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OBJECTIVE@#To systematically evaluate the protective effects of Humulus lupulus L. extract (HLE) on osteoporosis mice.@*METHODS@#In vivo experiment, a total of 35 12-week-old female ICR mice were equally divided into 5 groups: the sham control group (sham); the ovariectomy with vehicle group (OVX); the OVX with estradiol valerate [EV, 0.2 mg/(kg•d)] the OVX with low- or high-dose HLE groups [HLE, 1 g/(kg•d) and 3 g/(kg•d)], 7 in each group. Treatment began 1 week after the ovariectomized surgery and lasted for 12 weeks. Bone mass and trabecular bone mircoarchitecture were evaluated by micro computed tomography, and bone turnover markers in serum were evaluated using enzyme-linked immunosorbent assay (ELISA) kits. In vitro experiment, osteoblasts and osteoclasts were treated with HLE at doses of 0, 4, 20 and 100 µg/mL. Biomarkers for bone formation in osteoblasts and bone resorption in osteoclasts were analyzed.@*RESULTS@#Compared with the OVX group, HLE exerted bone protective effects by the increase of estradiol (P<0.05), the improvement of cancellous bone structure, bone mineral density (P<0.01) and the reduction of serum alkaline phosphatase (ALP), tartrate resistant acid phosphatase (TRAP), bone gla-protein, c-terminal telopeptides of type I collagen (CTX-I) and deoxypyridinoline levels (P<0.01 for all). In vitro experiment, compared with the control group, HLE at 20 µg/mL promoted the cell proliferation (P<0.01), and increased the expression of bone morphogenetic protein-2 and osteopontin levels in osteoblasts (both P<0.05). HLE at 100 µg/mL increased the osteoblastic ALP activities, and HLE at all dose enhanced the extracellular matrix mineralization (both P<0.01). Furthermore, compared with the control group, HLE at 20 µg/mL and 100 µg/mL inhibited osteoclastic TRAP activity (P<0.01), and reduced the expression of matrix metalloproteinase-9 and cathepsin K (both P<0.05).@*CONCLUSION@#HLE may protect against bone loss, and have potentials in the treatment of osteoporosis.
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Objective To explore the effects of lupulone (LUP) and humulone (HUM) in Humulus lupulus L. on osteoblasts and osteoclasts of rats. Methods Osteoblasts and osteoclasts isolated from 24-h-old Wistar rats were studied and divided into control group, LUP-treated low (10－15 mol/L)-, medium (10－14mol/L)-and high (10－13 mol/L)-dose groups, and HUM-treated low (10－15 mol/L)-, medium (10－14 mol/L)-and high (10－13mol/L)-dose groups. After drug treatment, the proliferation, differentiation and bone mineralization of osteoblasts were determined by MTT assay, alkaline phosphatase (ALP) activity assay and alizarin red staining, respectively. Osteoclasts were counted and tartrate-resistant acid phosphatase (TRAP) activity was measured to evaluate the effects of LUP and HUM on the activity of osteoclasts. Osteocalcin (OCN) levels were measured by kit assay, and the expression levels of bone formation related proteins osteopontin (OPN), bone sialoprotein (BSP), bone morphogenetic protein 2 (BMP-2), bone resorption related proteins cathepsin K (CK) and matrix metalloproteinase 9 (MMP-9) were measured by Western blotting analysis to evaluate the effects of LUP and HUM on bone metabolism. Results At the osteoblast level, LUP at dosages of 10－15 and 10－14 mol/L could significantly promote the cell proliferation (P0.05). LUP at dosages of 10－14 and 10－13 mol/L could significantly improve ALP activity and bone mineralization (P0.05, P0.01). LUP at dosage of 10－13 mol/L could significantly induce the expression of OCN (P0.01). Furthermore, LUP at dosages of 10－14 and 10－13 mol/L could significantly increase the expression of BSP and BMP-2 (P0.05). HUM at dosages of 10－15-10－13 mol/L could also significantly promote the osteoblastic proliferation, ALP activity and bone mineralization (P0.01), and could significantly increase the expression of OCN and OPN (P0.05, P0.01). Additionally, HUM at dosages of 10－14 and 10－13 mol/L could significantly increase the expression of BSP and BMP-2 (P0.05). At the osteoclast level, both LUP and HUM at dosages of 10－15-10－13 mol/L could significantly reduce the number of osteoclasts (P0.01) and could significantly inhibit the expression of CK (P0.05, P0.01). HUM at dosages of 10－15-10－13 mol/L could also significantly inhibit the expression of MMP-9 (P0.05, P0.01). Conclusion This study preliminarily clarifies that LUP and HUM can prevent bone loss by promoting bone formation and inhibiting bone resorption, which provides a new reference for the development of osteoporosis drugs.
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Tissue culture seedlings of Bletilla striata were treated with MeJA, SA and two kinds of endophytic fungi in order to study the effects of those treatments on the physiology and total phenols content. The method of tissue culture was used to culture seeds into seedlings, and then different treatments were applied on them to observe and measure the changes of physiology and total phenols content. We find that the growth of seedlings treated with SA was poor, which treated with 40 μmol•L⁻¹ MeJA, 50 mL•L⁻¹ Hypocrea koningii and 10 mL•L⁻¹ Trichoderma koningiopsis showed better. The activity of SOD, POD and CAT was at a high level under SA treatment of each concentration. The activity of SOD and POD increased as the rise of MeJA concentration, while CAT was highest at 80 μmol•L⁻¹. The activity of SOD and POD increased with the increasing of the concentration of H. koningii treatment, while CAT reached the highest at 1 mL•L⁻¹. The activity of SOD, POD and CAT increased first and then declined with the concentration of T. koningiopsis increasing, and the highest activity was at 10 mL•L⁻¹. The contents of MDA, soluble protein and proline were increased more or less under the four treatments. The content of polysaccharide was at a high level under 60 μmol•L⁻¹ of MeJA. The total phenols content was at a high level under 40 μmol•L⁻¹ of MeJA, 60 μmol•L⁻¹ of SA, 1 mL•L⁻¹ of H. koningii and 10 mL•L⁻¹ of T. koningiopsis. The results indicated that the addition of exogenous MeJA, SA and endophytic fungi under certain concentrations could improve the resistance of B. striata and increase the content of total phenols at some degree and the trearment of MeJA, H. koningii and T. koningiopsis could promote the growth of seedlings under certain concentrations.