Methods

Extraction and cultivation of ADSCs: All animal experiments were performed under the approval of the Institutional Animal Care and Use Committee of Yangzhou University (Approval No. DWLL-202112-201). Five two weeks old male SD rats were euthanized with excessive ether asphyxiation. The rats were routinely disinfected by 75% ethyl alcohol, and their blood vessels, fascia and other tissues were removed. The harvested inguinal adipose tissue was cut into 1 mm³ pieces and then placed into a 15 ml centrifuge tube. 0.1% type I collagenase was used to digest tissue for 40∼60 min. After filter to remove nondigested tissue, filtrate was collected and seed into tissue culture plastic in culture medium with 10% fetal bovine serum. The incubation was conducted at 37℃ with a volume fraction of 5% CO₂, and the liquid was altered after 24 h, 48 h and 72 h. Under the cell concentration of 80%, 0.25% trypsin-EDTA was employed for digestion and subculture. The 3rd generation cells were employed for the immunocytochemical identification and subsequent tests.

Bioinformatics analysis: Data retrieved was performed in the GEO database by search the key words “Hydrogen”. And finally {“type”:”entrez-geo”,”attrs”:{“text”:”GSE62434″,”term_id”:”62434″}}GSE62434 was obtained to analyze the expression of fibroblasts cultured in hydrogen. Fibroblasts were divided into groups according to whether they were cultured or not. The differentially expressed genes were obtained by GEO2R online tool. KEGG enrichment analysis was performed based on the differentially expressed genes in the DAVID online website. GSEA enrichment analysis of differentially expressed genes was subsequently performed using GSEA software.

Identification of ADSCs: ADSCs were cultured and then identified when 90% of the cells were covered. The cells were digested by trypsin at ambient temperature for 10 min, and it was collected after being centrifuged. Subsequently, the cells were washed with PBS and then prepared into 1×10⁵/ml cell number suspension. 100 μl of 1×10⁵ cell suspension was added with 10 μl of fluorescently labeled phenotypic antibody (CD45, CD44, CD90 and CD31) respectively for incubation. After the washing process, the expressions of CD45, CD44, CD90 and CD31 surface antigens were detected by performing the flow cytometry.

Identifying process for three-line differentiation of ADSCs: The multilineage differentiating potentials of ADSCs were characterized by osteogenic, adipogenic, and chondrogenic differentiation. Briefly, according to 5×10⁴ cells/cm² cell density, cells received the collecting and inoculating processes inside the orifice plate. The cells received the culturing process with 5% CO₂ at 37℃. Under the cell confluence of 90%, The cells were cultured in osteogenic, adipogenic and chondrogenic differentiation media and identified and analyzed by alizarin red (Solarbio, G8550, Beijing, China), oil red O (Solarbio, G1262, Beijing, China), toluidine blue staining (Solarbio, G3661, Beijing, China). Stained areas were observed under a fluorescence microscope (OLYMPUS, IX71).

Groups and cell proliferation of ADSCs: The cells were seeded in 96-well plates (Thermo Scientific, USA) at 1×10⁴/well in 100 μl of DMEM with FBS. The groups (control, H₂, 5-Aza and H₂＋5-Aza) were tested at days 1, 2 and 3. Furthermore, the control and 5-Aza groups were incubated in a conventional cell incubator (5% CO₂, 95% air) and the H₂ and H₂＋5-Aza groups were incubated in a hydrogen incubator (70% hydrogen, 21% oxygen, 5% CO₂, 4% nitrogen). The cell proliferation ability of H₂ and 5-Aza was compared by MTT and Alamar blue staining respectively. Cells were stained with MTT (20 μl; 5 mg/ml) for 4 h at 37℃. Dimethyl sulfoxide was added to samples and incubated for 30 min at 37℃. The absorbance was measured using a microplate reader (Thermo MK3 type) at 492 nM.

Cell samples were taken, and 10 μl Alamar blue staining was introduced to 100 μl cell suspension at 1- and 3-day induction time. Incubating in an incubator away from light for 6 h, and detecting with microplate reader with a wavelength of 600 nm. The diluted AM/PI kit (Biovision, K501-100, USA) was added and incubated for 30 min before washing. Observed by means of fluorescence microscope (OLYMPUS, IX71). Red and green denote dead cells and live cells, respectively. With ImageJ software (National Institutes of Health, Bethesda), this study determined the fluorescence intensity.

