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. 2024 Jul 19;16(14):2333.
doi: 10.3390/nu16142333.

Astragalus Extract Mixture HT042 Alleviates Dexamethasone-Induced Bone Growth Retardation in Rat Metatarsal Bones

Chae Yun Baek  1 , JunI Lee  1 , Donghun Lee  1 , Hocheol Kim  2
Affiliations

Astragalus Extract Mixture HT042 Alleviates Dexamethasone-Induced Bone Growth Retardation in Rat Metatarsal Bones

Chae Yun Baek et al. Nutrients. .

Abstract

The most widely used synthetic glucocorticoid, dexamethasone (DEX), causes stunted growth in children when used excessively or for long periods of time; however, there are still plenty of pediatric patients require long-term treatment with DEX. As an alternative, growth hormone is used in combination, but it has side effects, a high cost, and psychological factors, and it is not satisfactory in terms of effectiveness. It is necessary to develop a safe and affordable treatment that can replace it. The Korean Food and Drug Administration approved HT042, a standardized functional food ingredient, with the claim that it can help height growth of children. In this study, it was found that HT042 activated the Indian hedgehog/parathyroid hormone-related protein signaling pathway and enhanced the number of growth hormone receptors and insulin-like growth factor-1 receptors on the growth plate surface, which were reduced by DEX treatment, and restored growth retardation. In metatarsal bone and primary chondrocyte models, it was found that HT042 can promote the length of growth plate and recover DEX-induced growth retardation. It was also found that HT042 promotes cell proliferation using bromodeoxyuridine and terminal deoxynucleotidyl transferase dUTP nick end labeling assays; moreover, we verified increased expression of GHR/IGF-1R and Ihh/PTHrP pathway activity using qRT-PCR, western blotting, and siRNA analyses to verify its direct action on the growth plate. The anti-apoptotic effect of HT042 was identified by regulating the expression of apoptotic factors such as caspase-3, Bcl2, Bclx, and Bax. These results were identified using both ex vivo and in vitro models. Our study verified that co-administration of HT042 could recover the DEX induced growth retardation.

Keywords: HT042; apoptosis; dexamethasone; growth retardation; hypertrophy; proliferation.

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Conflict of interest statement

The authors confirm that there are no known conflicts of interest regarding the publication of this publication. In addition, there is no significant financial support affecting the outcome of this study.

