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Eucommia leaves are dry leaves of Eucommia ulmoides which have long been considered as a functional health food for the treatment of hypertension, hypercholesterolemia, fatty liver, and osteoporosis. With the recent development of Chinese medicine, Eucommia leaves are widely used for tonifying the kidneys and strengthening bone. However, the specific molecular mechanism of Eucommia leaves for strengthening bone remains largely unknown. Osteoblasts are the main functional cells of bone formation; thus, it is essential to study the effect of Eucommia leaves on osteoblasts to better understand their mechanism of action. In the present study, we prepared an aqueous extract of Eucommia leaves (ELAE) and determined its content by high-performance liquid chromatography (HPLC). The effects of ELAE on MC3T3-E1 cells were investigated by CCK-8 assay, alkaline phosphatase (ALP), and Alizarin red S staining assays, combined with RNA sequencing (RNA-seq) and qRT-PCR validation. We demonstrated that ELAE had a significant promoting effect on the proliferation of MC3T3-E1 cells and significantly enhanced extracellular matrix synthesis and mineralization, which were achieved by regulating various functional genes and related signaling pathways. ELAE significantly increased the expression level of genes promoting cell proliferation, such as Rpl10a, Adnp, Pex1, Inpp4a, Frat2, and Pcdhga1, and reduced the expression level of genes inhibiting cell proliferation, such as Npm1, Eif3e, Cbx3, Psmc6, Fgf7, Fxr1, Ddx3x, Mbnl1, and Cdc27. In addition, ELAE increased the expression level of gene markers in osteoblasts, such as Col5a2, Ubap2l, Dkk3, Foxm1, Col16a1, Col12a1, Usp7, Col4a6, Runx2, Sox4, and Bmp4. Taken together, our results suggest that ELAE could promote osteoblast proliferation, differentiation, and mineralization and prevent osteoblast apoptosis. These findings not only increase our understanding of ELAE on the regulation of bone development but also provide a possible strategy to further study the prevention and treatment of osteogenic related diseases by ELAE.

Background: Panax notoginseng (Burk.) F.H. Chen has long been used to stop bleeding for hundreds of years in China. At present, only dencichine and notoginsenoside Ft1 showed the hemostatic effect. Other ingredients from Panax notoginseng need to be further investigated. This study evaluates the hemostatic effect of 20(S)-panaxadiol (PD) and reveals its mechanism. Methods: We performed an in vivo study to measure PD on the hemostatic effect of mouse tail amputation and liver scratch models, and routine blood. Plasma coagulation parameters were measured using a blood analyzer. Platelet aggregation rate and adenosine triphosphate (ATP) release were analyzed by platelet aggregometer. Subsequently, degranulation marker P-selectin (CD62P), PAC-1 (activated GP IIb/IIIa receptor marker), the concentrations of cytosolic Ca2+ ([Ca2+]i) and cyclic adenosine monophosphate (cAMP) were also assessed. Results: PD shorted bleeding time on the mouse tail amputation and liver scratch models and mainly increased blood platelet count in the rats after subcutaneous injection of 4 h. Meanwhile, PD decreased APTT, increased FIB content, and directly induced platelet aggregation. In the absence of Ca2+, PD promoted the increase of [Ca2+]i and ATP, slightly increased CD62P expression and PAC-1 binding of platelets. After the addition of Ca2+, PD treatment markedly promoted platelet activation by releasing ATP level, increasing CD62P expression and PAC-1 binding, and decreasing cAMP level in platelets. Besides, PD increased phosphorylation of phosphoinositide 3-kinase (PI3K), protein kinase B (PKB or Akt), and glycogen synthase kinase 3β (GSK3β) in human platelets. Excitingly, PD-induced changes included platelet aggregation, a decrease of the cAMP content, and the increases of ATP, CD62P and PAC-1, which were significantly reversed by vorapaxar, which showed a similar function as thrombin. Conclusions: PD is an essential hemostatic ingredient in Panax notoginseng for promoting hematopoiesis and thrombopoiesis. PD induces platelet aggregation by affecting calcium signaling and activating PI3K/Akt/GSK3β signaling pathway, which could contribute to the new insight for the treatment of hemorrhagic disease.

