Hydrogen sulfide promotes the osteogenesis of bone marrow mesenchymal cells through hypoxia-inducible factor-1 alpha under tensile stress

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1 Chinese Journal of Tissue Engineering Research July 28, 2017 Vol.21, No.21 1α ( ). 1α [J] (21): DOI: /j.issn ORCID: () 1α (5 7 d)( mm/d) :R318 :A : (2017) α µstrain 1α 1α 1α GYY4137 YC-1 1 α ( )(N ) Hydrogen sulfide promotes the osteogenesis of bone marrow mesenchymal cells through hypoxia-inducible factor-1 alpha under tensile stress Jiang Xiao-wen, Zhang Yi, Fan Xiao-sheng, Chen Yan-ze (Department of Stomatology, the First People s Hospital of Chenzhou & Institute of Translation Medicine, University of China South, Chenzhou , Hunan Province, China) Abstract BACKGROUND: Hydrogen sulfide signaling has been proved to promote distraction osteogenesis; however, the underlying mechanism remains unclear. OBJECTIVE: To evaluate the relationship between hydrogen sulfide and hypoxia-inducible factor-1α (HIF-1α) Jiang Xiao-wen, Associate chief physician, Department of Stomatology, the First People s Hospital of Chenzhou & Institute of Translation Medicine, University of China South, Chenzhou , Hunan Province, China Corresponding author: Jiang Xiao-wen, Department of Stomatology, the First People s Hospital of Chenzhou & Institute of Translation Medicine, University of China South, Chenzhou , Hunan Province, China ISSN CN /R CODEN: ZLKHAH 3281

2 . 1α during the osteogenesis of rat bone marrow mesenchymal cells under tensile stress. METHODS: µ strain was loaded on the in vitro cultured rat bone marrow mesenchymal cells by a four-point bending apparatus, and hydrogen sulfide donor or HIF-1α inhibitor was adopted in the tensile unit. Subsequently, the levels of osteogenic markers were detected. RESULTS AND CONCLUSION: Exogenous hydrogen sulfide signaling could promote the osteogenesis of rat bone marrow mesenchymal cells under tensile stress. However, this promotion was obviously eliminated when the endogenous HIF-1α expression was inhibited. These results show that hydrogen sulfide signaling system promotes the osteogenesis of rat bone marrow mesenchymal cells under tensile stress probably through HIF-1α. Subject headings: Tissue Engineering; Hydrogen Sulfide; Hypoxia-Inducible Factor 1, alpha Subunit Funding: the National Natural Science Foundation of China, No ; the Key Project of the First People s Hospital of Chenzhou, No. N Cite this article: Jiang XW, Zhang Y, Fan XS, Chen YZ. Hydrogen sulfide promotes the osteogenesis of bone marrow mesenchymal cells through hypoxia-inducible factor-1 alpha under tensile stress. Zhongguo Zuzhi Gongcheng Yanjiu. 2017;21(21): Introduction (distraction osteogenesis DO) [1] [1] [2-10] [11] (H 2 S) L [12] [13-14] [15] H 2 S3 H 2 S β 3 L H 2 S [16] H 2 S H 2 S H 2 S [17-19] (hypoxia-inducible factors HIFs) [20] H 2 S [21-22] H 2 S (bone marrow mesenchymal cell BMMC) 1α 1α H 2 S 1Materials and methods SD g SYXK( ) Percoll( Gibco ) 5% PBS 3 10% LG-DMEM( Gibco ) 2 d 70% 80% 0.25% /cm 2 [1] 24 h 3282 P.O. Box 10002, Shenyang

3 . 1α 60 min( ( ) 0.5 Hz 1 mm µ strain0.2% α 1 α (YC-1) 80 µmol/l 3 H 2 S GYY41374 mmol/l 4 1α /H 2 S 3 YC-180 µmol/l 2 h h PBS H 2 S (10 ml 1 cm 0.8 cm ) 1 mol/l HCl 0.5 mlh 2 S 1 mol/l NaOH 0.5 ml H 2 S37 4 h H 2 S [17] h PBS 3 ml 2 ml PBS 90 µl PBS 3 ( )450 nm U/mg nm 2 h 1 ml 10 µl 40 µl min 50 µl min min 450 nm (PINP) ( ) 450 nm 2 h 1 ml 5 µl 45 µl PINP 30 min 150 µl 30 min 30 min 450 nm PINP Real time-pcr 2 h Trizol(Gibco) RNA; (TaKaRa) BioRad TM cdna 1 cdna SYBR Green RT-PCR GAPDH PCR 5 -CTT CGT CAG CGT CCT ATC AGT TC-3 5 -CAG CGT CAA CAC CAT CAT TCT G-3 2 (runt-related transcription factor 2 Runx2) mrna 5 -TAT GAC TCT ACC CAC GGC AAG T-3 5 -ATA CTC AGC ACC AGC ATC ACC-3 PCR95 30 s 95 5 s s 40 Runx2 GAPDH Western Blot 1α BCA ( Gibco ) 20 µg SDS-PAGE PVDF (1α1 500 Abcam ) (lgg )37 1 h 1.5 H 2 S (PINP) Runx2 1α 1.6 SPSS 13.0 x _ ±s LSD P < Results h 4 (P > ) 2.2 H 2 S 4 2 h H 2 S 1α/ H 2 SH 2 S 2(P < ) 2.3 H 2 S 1α/H 2 S (P < ) H 2 S 2.4 H 2 S 3 (P < ) 2.5 PINP 1α /H 2 S (P < ) H 2 S PINP 2.6 Runx2 mrna H 2 S Runx2 mrna 4 (P < ) ISSN CN /R CODEN: ZLKHAH 3283

4 . 1α 1 H 2S Table 1 Levels of osteogenic markers and hydrogen sulfide at the end of tensile stress loading (x _ ±s n=8) 1α H 2S 1α / H 2S (10 6 ) 6.22± ± ± ±0.61 H 2S(U/mg) 17.84± ± ±3.29 a 35.43±3.44 a (U/mg) ± ± ±26.81 a ±27.18 a (U) 1.48± ± ±0.25 a 1.28±0.18 PINP(µg/L) 8.00± ± ±0.57 a 3.16±0.37 Runx2 mrna 10.97± ± ±2.65 a 11.13±0.81 1α a P < 0.05 PINP A B C D M r α β-actin α Figure 1 Protein expression of hypoxia-inducible factor-1α in rat bone marrow mesenchymal cells at the end of tensile stress loading A D 1α H 2S 1α /H 2S B D 1α 2.7 1α Western blot4 hh 2 S 1α H 2 S 1α 1α1α /H 2 S ( 1) 3Discussion H 2 S 3 H 2 S [23-25] H 2 S H 2 S [17-18] GYY4137 H 2 S H 2 S [19] [26] [20] H 2 S H 2 S H 2 S Runx2 PINP Runx2 PINPH 2 S H 2 S H 2 S H 2 S H 2 S [27-28] α 3 α (1α 2α 3α) 1α H 2 S [29] Liu [21] H 2 S Bir [22] H 2 S [30] H 2 S [31] H 2 S 1α 1α mrna 1α [32-51] Wan [52] 3284 P.O. Box 10002, Shenyang

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