spine, femur and tibia was monitored weekly using peripheral quantitative computed tomography. Bone microarchitecture in the left distal femur was analyzed using micro-CT, while serum biochemical levels were measured using ELISA kits at the end of the study. GTE alone effectively mitigated BMD loss and improved bone microarchitecture in rats. The co-administration of GTE and alendronate increased total BMD in the lumbar spine, femur and tibia. Particularly, GTE synergistically enhanced the effect of alendronate at a low dose on bone microarchitecture and decreased serum tartrate-resistant acid phosphatase. These findings imply that the dosage of certain antiresorptive agents could be reduced when they are administrated simultaneously with GTE, so that their adverse effects are minimized. The findings may be used to support the development of a new synergistic intervention between food therapy and pharmacotherapy on the management of osteoporosis in a long-term basis.

Influence of pH-responsive compounds synthesized from chitosan and hyaluronic acid on dual-responsive (pH/temperature) hydrogel drug delivery systems of Cortex Moutan.

The polysaccharide-based pH-responsive compounds, namely, N,N,N-trimethyl chitosan (TMC), polyethylene glycolated hyaluronic acid (PEG-HA), and polysaccharide-based nano-conjugate of hyaluronic acid, chitosan oligosaccharide and alanine [HA-Ala-Chito(oligo)] were chemically synthesized using biopolymers chitosan and hyaluronic acid, and applied here to observe the changes in morphology, pH-stability, mechanical and drug-release behavior, and cytotoxicity of thermo-responsive polymer: Poloxamer 407 (PF127)-based drug delivery systems for traditional Chinese medicine Cortex Moutan (CM). The thermo-responsive hydrogel of PF127 loaded with CM (GelC) was used as control. The dual-responsive (pH/temperature) hydrogels: PF127/TMC/PEG-HA (Gel1) and PF127/HA-Ala-Chito(oligo) (Gel2) showed improved mechanical behavior as obtained by rheology and mechanical agitation study, and pH-stability under various external pH conditions, and those improvements occurred due to the addition of polysaccharide-based pH-responsive compounds in the systems. Both, Gel1 and Gel2 showed better morphology than GelC as obtained by SEM or TEM suggesting that interaction of polysaccharide-based pH-responsive compounds with PF127 in either gel or sol state gave better porous network structure in the hydrogels or more dispersed micellar arrangements in sol-state, respectively. Gel1 showed the highest cumulative drug release (86.5%) after 5 days under mild acidic condition (pH 6.4) suggesting that release behavior of a hydrogel drug carrier was dependent on morphology, mechanical behavior, and pH-stability. The transdermal release (ex-vivo) results indicated that gallic acid, the active marker of CM passed through porcine ear skin and all the formulations showed more or less similar transdermal release properties. The hydrogels loaded with CM showed no cytotoxicity (cell viability >90.0%) on human HaCaT keratinocytes within concentration range of 0.0-20.0 µg/ml as obtained by MTT assay, and cell viability was more than 100% at a concentration of 20.0 µg/ml for Gel2. The formulations without loaded drug namely, Gel1-CM and Gel2-CM exhibited strong anti-bacterial action against gram positive bacteria Staphylococcus aureus.

Adipose Tissue-Derived Mesenchymal Stem Cells (ADMSCs) and ADMSC-Derived Secretome Expedited Wound Healing in a Rodent Model - A Preliminary Study.

INTRODUCTION: The biological role of mesenchymal stem cells (MSCs) in wound healing has been demonstrated. However, there were limited studies on the healing effect of secretome which consists of many biological factors secreted by MSCs. In this study, we aimed to compare the therapeutic effects of secretome with MSCs on facilitating wound healing. METHODS: Green fluorescent protein labelled adipose-derived MSCs (GFP-ADMSCs) or secretome was injected in the full-thickness skin excision model on SD rats. The wound healing process was evaluated by calculating the healing rate and the histological examinations on skin biopsy. The cell viability, proliferation and mobility of the rat dermal fibroblasts were compared after different treatments. The inflammatory response in macrophages was indicated by the level of nitric oxide (NO) and inflammatory cytokines through NO assay and ELISA. RESULTS: On day 5 and day 14, both MSCs and secretome accelerated the wound healing, secretome further enhanced the process. GFP-MSCs were detected 10 days after transplantation. The level of IL-6 and TNF-α in blood was reduced after MSCs and secretome treatments. The expressions of VEGF and PCNA were increased after treatment, higher intensity of VEGF was observed in secretome-injected tissue. The concentrations of total protein and VEGF in secretome were 2.2 ± 0.5 mg/mL and 882.0 ± 72.7 pg/mL, respectively. The cell viability and proliferation of FR were promoted significantly after the treatment. The scratch test showed that secretome accelerated the wound healing speed. Secretome reduced the metabolism of macrophages remarkably, but it did not decrease the level of macrophage-secreted NO. The expression of the pro-inflammatory cytokines (IL-6, MCP-1 and TNF-α) was downregulated significantly. CONCLUSION: Our study indicated both MSCs and MSCs-derived secretome enhanced the wound healing process in early phase. Secretome further promoted the healing effects through promoting the fibroblast proliferation and migration and suppressing the inflammatory response.