Figures

Figure 1
Figure 1
The 3D high-performance liquid chromatography chromatograms (HPLC) of formononetin, eleutheroside E, and shanzhiside methyl ester (A) in HT042. The HPLC shows formononetin (B), eleutheroside E (C) and shanzhiside methyl ester (D) as the compounds of Astragalus membranaceus, Eleutherococcus senticosus, and Phlomis umbrosa in HT042, respectively.
Figure 2
Figure 2
Evaluation of MTB length during 0 to 21 days. (A) Representative picture; (B–E) analysis of MTB length. Cells were treated with HT042 (30 µg/mL), DEX (100 ng/mL), and HT042+DEX (30 µg/mL + 100 ng/mL). ### p < 0.001 vs. NT by one-way ANOVA and Dunnett’s test; ** p < 0.01 vs. DEX; *** p < 0.001 vs. DEX by Student’s t-test. NT: Non-treated; MTB: metatarsal bone.
Figure 3
Figure 3
Photography of CV- and TB-stained growth plate of MTB. Representative photographs are shown from each treatment of MTB stained with (A) CV and (B) TB. Analysis of HZ and PZ length in each group (C,D). Cells were treated with HT042 (30 µg/mL), DEX (100 ng/mL), and HT042+DEX (30 µg/mL + 100 ng/mL). ### p < 0.001 vs. NT by one-way ANOVA and Dunnett’s test; and *** p < 0.001 vs. DEX by Student’s t-test. CV: Cresyl violet; HZ: hypertrophic zone; PZ: proliferative zone; TB: toluidine blue; NT: non-treated.
Figure 4
Figure 4
Immunohistochemical localization of Ihh in the growth plate. (A) representative photo of HZ of MTB. (B) analysis for HZ height of growth plate. Cells were treated with HT042 (30 µg/mL), DEX (100 ng/mL), and HT042+DEX (30 µg/mL + 100 ng/mL). ### p < 0.001 vs. NT by one-way ANOVA and Dunnett’s test; and *** p < 0.001 vs. DEX by Student’s t-test. IHC: immunohistochemistry; Ihh: Indian hedgehog; MTB: metatarsal bone, NT: non-treated; HZ: hypertrophic zone.
Figure 5
Figure 5
Effect of HT042 on anti-apoptosis in MTB. Apoptotic cells in the MTB were evaluated by the TUNEL assay. Representative photomicrograph indicated apoptosis in the MTB culture. Cells were treated with HT042 (30 µg/mL), DEX (100 ng/mL), and HT042+DEX (30 µg/mL + 100 ng/mL). MTB: Metatarsal bone, PZ: proliferative zone, TUNEL: terminal deoxynucleotidyltransferase-mediated dUTP end labeling.
Figure 6
Figure 6
Effect of HT042 on cell proliferation in the MTB. (A) Chondrocytes were injected with BrdU in NT, HT042, DEX, and HT042+DEX. (B) Checking the cell proliferative effect using DAPI staining. (C) Analyzed for BrdU positive cell by % of NT. Cells were treated with HT042 (30 µg/mL), DEX (100 ng/mL), and HT042+DEX (30 µg/mL + 100 ng/mL). ### p < 0.001 vs. NT by one-way ANOVA and Dunnett’s test; *** p < 0.001 vs. DEX by Student’s t-test. BrdU: Bromodeoxyuridine; MTB: metatarsal bone; NT: non-treated.
Figure 7
Figure 7
mRNA expression analysis in the chondrocyte. (A–H) mRNA expression of GHR, IGF-1R, PTHrP, Ihh, caspase-3, BCl-X, BAX, and BCl-2 as determined by qRT-PCR. Cells were treated with HT042 (30 µg/mL), DEX (100 ng/mL), and HT042+DEX (30 µg/mL + 100 ng/mL). ## p < 0.01 vs. NT, ### p < 0.001 vs. NT by one-way ANOVA and Dunnett’s test; * p < 0.05 vs. DEX and *** p < 0.001 vs. DEX by Student’s t-test. GHR: GH receptor; IGF-1R: insulin-like growth factor-1 receptor; NT: non-treated.
Figure 7
Figure 7
mRNA expression analysis in the chondrocyte. (A–H) mRNA expression of GHR, IGF-1R, PTHrP, Ihh, caspase-3, BCl-X, BAX, and BCl-2 as determined by qRT-PCR. Cells were treated with HT042 (30 µg/mL), DEX (100 ng/mL), and HT042+DEX (30 µg/mL + 100 ng/mL). ## p < 0.01 vs. NT, ### p < 0.001 vs. NT by one-way ANOVA and Dunnett’s test; * p < 0.05 vs. DEX and *** p < 0.001 vs. DEX by Student’s t-test. GHR: GH receptor; IGF-1R: insulin-like growth factor-1 receptor; NT: non-treated.
Figure 8
Figure 8
Protein expression of (A–I) GHR, IGF-1R, Ihh, PTHrP, caspase-3, BAX, BCl-X, BCl-2, and GAPDH in primary chondrocytes. Cells were treated with HT042 (30 µg/mL), DEX (100 ng/mL), and HT042+DEX (30 µg/mL + 100 ng/mL) for 72 h. ## p < 0.01 vs. NT, ### p < 0.001 vs. NT by one-way ANOVA and Dunnett’s test; *** p < 0.001 vs. DEX by Student’s t-test. GHR: GH receptor; IGF-1R: insulin-like growth factor-1 receptor; NT: non-treated.
Figure 9
Figure 9
Effects of HT042 on Ihh/PTHrP signaling on Ihh target gene function. A representative interacted cell is indicated by the arrow. Cells were treated with HT042 (30 µg/mL), DEX (100 ng/mL), and HT042+DEX (30 µg/mL + 100 ng/mL). Ihh: Indian hedgehog; NT: non-treated.
Figure 10
Figure 10
Effects of HT042 on chondrocyte apoptosis. A representative apoptotic cell is indicated by the arrow. Cells were treated with HT042 (30 µg/mL), DEX (100 ng/mL), and HT042+DEX (30 µg/mL + 100 ng/mL). NT: Non-treated; DEX: dexamethasone.
Figure 11
Figure 11
The effect of HT042 and DEX on chondrocyte proliferation and Ihh expression in cultured growth plate chondrocytes using transfected GHR siRNA. Chondrocytes were transfected with siRNA targeted for GHR and cultured in DMEM containing 10% FBS for 72 h after transfection. Both mRNA and protein expression were analyzed by real-time PCR (A) and Western blotting (B), respectively. *** p < 0.001 vs. NT by one-way ANOVA, Dunnett’s test. Cells were treated with HT042 (30 µg/mL), DEX (100 ng/mL), and HT042+DEX (30 µg/mL + 100 ng/mL). Ihh mRNA expression was detected in cultured chondrocytes by real-time PCR. Results are expressed as the as-fold change compared with untreated control chondrocytes (C). GHR: GH receptor; IGF-1R: insulin-like growth factor-1 receptor; NT: non-treated.
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References

    1. Tyler A., Bryan M.A., Zhou C., Mangione-Smith R., Williams D., Johnson D.P., Kenyon C.C., Rasooly I., Neubauer H.C., Wilson K.M. Variation in Dexamethasone Dosing and Use Outcomes for Inpatient Croup. Hosp. Pediatr. 2022;12:22–29. doi: 10.1542/hpeds.2021-005854. - DOI - PMC - PubMed
    1. Allen D.B., Julius J.R., Breen T.J. Treatment of Glucocorticoid-Induced Growth Suppression with Growth Hormone. The J. Clin. Endocrinol. Metab. 1998;83:2824–2829. doi: 10.1210/jcem.83.8.5036. - DOI - PubMed
    1. Wong S.C., Dobie R., Altowati M.A., Werther G.A., Farquharson C., Ahmed S.F. Growth and the Growth Hormone-Insulin Like Growth Factor 1 Axis in Children With Chronic Inflammation: Current Evidence, Gaps in Knowledge, and Future Directions. Endocr. Rev. 2016;37:62–110. doi: 10.1210/er.2015-1026. - DOI - PubMed
    1. Mazziotti G., Giustina A. Glucocorticoids and the Regulation of Growth Hormone Secretion. Nat. Rev. Endocrinol. 2013;9:265–276. doi: 10.1038/nrendo.2013.5. - DOI - PubMed
    1. Hyams J.S. Corticosteroids and Growth. J. Pediatr. 1988;113:249–254. doi: 10.1016/S0022-3476(88)80260-9. - DOI - PubMed

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