Combined pre‐conditioning with salidroside and hypoxia improves proliferation, migration and stress tolerance of adipose‐derived stem cells

Abstract Oxidative stress after ischaemia impairs the function of transplanted stem cells. Increasing evidence has suggested that either salidroside (SAL) or hypoxia regulates growth of stem cells. However, the role of SAL in regulating function of hypoxia‐pre–conditioned stem cells remains elusive. Thus, this study aimed to determine the effect of SAL and hypoxia pre‐conditionings on the proliferation, migration and tolerance against oxidative stress in rat adipose‐derived stem cells (rASCs). rASCs treated with SAL under normoxia (20% O2) or hypoxia (5% O2) were analysed for the cell viability, proliferation, migration and resistance against H2O2‐induced oxidative stress. In addition, the activation of Akt, Erk1/2, LC3, NF‐κB and apoptosis‐associated pathways was assayed by Western blot. The results showed that SAL and hypoxia treatments synergistically enhanced the viability (fold) and proliferation of rASCs under non‐stressed conditions in association with increased autophagic flux and activation of Akt, Erk1/2 and LC3. H2O2‐induced oxidative stress, cytotoxicity, apoptosis, autophagic cell death and NF‐κB activation were inhibited by SAL or hypoxia, and further attenuated by the combined SAL and hypoxia pre‐treatment. The SAL and hypoxia pre‐treatment also enhanced the proliferation and migration of rASCs under oxidative stress in association with Akt and Erk1/2 activation; however, the combined pre‐treatment exhibited a more profound enhancement in the migration than proliferation. Our data suggest that SAL combined with hypoxia pre‐conditioning may enhance the therapeutic capacity of ASCs in post‐ischaemic repair.


| INTRODUC TI ON
Adipose-derived stem cells (ASCs) can be primarily harvested from adipose tissues by using a simple, minimally invasive method, and are easily cultured and expanded in vitro. 1 A number of studies have revealed that ASCs have multi-lineage potential as they can differentiate into multiple types of somatic cells, such as nerve cells, bone cells, endothelial cells and cardiomyocytes, under specific conditions. 2,3 Due to the cellular plasticity, ASCs are considered a promising cell source for regenerative medicine.
Transplantation of ASCs has been applied to the studies of post-ischaemic repair. [4][5][6] However, oxidative stress after ischaemia results in the production of reactive oxygen species (ROS) and inflammatory factors, 7,8 which can cause dysfunction of transplanted stem cells. [9][10][11] The accumulation of ROS also contributes to stem cell ageing and various types of cell death including apoptosis, necrosis and autophagic cell death (ACD; also known as type 2 programmed cell death [PCD]). Thus, enhancing the tolerance of ASCs against oxidative injury is critical to cell transplantation. Besides, generation of a sufficient number of transplanted cells can also increase the efficiency of post-implantation survival and proliferation capacity of stem cells in vivo. 12 Autophagy is an evolutionarily conserved catabolic process that decomposes cytosolic proteins and organelles by forming autophagosomes to load cargo and subsequently fuse with lysosomes. 13 Accumulating evidence has demonstrated that autophagy plays a cytoprotective role in response to cellular stress. Specifically, Liu et al reported that autophagy actually promotes hypoxia pre-conditioning improving the viability of marrow mesenchymal stem cells (MSCs). 14 However, excessive autophagy may lead to ACD. 15 Several studies revealed that autophagy is involved in the regulatory mechanism of stem cell death and survival under stressed conditions. [16][17][18][19][20][21] Hypoxia (1%-5% O 2 ) pre-conditioning is a promising strategy to optimize or increase the self-renewal efficacy of MSCs, including bone marrow mesenchymal stem cells (BMSCs) and ASCs, 22,23 indicating that hypoxia pre-conditioning could be an approach to increase cell yield for clinical-scale ASC expansion. Moreover, hypoxia pre-conditioning enhances the survival of BMSCs in ischaemic tissues by increasing autophagy and decreasing apoptosis, suggesting that hypoxia may provide a protective effect on stressed injury in MSCs. 14 A similar stimulatory effect of hypoxia pre-conditioning was observed on BMSC survival in vivo, with about 5% of the transplanted BMSCs remaining alive on day 14, which implies that there is still a great room to improve stem cell function under stressed or pathological conditions. Salidroside (SAL) one of the main effective constituents of traditional Chinese herb Rhodiola possesses diverse pharmacological effects. 25 Indeed, SAL can promote the proliferation, differentiation, anti-apoptosis, anti-oxidation and anti-inflammation activities of MSCs. [26][27][28][29] Therefore, SAL may further enhance the function of hypoxia-pre-conditioned MSCs.
In this study, we determined the roles of SAL pre-conditioning on hypoxia-mediated proliferation and migration of rat ASCs (rASCs) by detecting the cell viability, cell proliferation, migratory ability and the activation of Akt, Erk1/2 and LC3. Furthermore, we also determined whether H 2 O 2 -mediated cytotoxicity, cell death, redox disequilibrium and NF-κB activation contribute to the resistance of pre-conditioned rASCs against oxidative stress.