Effects of topical application of a tri-herb formula on inflammatory dry-skin condition in mice with oxazolone-induced atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a chronic relapsing inflammatory and pruritic skin disease, affecting 10-20% of the population worldwide. Paeonia suffruticosa Andrews (Paeoniaceae) (Cortex Moutan) and Mentha haplocalyx Briq. (Labiatae) (Herba Menthae) have shown beneficial effects on AD. Calendula officinalis L. (Asteraceae) is commonly used for treating skin rashes and wounds. PURPOSE: In the present study, a three-herbs formula including Cortex Moutan and Herba Menthae, together with C. officinalis at 1:1:1 weight ratio was used as a topical agent and its therapeutic effects on AD was investigated. METHODS: In vitro effects of individual herbs and three-herbs formula (0.125-1 mg/ml) were examined using cytokine release assay on human mast HMC-1 cells, inflammation test on murine macrophage RAW cells and human keratinocyte (HaCaT) cells, and migration scratch assay on human umbilical vein endothelial cells (HUVEC). The contributing functional pathway of three-herbs formula in AD was explored using Western Blot assay in HMC-1 cells. Oxazolone-induced AD-like mice model was also used to investigate the in vivo therapeutic effect of the topical application of the three-herbs formula. RESULTS: Herba Menthae, Cortex Moutan, and three-herbs formula significantly reduced the production of IL-6 and tumor necrosis factor (TNF)-α in HMC-1 cells, inhibited the expression of IL-6, IL-8 and CCL2 in TNF-α/IFN-γ stimulated HaCaT cells, and suppressed the lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 cells. Moreover, Herba Menthae and three-herbs formula significantly suppressed CCL2 and TNF-α production in LPS-induced RAW 264.7 cells. C. officinalis and three-herbs formula promoted wound healing in HUVEC. For intracellular mechanisms, three-herbs formula inhibited the expressions of molecules in STAT1 and STAT3-dependent pathways. In vivo model showed that topical application of three-herbs formula on challenged ear reduced ear swelling and mice scratching frequencies. H&E and toluidine blue staining of the challenged ear tissue demonstrated that three-herbs formula reduced the epidermal thickness and mast cell infiltration, respectively. CONCLUSION: The three-herbs formula of Cortex Moutan, Herba Menthae and C. officinalis at 1:1:1 (w/w) exhibited anti-inflammatory effect and promotion of cell migration in vitro. It also alleviated ear redness, swelling, epidermal thickness and inflammation of the OXA-induced AD mice. These findings suggest a potential beneficial role of the topical application of the three-herbs formula for treatment of AD.

Topical application of Chinese herbal medicine DAEP relieves the osteoarthritic knee pain in rats.

BACKGROUND: The potential adverse effects of conventional oral pharmacotherapy of osteoarthritis (OA) restrict their long-term use. Topical application of a Chinese herbal paste for relieving OA knee pain can be effective and safe. However, evidence-based scientific research is insufficient to support its application worldwide. The aim of this study was to investigate the in vivo efficacy of a topical Chinese herbal paste on relieving OA knee pain and its underlying mechanism. METHODS: An OA rat model was developed by anterior cruciate ligament transection (ACLT) followed by treadmill running. A herbal paste including Dipsaci Radix, Achyranthis Bidentatae Radix, Eucommiae Cortex and Psoraleae Fructus, named as DAEP, was applied topically on the knee joint of the rats (DAEP). The rats without DAEP treatment served as Control. Rats with surgery but without ACLT, treadmill running and DAEP treatment acted as Sham. The morphologic change of the knee joint was observed radiographically. Nociception from the knee of the rats was assessed using Incapacitent test and CatWalk gait system. The therapeutic mechanism was investigated by analyzing the gene and protein expression of inflammatory markers via qPCR and Western blot, respectively. RESULTS: Radiographic images showed less destruction at the posterior tibial plateau of the DAEP group compared with the Control after 2 weeks of treatment. The static weight ratio and the gait parameters of the Control were reduced significantly via Incapacitance test and CatWalk gait analysis, respectively. DAEP treatment increased the Print Area and Maximum Intensity significantly compared with the Control. DAEP significantly suppressed the upregulation of gene expression of interleukin (IL)-6, tumor necrosis factor (TNF)-α, and inducible nitric oxide synthase (iNOS). CONCLUSIONS: DAEP exhibited its effect via the nuclear factor (NF)-κB pathway by suppressing the phosphorylation of IκB kinase αß (p-IKKαß) and cyclooxygenase-2 (COX-2) protein expression. This study provides scientific evidence to support the clinical application of the Chinese herbal paste on reliving OA pain.

