Screening Research On Anti-osteoporosis Chinese Medicine Based On Cellular Level

Osteoporosis is a leading cause imperiling human health.Seeking effective anti-osteoporosis drugs has been an important issue in medicine.With the modernization of Traditional Chinese Medicine,treatment of Traditional Chinese medicine on osteoporosis is expected to go deep into the cellular and molecular level.In order to achieve the effect of treating the symptoms and take full advantage of Chinese medicine to prevent osteoporosis,it is expected to screened out efficient,stable,low toxicity of traditional Chinese medicine monomer or compound component based on cellular and molecular level.The purpose of this study is to screen traditional Chinese medicine which can inhibit bone resorption and promote bone formation based on cellular level.The first part of the study,we cultured osteoclasts and osteoblasts in vitro.We cultured osteoclasts which obtained from 1α,25-（OH）₂VitD₃ inducing rabbit bone marrow cells and identified them with morphological observation,HE staining,tartrate-resistant acid phosphatase（TRAP）staining and lacunae toluidine blue staining.We cultured MC3T3-E1Subclone 14 cells and identified them with alkaline phosphatase staining and alizarin red staining.The Second part of the study,we selected more than 20 kinds of Chinese medicine with potential anti-osteoporosis pharmacological effect from more than 30 kinds of traditional Chinese medicine prescriptions which have the functions of bu-gan-shen,qiang-jin-gu,huo-xue-hua-yu,he-wei-jian-pi and so on,according to traditional Chinese medicine pharmacological prevention of osteoporosis.Then we mademore than 20 kinds of of extracts of the medicines of 75%ethanol and water.The third part of the study,We adapted three indicators-TRAP staining,TRAP enzyme activity and the number and area of bone resorption pits to detect the effect of Traditional Chinese Medicine which has three doses-low,middle,high（2 mg/L,20mg/L,200 mg/L）of each alcohol extract and water extract on osteoclasts differentiation and bone resorption ability.The results showed that:alcohol extract and water extract of Phellodendri chinensis cortex,Sargentodoxae caulis,Siegesbeckiae herba,Visci herba,Cynomorii herba,Angelicae sinensis radix,Euodiae fructus,Allii tuberosi semtn,Cibotii rhizoma,Clematidis radix et rhizoma,Dipsaci radix,can significantly inhibit the osteoclast activity in a dose-dependent manner.The water extract of Ecliptae herba,Carthami flos,Cistanches herba,Astragali radix,Ligustri lucidi fructus,have significant inhibition on osteoclast activity in a dose-dependent manner.The alcohol extract of Chaenomelis fructus and the middle-dose and high-dose alcohol extract of Ligustri lucidi fructus,can significantly inhibit the osteoclast activity in a dose-dependent manner.The alcohol extract and water extract of Polygoni multiflori radix praeparata have significant inhibition on osteoclast activity.Angelicae pubescentis radix and Achyranthis bidentatae radix basically have no inhibition on osteoclast activity.The fourth part of the study,we detected the effect of Traditional Chinese Medicine on proliferation and differentiation of osteoblasts.Firstly we detected the effect of Traditional Chinese Medicine onproliferation of osteoblasts with MTT.The results showed that:the high-dose of alcohol extract and water extract of Phellodendri chinensis cortex,Sargentodoxae caulis,Ecliptae herba,Carthami flos,Chaenomelis fructus,Dipsaci radix,Ligustri lucidi fructus,Achyranthis bidentatae radix,had significant inhibitory effect on the proliferation of osteoblasts,and the low-dose and middle-dose of them could significantly promote the proliferation of osteoblasts.All of Visci herbai,Cistanches herba,Polygoni multiflori radix praeparata and Euodiae fructus could promote the proliferation of osteoblasts.The high-dose of alcohol extract and water extract of Visci herba had significant inhibitory effect on the proliferation of osteoblasts.All doses of alcohol extract and high-dose of water extract of Cistanches herba could significantly promote the proliferation of osteoblasts.The low-dose and middle-dose of alcohol extract and water extract of Polygoni multiflori radix praeparata had significant promoted effect on the proliferation of osteoblasts.The high-dose of alcohol extract and all doses of water extract of Euodiae fructus could significantly promote the proliferation of osteoblasts.The alcohol extract of Clematidis radix et rhizoma could promote the proliferation of osteoblasts but non-significantly,water extract of which had inhibitory effect on the proliferation of osteoblasts.Angelicae sinensis Radix could promote the proliferation of osteoblasts except thehigh-dose of alcohol extract,the middle-dose of alcohol extract and high-dose of water extract of which had significant promoted effect on the proliferation of osteoblasts.The high-doses of alcohol extract and high-dose of water extract of Cibotii rhizoma,Astragali radix and Angelicae sinensis radix and middle-dose of alcohol extract of Astragali radix and Angelicae sinensis radix had inhibitory effect on the proliferation of osteoblasts,and other doses of Cibotii rhizoma,Astragali radix and Angelicae sinensis radix could promote the proliferation of osteoblasts,and The low-dose of alcohol extract and water extract of Cibotii rhizoma and Angelicae sinensis radix had significantly promoted effect.Siegesbeckiae herba,Cynomorii herba and Allii tuberosi semtn had inhibitory effect on the proliferation of osteoblasts.The we detected the effect of Traditional Chinese Medicine ondifferentiation of osteoblasts with ALP activity.The results showed that:All doses of alcohol extract and water extract of Phellodendri chinensis cortex,Sargentodoxae caulis,Cibotii rhizoma,Carthami flos,Astragali radix,Dipsaci radix,Achyranthis bidentatae radix,could significantly promote differentiation of osteoblasts,the middle-dose of which had the best effect.All doses of alcohol extract and water extract ofVisci herba,Euodiae fructus,Cynomorii herba,Ecliptae herba and Chaenomelis fructus could significantly promote differentiation of osteoblast in a dose-dependent manner.All doses of alcohol extract and water extract of Cibotii rhizoma and Cistanches herba could significantly promote differentiation of osteoblasts,the middle-dose alcohol extract of which had the best effect,the water extract of which had the effect a dose-dependent manner.All doses of alcohol extract and water extract of Ligustri lucidi fructus could significantly promote differentiation of osteoblasts,the alcohol extract of which had the effect a dose-dependent manner,the middle-dose water extract of which had the best effect.The low-dose and middle-dose of alcohol extract and water extract of Siegesbeckiae herba and Polygoni multiflori radix praeparata could significantly promote differentiation of osteoblasts,the middle-dose of which had the best effect.The low-dose and middle-dose of alcohol extract and middle-dose of water extract of Angelicae pubescentis radix could significantly promote differentiation of osteoblasts,the middle-dose of which had the best effect.The alcohol extract and water extract of Allii tuberosi semtn and Clematidis radix et rhizoma could promote differentiation of osteoblasts but non-significantly.In summary,through osteoclast model and osteoblast model in vitro,we found that in this study,Phellodendri chinensis cortex,Sargentodoxae caulis,Ecliptae herba,Carthami flos,Chaenomelis fructus,Dipsaci radix,Ligustri lucidi fructus,Visci herba,Cistanches herba,Polygoni multiflori radix praeparata,Euodiae fructus,Angelicae sinensis radix,Astragali radix,Cibotii rhizoma and Cynomorii herba could both inhibit bone resorption and promote bone formation.Siegesbeckiae herba,Allii tuberosi semtn and Clematidis radix et rhizoma could inhibit bone resorption.Achyranthis bidentatae radix and Angelicae pubescentis radix could promote bone formation.