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To determine the absorption rate and anti-osteoporosis efficacy of calcium zinc vitamin D vitamin K oral solution (YQJ) and its components in Caco-2 cells and zebrafish, and explore its potential mechanism based on transcriptome sequencing.
We compared the calcium-ion absorption rates of YQJ and its components in Caco-2 cells, to simulate gastrointestinal absorption. An osteoporosis model was induced using dexamethasone. Bone density was detected by Alizarin Red staining and dual validation of drug efficacy was analyzed using transgenic (ola. sp7: nlsGFP) osteoblast fluorescence. Zebrafish were divided into YQJ 250 μg/mL, MC, and NC groups (3 biological replicates each). Transcriptome sequencing was carried out and differentially expressed genes at the intersection of the three groups were screened using | log2 fold-change| ≥ 2 and Q value ≤ 0.001. Differential genes underwent functional annotation and enrichment analyses to screen out the expression pathway of YQJ anti-osteoporosis genes, followed by quantitative polymerase chain reaction validation.
The absolute calcium-absorption rate in YQJ-treated Caco-2 cells was 42.6%, which was 1.70 fold higher than that of the calcium citrate-alone group, 1.59, 1.58, and 1.56 fold higher than the calcium citrate + vitamin (V) D3, calcium citrate + VK2, and calcium citrate + (casein phosphopeptides) CPP groups, respectively, and 1.33 fold higher than the calcium citrate + zinc gluconate + D3 group, indicating a significant synergistic effect of YQJ on promoting calcium absorption. YQJ significantly restored zebrafish skull density and increased osteogenic fluorescence intensity, and 125 μg/mL YQJ increased the anti-osteoporosis effect by 24% ~ 25% compared with calcium citrate alone. Sequencing identified 335 and 231 differentially expressed genes in MC group/NC group and YQJ group/MC group, respectively, with 58 overlapping genes at the intersection of the three groups. Functional enrichment of overlapping genes revealed that YQJ's anti-osteoporosis effect involved four pathways and 11 key genes. Using qPCR verification, YQJ significantly restored the mRNA expression levels of c3a. 1, cp, coro1a, cldni, and bmpr1aa.
The in vitro calcium-absorption rate and anti-osteoporosis efficacy of YQJ both demonstrated synergistic effects, with its anti-osteoporosis effect specifically increasing skull density and osteoblast signaling intensity. Transcriptome sequencing revealed that YQJ primarily functions by modulating the complement and coagulation cascade, ferroptosis, phagosome, oxidative stress response signaling pathways, and other functional proteins, involving the regulation of key genes such as c3a. 1, c3a. 2, c3a. 6, c9, c5, cfb, coro1a, cp, mpx, cldni, and bmpr1aa.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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