Effect of hydrogen on mitochondria by mito-tracker green fluorescent staining: To investigating the influence exerted by H₂ for mitochondria. First, ADSCs received the inoculating process on confocal petri dishes and the culturing process inside normal medium for 12 h. Next, the cells received the seven-day incubation with control, H₂, 5-Aza, H₂＋5-Aza. Then, the medium received the removal and rinsing process by applying PBS, and then it was incubated with the mito-tracker (1：5,000∼1：50,000) at 37℃ for 15∼45 min. Lastly, under a Laster confocal fluorescence microscope, the observation of cells was conducted.

Immunofluorescence detecting process of the expressions of Desmin, Myosin and β-actin in cells: After incubation with control, H₂, 5-Aza, H₂＋5-Aza for 7 days, ADSCs were fixed in 4% paraformaldehyde for 15 min. Then, 0.1% Triton was used for stabilization at ambient temperature for 15 min. The cells were sealed with 5% FBS for 15 min under the temperature of the ambient. Subsequently, the cells were incubated by using primary antibody at 4℃ throughout the night. After washing, it was incubated at 37℃ for 1 h by using secondary antibody. Hochest received the incubating process under ambient temperature and the isolating process away from light for 15 min. Lastly, with the use of a fluorescence microscope (OLYMPUS, IX71), the samples were observed.

Real-time PCR analysis of Myod, Mhc and Myogenin: Under the p38 MAPK signal pathway inhibitor (SB203580), the cells were collected after being inducted with control, H₂, 5-Aza, H₂＋5-Aza for 7 days. Overall RNA was extracted from the cells under the transfection with the Trizol extraction tool in accordance with the guidelines of the producer. According to the guidelines of the producer, cDNA received the synthesis based on reversely transcribing process with the first strand cDNA synthesis tool. Primers for reverse transcription PCR received the designing and synthesizing processes with Primer Premier 5.0 software (Shanghai Biotechnology, China) based on internal reference of housekeeper gene GAPDH. After PCR amplification, the results were automatically analyzed by using the fluorescence quantitative PCR tool in the real time, the baseline and threshold were regulated in accordance with the negative control to determine the Ct value of the respective specimen, as well as whether the Ct value was effective based on the fusion curve. For the export, the 2^−ΔΔCt approach inside gene expressing state differences of control and the concentration groups is: ΔCt=Ct gene－Ct inside. Subsequently the control group ΔCt remember was obtained for ΔCt contrast, ΔCt contrast average was achieved, in which the respective group of ΔCt minus ΔCt contrast average, calculated by ΔΔCt value, i.e., the ΔΔCt=ΔCt sample-ΔCt contrast, next, the respective group 2^−ΔΔCt value was calculated, which indicated the comparative expressions of genes.

Observation and analysis of myotube: To investigate the effect of H₂ combined with 5-Aza on the myogenic differentiation system of ADSCs, the authors introduced the SB203580 (5 uM) to the culture medium (DMEM). Under the p38 MAPK signal pathway inhibitor (SB203580), cells were collected after being inducted with control, H₂, 5-Aza, H₂＋5-Aza for 7 days. First, ADSCs received the inoculating process on 24-well plates under a density of 8000 cells/cm for 12 h. Subsequently, the cells were further cultured at a concentration of 70% H₂, and the medium received the renewal every two days. Myotube formation in ADSCs regulated by H₂ combined with 5-Aza was measured by immunofluorescence staining. ImageJ software was used to obtain the index maturation of myotube (National Institutes of Health, Bethesda, USA).

Western blotting assay: ADSCs were placed in 3.5 cm culture dishes with or without SB203580 and cultured with control, H₂, 5-Aza and H₂＋5-Aza for 7 days. Next, cell mass was washed three times with PBS, lysed for 30 min in ice-cold RIPA lysis buffer, and then its protein was quantified. According to the number of samples, the authors introduced the 200 μl BCA working solution (KGPBCA) in samples, and determined the absorbance at 562 nm. After being denaturalized, the protein was administrated with polyacrylamide gel and then transferred to the methanol-activated PVDF membrane. After being blocked, the primary antibody was incubated at 4℃ throughout the night: Myod (Proteintech, 1：1,000∼1： 6,000), Mhc (Bioss, bs-10904R, 1：500), p-P38 (Affinity, 1：500∼1：2,000), GAPDH (Proteintech, 1：5,000). The PVDF membrane was introduced into rabbit secondary antibody or rat secondary antibody (ZSGB-BIO, 1：5,000) solution under the dilution with 1∶5,000 HRP-labeled solution and was incubated at 37℃ under L h slow oscillation. By employing ECL luminescent solution (Beijing Dingguo ECL-0011), the imprints were developed, and by adopting Image J (NIH, Bethesda, USA), the immunoreaction bands received the quantification.