Chinese topical herbal medicine gives additive effect on pharmaceutical agent on fracture healing.

OBJECTIVE: To investigate the efficacy on the combination of oral strontium ranelate (SrR) with a topical Chinese herbal paste on facilitation of fracture healing. METHODS: An open fracture was created at the mid-shaft of the right tibia of rat. A herbal paste called CDR containing Honghua (Flos Carthami), Chuanxuduan (Radix Dipsaci Asperoidis) and Dahuang (Radix Et Rhizoma Rhei Palmati) was prepared. The rats were treated with either CDR topically on the fracture site, or SrR orally, or their combinations. Bone turnover biochemical markers in serum were measured. Microarchitecture of the fracture was analyzed using micro-CT after 14 and 28 d, followed by histomorphometrical analysis. RESULTS: Micro-computed tomography analysis revealed that the combined treatment of CDR with 600 mg/g SrR significantly increased the total callus density, mineralized callus volume fraction, mineralized callus mineral content and mineralized callus density of the callus after 28 d of treatment. This result was consistent with the histomorphometrical analysis on the osteoid volume. Analysis of biochemical markers showed that the combined treatments reduced the bone resorption that occurs temporarily after fracture. CONCLUSION: This study demonstrated that the combined treatment of oral SrR and topical CDR is effective to promote fracture healing by their additive effect on promoting bone formation and retarding bone resorption.

Inhibitory effect of different Dendrobium species on LPS-induced inflammation in macrophages via suppression of MAPK pathways.

Dendrobii Caulis (DC), named 'Shihu' in Chinese, is a precious herb in traditional Chinese medicine. It is widely used to nourish stomach, enhance body fluid production, tonify "Yin" and reduce heat. More than thirty Dendrobium species are used as folk medicine. Some compounds from DC exhibit inhibitory effects on macrophage inflammation. In the present study, we compared the anti-inflammatory effects among eight Dendrobium species. The results provided evidences to support Dendrobium as folk medicine, which exerted its medicinal function partially by its inhibitory effects on inflammation. To investigate the anti-inflammatory effect of Dendrobium species, mouse macrophage cell line RAW264.7 was activated by lipopolysaccharide. The nitric oxide (NO) level was measured using Griess reagent while the pro-inflammatory cytokines were tested by ELISA. The protein expressions of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2) and mitogen-activated protein kinases (MAPKs) phosphorylation were evaluated by Western blotting analysis. Among the eight Dendrobium species, both water extracts of D. thyrsiflorum B.S.Williams (DTW) and D. chrysotoxum Lindl (DCHW) showed most significant inhibitory effects on NO production in a concentration-dependent manner. DTW also significantly reduced TNF-α, MCP-1, and IL-6 production. Further investigations showed that DTW suppressed iNOS and COX-2 expression as well as ERK and JNK phosphorylation, suggesting that the inhibitory effects of DTW on LPS-induced macrophage inflammation was through the suppression of MAPK pathways. In conclusion, D. thyrsiflorum B.S.Williams was demonstrated to have potential to be used as alternative or adjuvant therapy for inflammation.

Dihydrofisetin exerts its anti-inflammatory effects associated with suppressing ERK/p38 MAPK and Heme Oxygenase-1 activation in lipopolysaccharide-stimulated RAW 264.7 macrophages and carrageenan-induced mice paw edema.

