| Osteoporosis,as a systemic metabolic disease,is common in the elderly due to its onset characteristics.It has the characteristics of bone loss,increased bone fragility,and damaged bone microstructure.As my country enters a deeply aging society,the elderly population Increasingly,osteoporosis has become a major public disease that threatens the life safety of the elderly.According to statistics,the low bone mass rate of people over 50 years old in my country has reached 46.4%,and the prevalence of osteoporosis of people over 65 years old has reached 32.0%.The fatality rate and disability rate of fractures caused by osteoporosis are also increasing year by year,and the attention and research on osteoporosis are urgent.At present,western medicines for the treatment of osteoporosis are mainly divided into three categories: inhibiting bone resorption,promoting bone formation and biphasic promoting.However,these drugs are accompanied by different degrees of side effects,while treating the disease,it also brings pain to the patient.With the country’s gradual emphasis on traditional Chinese medicine and the continuous efforts of traditional Chinese medicine practitioners,the motherland’s medicine has made great strides and found its own advantages.Compared with Western medicine,traditional Chinese medicine has a significant effect on osteoporosis.Traditional Chinese medicine in the treatment of osteoporosis has the characteristics of obvious effect and small side effects,and it can be treated by stimulating the patient’s own body potential.The combination of Chinese and Western medicine has been used for a long time and is becoming more and more common.Especially during the COVID-19 epidemic,Academician Zhang Boli used the combination of Chinese and Western medicine to treat the COVID-19 virus and achieved very good results,which further shows that the combination of Chinese and Western medicine has unexpected therapeutic effects.Osteoporosis is classified into the category of ’bone withered’ and ’bone atrophy’ in traditional Chinese medicine.Traditional Chinese medicine believes that the connection between the kidney and the bone is very close.According to the "Essence of the Classic of Medicine" : "The kidney stores the essence,the essence produces the marrow,and the marrow produces the bone.Therefore,the bone is where the kidney is combined.The marrow is produced by the kidney essence,the essence is sufficient,the marrow is full,and the marrow is full,the bone is strong." And when the kidney essence is deficient,it will lead to a decrease in bone mass,which in turn leads to osteoporosis.Cistanche and Epimedium have been used as kidney-tonifying drugs in traditional Chinese medicine to treat kidney-yang deficiency.Chondroitin sulfate and glucosamine are used together to treat osteoarthritis and can also increase bone density.This topic uses animal experiments,network pharmacology,western blotting to explore the effect and mechanism of the combination of Cistanche,Epimedium,chondroitin sulfate and glucosamine in the treatment of osteoporosis,and to provide a basis for further new drug research and development.The experimental content of this subject is divided into the following parts:1.In experiment 1,the method of injecting D-galactose for 8 consecutive weeks was selected to establish a rat model of osteoporosis,and the experimental rats were divided into blank group,model group,positive drug group,Cistanche and Epimedium(traditional Chinese medicine)group,Chondroitin Sulfate and Glucosamine(Western Medicine)Group,Cistanche,Epimedium,Chondroitin Sulfate,Glucosamine(combination of traditional Chinese and western medicine)group,8 animals in each group were given the corresponding drug-positive drug alendronate sodium(0.9 mg)/kg),Cistanche extract(1g/kg),icariin(100mg/kg),Chondroitin Sulfate(102mg/kg),Glucosamine(128mg/kg),the control group and the model group were given an equal volume of 0.5% normal saline,8 in each group,intraperitoneal injection of D-galactose in the morning and the corresponding drugs in the afternoon for 8 weeks.After 8 weeks,blood was collected from the abdominal aorta of the experimental rats,and serum biochemical indexes were detected(alkaline phosphatase ALP,osteocalcin BGP,type I procollagen N-terminal propeptide PINP),and the right femur was taken for bone mineral density analysis.The right tibia was taken for histopathological evaluation.The results of serum biochemical indicators showed that after 8 weeks of administration,compared with the blank group,the ALP in the model group was significantly increased,with a statistical difference(P<0.05).Compared with the model group,the ALP in each administration group was significantly decreased,there was a statistical difference(P<0.05);compared with the blank group,the BGP of the model group was significantly decreased,but there was no statistical difference(P>0.05).Compared with the model group,the BGP of the positive drug group and the combination group of traditional Chinese and western medicine were increased,but there was no statistical difference(P>0.05).BGP in both the traditional Chinese medicine group and the western medicine group was significantly increased,with a statistical difference(P<0.05).Compared with the blank group,the PINP in the model group was significantly increased,with statistical