The evaluation of ADSCs

ADSCs were identified by flow cytometry: ADSCs have no specific surface antigens. Accordingly, the analysis of multiple surface antigens simultaneously to determine the characteristics of adipose stem cells. By flow cytometry, the ADSCs surface markers was tested. We examined the expression of CD31, CD44, CD45 and CD90 in ADSCs. Flow cytometry results showed that 1.6% expressed CD31, 95.2% expressed CD44, 1.1% expressed CD45, and 97.4% expressed CD90 (Fig. 1G). CD44 (95.2%) and CD90 (97.4%) express stem cells. The low expressions of CD31 (1.6%) and CD45 (1.1%) excluded epidermal cells and vascular endothelial cells, respectively. This characteristic meet the criteria stated by the International Society for Cellular Therapy (ISCT) for MSC.

ADSCs were identified by three-line differentiation: The multilineage differentiating potentials of ADSCs were characterized through the osteogenic, adipogenic and chondrogenic differentiation. Alizarin red staining under the induction by ADSCs osteogenesis are presented in Fig. 1H (a), demonstrating the appearance of considerable osteoblasts when the induction was achieved. Fig. 1H (b) shows the staining process of oil red O when the adipogenic induction was achieved, and the stained cells were observed as well. Fig. 1H (c) shows the staining of toluidine blue when the chondroblast induction was achieved, and many chondrocytes were stained. In brief, the mentioned cell could achieve a three-line differentiation, which demonstrated that it acts as a rat ADSCs.

Biocompatibility evaluation of hydrogen

The optimal concentration of hydrogen: Whether H₂ could promote cell viability and proliferation was evaluated by MTT assay (Fig. 2F). After being cultured with H₂ at different concentrations (20%, 50%, 70%), ADSCs showed different cell viability and proliferation. On day 1, no significant difference was fund between the four groups (Control, 20%H₂, 50%H₂, 70%H₂) (p>0.05). As compared with other three groups, cells displayed a remarkably high proliferation in 70%H₂ group (p<0.01) on day 2 and 3. Subsequent experiments were performed at 70% concentration of H₂.

Assessment of cell proliferation by hydrogen: First, we evaluated whether H₂ could promote cell viability and proliferation by MTT and Alamar blue Staining (Fig. 2A∼E). In H₂ group, when cultivated for 1 and 3 days, the cells were primarily alive (green), while few had death (red) (Fig. 2A and ​and2C).2C). In addition, the cell proliferation characteristic received the quantitative analysis of Live-Dead Cell Staining. As compared with the 5-Aza group, cells displayed a remarkably high proliferation in H₂ group (p<0.01) on day 1. There was no significant difference between the other three groups (Control, H₂, 5-Aza＋H₂) (p>0.05). On day 3, compared with 5-Aza and 5-Aza＋H₂ groups, H₂ group displayed the highest expression (p< 0.01). Compared with the 5-Aza group, the survival rate of cells in the 5-Aza＋H₂ group was still higher (p<0.01).

Furthermore, the cell proliferation behavior was quantitatively analyzed by Alamar blue assay. On day 1, the cell viability in H₂ group was significantly higher than other groups (5-Aza, 5-Aza＋H₂) (p<0.01). And, compared with the control group, only the H₂ group showed high proliferation (p<0.05). Interestingly, compared with the 5-Aza group, the 5-Aza＋H₂ group also showed significantly higher proliferation (p<0.01). On day 3, the cell viability in H₂, 5-Aza and 5-Aza＋H₂ groups maintained the same trend as day 1 (Fig. 2B). Based on MTT analysis (Fig. 2D and ​and2E),2E), compared with the 5-Aza group, the H₂ group significantly promoted cell proliferation in the first three days (p<0.01). At the same time, compared to the 5-Aza group, the 5-Aza＋H₂ group also increased cell proliferation (p<0.05). As demonstrated by all the mentioned results, H₂ remarkably enhanced the ADSCs proliferation.

Mito-tracker green fluorescent staining: The green mito-tracker staining was performed for explaining the effect of H₂ on the mitochondria function of ADSCs (Fig. 2G and ​and2H).2H). Obviously, compared with the other three groups, H₂ group expressed significant single-cell mitochondrial fluorescence intensity (p<0.01). Compared with the control group, the intensity of green fluorescence was significantly decreased in the 5-Aza group (p<0.01), and the fluorescence expression of single-cell mitochondria was also significantly increased in the 5-Aza＋H₂ group compared with the 5-Aza group (p<0.01).