Dihydrofisetin is a flavanonol derived from some edible wild herbs and traditional Chinese medicines. It has been found to possess many biological activities. However, the anti-inflammatory potential of Dihydrofisetin remains uncharacterized. The aim of the present study was to investigate the anti-inflammatory activity of Dihydrofisetin and its underlying mechanisms. We found that Dihydrofisetin dose-dependently inhibited lipopolysaccharide-induced productions of nitric oxide (NO) and prostaglandin E2 (PGE2) in RAW 264.7 macrophages, probably through suppressing the protein expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). The expressions of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and monocyte chemotactic protein (MCP-1) were also suppressed. We further demonstrated that Dihydrofisetin inhibited the activation of mitogen-activated protein kinases (MAPKs) pathway and phosphorylation of IκB-α whereas upregulated the expression of heme oxygenase-1 (HO-1). The in vivo carrageenan-induced mice paw edema study also indicated that treatment with 100 mg/kg of Dihydrofisetin could significantly inhibit carrageenan induced paw edema, decrease the levels of TNF-α, IL-6 and MDA, and increase the activity of GSH-Px in paw tissues. Taken together, Dihydrofisetin may act as a natural agent for treating inflammatory diseases by targeting MAPK, NF-κB and HO-1 pathways.

Integrative Approach to Facilitate Fracture Healing: Topical Chinese Herbal Paste with Oral Strontium Ranelate.

Strontium ranelate (SrR) is one of the pharmaceutical agents reported to be effective on the promotion of fracture healing. This study aimed to evaluate the integrative effect of the oral SrR with a topical Chinese herbal paste, namely, CDR, on facilitation of bone healing. The in vivo efficacy was evaluated using rats with tibial fracture. They were treated with either CDR topically, or SrR orally, or their combined treatments. The in vivo results illustrated a significant additive effect of CDR on SrR in increasing the yield load of the fractured tibia. The in vitro results showed that neither SrR nor CDR exhibited a cytotoxic effect on UMR106 and bone-marrow stem cell (BMSC), but both of them increased the proliferation of BMSC at low concentrations. The combination of CDR at 200 µg/mL with SrR at 200 or 400 µg/ml also showed an additive effect on increasing the ALP activity of BMSC. Both SrR and CDR alone reduced osteoclast formation, and the effective concentration of SrR to inhibit osteoclastogenesis was reduced in the presence of CDR. This integrative approach by combining oral SrR and topical CDR is effective in promoting fracture healing properly due to their additive effects on proosteogenic and antiosteoclastogenic properties.

Seropharmacological study on osteogenic effects of post-absorption ingredients of an osteoprotective herbal formula.

OBJECTIVE: To further investigate the {ptin vitro} effects of an osteoprotective herbal formula "ELP" (Herba Epimedii, Fructus Ligustri Lucidi and Fructus Psoraleae) using seropharmacological approach. METHODS: Rats were fed with ELP or its individual component herbs for 2 days. The serum containing the postabsorbed ingredients of the herbal items were collected for cell culture using UMR106 cell, RAW264.7 cell and mesenchymal stem cell (MSC) isolated from the bone marrow of the rats. The effects of the herbal-containing serum on cell toxicity were detected by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay; bromodeoxyuridine assay was conducted to measure the cell proliferation of UMR106 cell and MSC; cell activity was measured using colorimetric method, and mRNA expression of runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and osteopontin (OPN) of UMR106 and MSC as well as matrix metalloproteinase 9 (MMP-9), tartrate-resistant acid phosphatase (TRAP) and cathepsin K of RAW264.7 were analyzed using real-time reverse-transcription polymerase chain reaction. RESULTS: ELP and its component serum exhibited no cytotoxic effects on the cells. The ELP-containing serum increased the proliferation of UMR106 cell and MSC by 25.7% and 14.4 %, respectively and the alkaline phosphatase activity of MSC was increased by 42.6%. On the contrary, it inhibited the RAW264.7 cell differentiation by 29.2 %. ELP serum upregulated the Runx2 expression of UMR and MSC by 1.18 fold and 1.27 fold, respectively. It also upregulated ALP and OPN expression in MSC by 1.69- and 2.12-fold, respectively. On the other hand, ELP serum down-regulated MMP-9 and cathepsin K expression of RAW264.7 cell by 0.46- and 0.36-fold, respectively. CONCLUSIONS: The serum of the animals fed with ELP contains active ingredients which are effective in promoting osteogenesis and inhibiting osteoclastogenesis.