Panax notoginseng (Burk.) F.H. Chen has long been used to stop bleeding for hundreds of years in China. At present, only dencichine, notoginsenoside Ft1, and 20(S)-protopanaxadiol (PPD) showed hemostatic effect. However, the molecular mechanism of PPD on the platelet aggragetion needs to be further investigated. The study aims to evaluate the hemostatic effect of PPD and reveal its interacting targets using a series of experiments. In this study, the bleeding time was measured in mouse tail amputation and liver scratch models to evaluate hemostatic effect of PPD. The routine blood and plasma coagulation parameters in NS, HC, and PPD (2, 4, and 8 mg/kg) groups were measured using a blood analyzer. Platelet aggregation rate and ATP release were analyzed by a platelet aggregometer. Subsequently, the degranulation marker CD62P and PAC-1, and the concentrations of cytosolic Ca2+ ([Ca2+]i), cAMP, cGMP, and PAC-1 expressions were also assessed. We found that PPD shorted the bleeding time on the mouse tail amputation and liver scratch models and mainly increased blood platelet count in the rats after subcutaneous injection for 4 h. Meanwhile, PPD decreased APTT, increased FIB content, and directly induced platelet aggregation in vitro. In the absence of Ca2+, PPD induced the increase of [Ca2+]i and slightly increased the levels of CD62P and PAC-1. After the addition of 1 mM Ca2+, PPD treatment markedly promoted platelet activation by promoting ATP level, releasing CD62P and increasing PAC-1 binding in washed platelets. Excitingly, PPD-induced changes including platelet aggregation, decreased cAMP content, and the increases of CD62P and PAC-1 were significantly reversed by protease-activated receptor 1 (PAR-1) antagonist, vorapaxar, which showed similar function as thrombin. In addition, molecular docking analysis and ELISA assay demonstrated that PPD had a promising docking score with -6.6 kcal/mol and increased PAR-1 expression in human platelets, which indicated that PAR-1 is involved in PPD-induced platelet aggregation by regulating calcium signaling. Collectively, our study could provide the new insights of PPD as an essential hemostatic ingredient in Panax notoginseng for the treatment of hemorrhagic disease.

Background Guzhi Zengsheng Zhitongwan (GZZSZTW) is an effective formula of traditional Chinese herbal medicine and has been widely applied in the treatment of joint diseases for many years. The aim of this study was to dissect the molecular targets and signaling pathways of Guzhi Zengsheng Zhitongwan based on the analysis of serum proteomics. Methods The Chinese herbs of GZZSZTW were immersed in 5 l distilled water and boiled with reflux extraction method. The extract was filtered, concentrated and freeze-dried. The chemical profile of GZZSZTW extract was determined by high-performance lipid chromatography (HPLC). The 7-week old Sprague-Dawley (SD) rats in GZZSZTW groups were received oral administration at doses of 0.8, 1.05, and 1.3 g/kg per day and the rats in blank group were fed with drinking water. Serum samples were collected from the jugular veins. Primary chondrocyte viability was evaluated by CCK-8 assay. A full spectrum of the molecular targets and signaling pathways of GZZSZTW were investigated by isobaric tags for relative and absolute quantitation (iTRAQ) analysis and a systematic bioinformatics analysis accompanied with parallel reaction monitoring (PRM) and siRNA validation. Results GZZSZTW regulated a series of functional proteins and signaling pathways responsible for cartilage development, growth and repair. Functional classification analysis indicated that these proteins were mainly involved in the process of cell surface dynamics. Pathway analysis mapped these proteins into several signalling pathways involved in chondrogenesis, chondrocyte proliferation and differentiation, and cartilage repair, including hippo signaling pathway, cGMP-PKG signaling pathway, cell cycle and calcium signaling pathway. Protein–protein interaction analysis and siRNA knockdown assay identified an interaction network consisting of TGFB1, RHO GTPases, ILK, FLNA, LYN, DHX15, PKM, RAB15, RAB1B and GIPC1. Conclusions Our results suggest that the effects of GZZSZTW on treating joint diseases might be achieved through the TGFB1/RHO interaction network coupled with other proteins and signaling pathways responsible for cartilage development, growth and repair. Therefore, the present study has greatly expanded our knowledge and provided scientific support for the underlying therapeutic mechanisms of GZZSZTW on treating joint diseases. It also provided possible alternative strategies for the prevention and treatment for joint diseases by using traditional Chinese herbal formulas.

Background The collagen hydrolysates as a cosmetic material have already been wide application. At present, few studies concern with transdermal behavior of collagen hydrolysates in vitro. Objective Deer sinew contains rich collagen with a content of 82.12%. Thus, this article mainly studies the transdermal effect of collagen hydrolysates of deer sinew (DSCH) on mouse skin, ex vitro, and to explore skincare protection of percutaneous proteins. Methods Collagen hydrolysates of deer sinew were extracted by 0.2% HCl and a two-step enzymatic method of pepsin-trypsin. The content of 17 amino acids of DSCH was detected by precolumn derivatization RP-HPLC. Using Franz diffusion cell systems studied the transdermal effect of DSCH and then examined the percutaneous rate and molecular weight distribution of percutaneous proteins (PP). Further, we studied the bioactivity of PP in vitro, such as the total antioxidant capacity and collagen secretion in NIH/3T3 cells. Results About 8.0% DSCH could penetrate skin of mouse, the molecular weight of PP mainly distributed in 5 ~ 13 kDa, accounted for 91.55%. Compared with the antioxidant activity of DSCH, PP had obvious antioxidant activity of scavenging radical cation. Meanwhile, PP promoted cell proliferation and collagen I secretion in fibroblast cells; however, level of type III collagen has no change. Conclusion Collagen hydrolysates of deer sinew may be used as cosmetic material to protect the skin from oxidative stress, to prevent premature skin aging.