Myogenic differentiating effect of hydrogen

Immunofluorescence detecting process of the expressions of Desmin, Myosin and β-actin in cells: To investigate the effect of H₂ combined with 5-Aza on myogenic differentiation at the myoblast stage, ADSCs were employed as an in vitro model of myogenic differentiation. The cells were respectively cultured with 5-Aza, H₂, H₂＋5-Aza for 7 days. Fig. 3A shows the results of immunofluorescence staining assay after incubation for 7 days. The immunofluorescence results showed that remarkably higher immunofluorescence expressing states of β-actin and desmin in the H₂ group and 5-Aza group than in the control (p<0.01). In addition, compared with the control group, the H₂ group showed increased myosin expression (p<0.05). Additionally, the expressing states of all test antibody were remarkably higher in the H₂＋5-Aza group than in the H₂ group (p<0.01). It is noteworthy that the expressing states of the β-actin in 5-Aza and H₂ group were found, whereas noticeable difference was identified between the two groups (p<0.01). Additionally, the expressing states of Mhc antibody were remarkably higher in the H₂＋5-Aza group than in the other group (5-Aza and H₂ groups) (p<0.05).

Observation and analysis of myotube: Mhc protein immunofluorescence staining assay was studied to observe the morphology of myotubes formed in ADSCs. Fig. 3B shows the results of immunofluorescence staining assay after 7 days of incubation. The number, length, diameter and maturation index of myotubes were quantified by morphological analysis. Myotube maturation was quantified by measuring the ratio of the number of cells with more than 2 nuclei to the total number of cells (matu-ration index). The remaining three groups (5-Aza, H₂ and 5-Aza＋H₂) showed statistically significant differences in myotube number, length, diameter and maturation index compared to Control (p<0.01). Notably, the 5-Aza＋H₂ group exhibited the best myotube number, length, diameter and maturation index compared to the 5-Aza and H₂ groups (p<0.05) (Fig. 3B a∼d). After the introduction of the p38 MAPK signaling pathway inhibitor (SB203580), the number, length, diameter and maturation index of myotubes were lower in the 5-Aza, H₂ and 5-Aza＋H₂ groups (Fig. 3B e∼h).

Real-time PCR analysis of Myod, Mhc and Myogenin: We further investigated the effect of the H₂ combined with 5-Aza on myogenic genes expression of ADSCs in vitro. Myod, Myogenin and Mhc, the early and late markers of myogenesis, were used to determine the myogenic differentiation at mRNA level (Fig. 3C). After being cultured for 7 days, the quantitative RT-PCR results showed remarkably higher mRNA expressing states of MyoD, Myogenin and Mhc in the H₂＋5-Aza＋SB203580 group than other four group (p<0.01). However, compared with the SB203580 group, the expression of MyoD, Myogenin and Mhc genes was significantly higher in the H₂ group (p<0.01). No noticeable difference was identified in MyoD, Myogenin and Mhc RNA expressions among the H₂＋SB203580 and 5-Aza＋SB203580 groups (p>0.05). As impacted by SB203580, the expressions of MyoD, Myogenin and Mhc of the inhibitor groups was also a little less than control group (p<0.05).

Western blotting assay of Myod, Mhc and Myogenin: In addition, under the expressing states of Myogenin and Myod (early myogenic marker) and the differentiated myotube marker myosin heavy chain (Mhc), we conducted the western blotting assay for assessing myogenic differentiation (Fig. 4A b∼d). As revealed from the results of the western blotting assay, remarkably higher stripe expressing states of Myod, Mhc and Myogenin were found in the H₂ group, 5-Aza group and 5-Aza＋H₂ group as compared with those in the control (p<0.01). The protein expression status of Myod, Myogenin and Mhc was significantly higher in the H₂＋5-Aza group than in the H₂ and 5-Aza groups (p<0.01).

Effect of hydrogen combined with 5-Aza on the phosphorylation of p38 in myogenic differentiation

According to the mentioned results, H₂ combined with 5-Aza significantly improves the proliferation and myogenic differentiation of ADSCs, whereas the molecular system was unclear. Through the analysis of bioinfor-matics and literature reports, it is known that p38 MAPK was indicated to participate in a range of biological processes of myogenic differentiation. Accordingly, We assessed whether phosphorylation levels of p38 MAPK are influenced by H₂ combined with 5-Aza regulation during myogenic differentiation. ADSCs were incubated in DMEM for 7 days. According to Fig. 4A, the level of p-P38 in the 5-Aza and H₂ cotreatment group was significantly greater than that in the control (p<0.01), which indicated an enhancement in the active form of p38 that promotes differentiation. This result suggests that 5-Aza and H₂ cotreatment during myogenic differentiation could improve p38 activity. It is noteworthy that the levels of p-P38 in the 5-Aza and H₂ single-treatment groups also significantly increased compared with those in the control, which indicated that either 5-Aza or H₂ treatment during myogenic differentiation increases p38 activity.