Polysaccharides of Dendrobium officinale Kimura & Migo protect gastric mucosal cell against oxidative damage-induced apoptosis in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Dendrobium officinale Kimura & Migo (DO) is a valuable Traditional Chinese Medicine to nourish stomach, in which polysaccharides are identified as active ingredients. However, limited scientific evidences have been reported on the gastroprotective efficacy of DO. The aim of the current study was to investigate the protective effects and underlying mechanism of polysaccharides from DO(DOP) on gastric mucosal injury. MATERIAL AND METHODS: For in vitro study, HFE145 cells were pretreated with DOP before induction of cell apoptosis by H2O2. Cell apoptosis and related proteins expression were detected. In the in vivo study, absolute ethanol was administered orally to induce gastric mucosal injury in rat. The gastric mucosal injury area and histological examination were used to evaluate the effects of DOP treatment on the recovery of the gastric mucosal injury. RESULTS: H2O2 treatment for 6h significantly induced cell apoptosis in HFE145 cells. However, the destructive effects of H2O2 on HFE 145 cells could be reversed by the pretreatment with DOP. The increased ROS level induced by H2O2 for 4h was reduced after DOP pretreatment. The number of apoptotic cells in both early and late apoptosis stages decreased significantly and the nuclei morphology changes were improved with DOP pretreatment. Furthermore, DOP inhibited caspase 3 activation and PARP cleavage, downregulated Bax expression and upregulated Bcl2 expression in cell model. Further study revealed that pretreatment of DOP inhibited p -NF-κBp65/NF-κBp65 level, indicating DOP inhibited H2O2-mediated apoptosis via suppression of NF-κB activation. In addition, DOP treatment could ameliorate gastric mucosal injury and inhibit mucin loss induced by ethanol in animal model. DOP treatment also interfered with ethanol-induced apoptosis process by downregulating Bax/Bcl2 ratio in gastric mucosa. CONCLUSIONS: The present study was the first one to demonstrate the gastroprotective effect of DOP through inhibiting oxidative stress-induced apoptosis. This study provided a solid evidence for the potential use of DO as a therapy or health supplement for gastric mucosal diseases.

Evaluation of the combined use of metronomic zoledronic acid and Coriolus versicolor in intratibial breast cancer mouse model.

ETHNOPHARMACOLOGICAL RELEVANCE: Coriolus versicolor (CV) is a mushroom traditionally used for strengthening the immune system and nowadays used as immunomodulatory adjuvant in anticancer therapy. Breast cancer usually metastasizes to the skeleton, interrupts the normal bone remodeling process and causes osteolytic bone lesions. The aims of the present study were to evaluate its herb-drug interaction with metronomic zoledronate in preventing cancer propagation, metastasis and bone destruction. MATERIALS AND METHODS: Mice inoculated with human breast cancer cells tagged with a luciferase (MDA-MB-231-TXSA) in tibia were treated with CV aqueous extract, mZOL, or the combination of both for 4 weeks. Alteration of the luciferase signals in tibia, liver and lung were quantified using the IVIS imaging system. The skeletal response was evaluated using micro-computed tomography (micro-CT). In vitro experiments were carried out to confirm the in vivo findings. RESULTS: Results showed that combination of CV and mZOL diminished tumor growth without increasing the incidence of lung and liver metastasis in intratibial breast tumor model. The combination therapy also reserved the integrity of bones. In vitro studies demonstrated that combined use of CV and mZOL inhibited cancer cell proliferation and osteoclastogenesis. CONCLUSIONS: These findings suggested that combination treatment of CV and mZOL attenuated breast tumor propagation, protected against osteolytic bone lesion without significant metastases. This study provides scientific evidences on the beneficial outcome of using CV together with mZOL in the management of breast cancer and metastasis, which may lead to the development of CV as adjuvant health supplement for the control of breast cancer.

Whole extracts of Radix Achyranthis Bidentatae and Radix Cyathulae promote angiogenesis in human umbilical vein endothelial cells in vitro and in zebrafish in vivo.

Although Radix Achyranthis Bidentatae (RAB) and Radix Cyathulae (RC) are from two different medicinal plants, they are both used as 'Niu-Xi', a widely used traditional Chinese medicine that is believed to stimulate menstruation and affect bone injury. Angiogenesis is actively involved in treating these illnesses. The aim of the present study was to investigate whether the whole extracts of RAB and RC possess pro-angiogenic effects. In order to examine this idea whole extracts of RAB and RC were extracted with boiling water followed by ethanol, respectively. Results from the MTT, wound healing and tube formation assays in human umbilical vein endothelial cells (HUVECs) in vitro revealed that the whole extracts of RAB and RC did not increase cell proliferation or tube formation, but enhanced cell migration. Their angiogenic effects were also confirmed in zebrafish in vivo via increasing the sprout numbers in the sub-intestinal vessel. As determined by quantitative polymerase chain reaction, the whole extracts of RAB and RC both regulated the expression of cell migration-related genes in zebrafish. It is concluded that the whole extracts of RAB and RC induced angiogenesis in HUVECs in vitro and in zebrafish in vivo via increasing cell migration.