significance.Compared with the model group,the PINP of the positive drug group decreased slightly,but there was no statistical difference(P>0.05).The PINP in the traditional Chinese medicine group,the western medicine group and the combined Chinese and western medicine group were significantly decreased,and there was a statistical difference(P<0.01).The results of bone mineral density showed that compared with the blank group,the bone mineral density of the model group was significantly decreased,and there was a statistical difference(P<0.01).Compared with the model group,the bone mineral density of combined Chinese and western medicine group was significantly increased,and there was a statistical difference(P<0.05).The histopathological results showed that compared with the blank group,the number of trabecular bone in the model group was significantly reduced,the trabecular bone gap increased,and the number of trabecular bone was greatly reduced;The number of specific trabecular bone increased to varying degrees,the morphological structure was relatively complete,and the gap was relatively reduced.2.In Experiment 2,the mechanism of the combination of Cistanche,Epimedium,Chondroitin Sulfate and Glucosamine in the treatment of osteoporosis was predicted by the method of network pharmacology.The database was standardized,and the disease target information was collected in the Gene Cards,Drug Bank and OMIM databases with’osteoporosis’ as the keyword,and the disease-component-target network diagram was drawn using Cytoscape software,and the drug targets and disease targets were obtained by Venny related software.The intersection targets were imported into the STRING database to obtain the PPI network map,and then the PPI network map was imported into the Cytoscape software for further analysis to obtain the core target protein.The Metascape database was used for GO and KEGG enrichment analysis,and the R language software was used for visualization processing.Then use Auto Dock and Py MOL software to preprocess the screened active ingredients and target proteins,and conduct molecular docking to verify the prediction results of network pharmacology.The interaction network of chondroitin and glucosamine in the treatment of osteoporosis was subjected to cluster analysis,and the Score≥4 category and RANK1 category were obtained.The MCL algorithm in Cluster in the STRING database was used to cluster the target proteins,and the cluster1 category was obtained,using the Metascape database to perform KEGG enrichment analysis on Score≥4categories,RANK1 categories and cluster1 categories to obtain the possible signaling pathways of Cistanche,Epimedium,Chondroitin Sulfate,and Glucosamine in the treatment of osteoporosis.The results showed that a total of 8 active ingredients of Cistanche,19 active ingredients of Epimedium,231 drug targets,1123 disease targets,and 135 intersection targets were obtained.GO analysis showed drug treatment of osteoporosis in biological process(BP),it mainly treated osteoporosis by reducing the response to lipopolysaccharide and inflammation,and promoting the proliferation of epithelial cells;in the cellular component(CC),the drug mainly acted on the cell membrane,nucleus,cytoplasm and endoplasmic reticulum,etc.;in molecular function(MF),drugs had important effects on DNA transcription,cytokines,phosphorylation,etc.KEGG analysis showed that the pathways involved in drug treatment of osteoporosis mainly include PI3K-Akt signaling pathway,apoptosis,TNF signaling pathway,etc.It showed that the active ingredient of the drug achieves the purpose of treating osteoporosis through these signaling pathways.In the molecular docking experiment,it was found that the binding energy of most of the screened active ingredients to the target protein was less than-5.0 kcal/mol,indicating that the screened active ingredients has good binding to the target protein,and the Score ≥ 4 categories based on the MCODE algorithm and the MCL algorithm KEGG enrichment analysis of RANK1,RANK1 and cluster1 categories indicated that PI3K/AKT signaling pathway and apoptosis may play a key role in the treatment of osteoporosis with Cistanche,Epimedium,Chondroitin Sulfate and Glucosamine.3.Experiment 3 verified the top-ranked PI3K/AKT and apoptosis signaling pathways in Experiment 2.The Western Blot method was used in the experiment.The protein in the femur sample was extracted and the concentration was determined.The gel plate was washed and the gel was prepared.The previously extracted protein was denatured and then loaded.Cut the gel for membrane transfer,block with 5% nonfat milk powder for 3 hours,incubate the primary antibody overnight,incubate the secondary antibody for 1.5 hours the next day,and finally use the imaging system to develop and analyze with Image J software.The results showed that the protein expressions of PI3 K,p-AKT and BCL2 in the model group were significantly decreased,and there were significant differences compared with the blank group.The effect of the use group was the most obvious,and there was a significant difference compared with the model group.The results showed that combined using of Cistanche,Epimedium,chondroitin sulfate,and glucosamine may treat osteoporosis by activating the PI3K/AKT/BCL2 signaling pathway. |
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