| MATERIAL S AND ME THODS
2.1 | Culture, identification and transfection of rASCs rASCs were purchased from Cyagen Biosciences Inc. rASCs were planted in a 75-cm 2 culture flask and maintained in basal medium, supplemented with 10% foetal bovine serum, 2 mmol/L glutamine and 1% penicillin-streptomycin solution. The cells were incubated in a humidified incubator with 5% CO 2 at 37°C. The culture media were changed every two days, and the adherent cells were passaged at a confluency of approximately 80%. P5-7 rASCs used in this study were identified by immunophenotyping and directed differentiation of specific lineages. rASCs for immunophenotyping by flow cytometry (FCM) were digested and resuspended in 100 μL antibody working solution (90 µL of PBS containing 5% FBS and 10 µL of fluorescein-conjugated monoclonal antibody or isotype control). The antibodies and isotype controls used for immunophenotyping were as follows: PE hamster anti-rat CD29, PE hamster IgM, PE mouse anti-rat CD45 and PE mouse IgG1, from BD Bioscience; CD90 monoclonal antibody (OX-7), PE and CD34 monoclonal antibody (QBEND/10), PE, from Thermo Fisher Scientific. After being incubated in the dark on ice with shaking for 1 hour, rASCs were washed 3 times with PBS and then analysed by FCM. rASCs for osteogenic and adipogenic differentiation were cultured in 6-well plates and orientiatedly induced using osteogenic differentiation medium and adipogenic differentiation medium, respectively. After the induction of differentiation, rASCs were stained with Alizarin red or Oil Red O and observed under an inverted phase-contrast microscope (Leica, DMI3000 B).
The lentivirus (LV) for stubRFP-sensGFP-LC3 overexpression was purchased from GeneChem. Cell transfection was performed following the protocol provided by the manufacturer. Polybrene (5 μg/ mL) was added to the medium for improving transfection efficiency.

| SAL and hypoxia pre-conditionings
rASCs in the logarithmic growth phase were seeded in cell culture plates at a density of 3 × 10 3 /well, followed by serum deprivation for 24 hours when cells reached confluence of 50%-60%. In order to explore the most optimal pre-conditioning conditions, rASCs were incubated at 37°C with different concentration of SAL (0, 25, 50, 100, 200 and 400 μmol/L, respectively) in a 5% CO 2 incubator (Thermo Fisher Scientific, 371, USA) or in a tri-gas incubator (Thermo Fisher Scientific, 3131, USA) that maintains a 5% O 2 level. rASCs were preconditioned for 1, 3 and 5 days.

| Cell viability analysis
The cell viability was measured using an enhanced Cell Counting Kit-8 (CCK-8, Beyotime). Briefly, 100 μL CCK-8 solution was added to each well. After 2 hours of incubation at 37°C, the optical density (OD) value was measured at A 450 nm. Three independent experiments were run.

| Observation of autophagosomes and autolysosomes
rASCs transfected with stubRFP-sensGFP-LC3 LV were seeded in 30-mm glass-bottom culture dishes. At the end of the experiments, rASCs were observed under a laser scanning confocal microscope (LSCM, Leica). The fluorescence of yellow dots (overlays of red and green channels) and red dots was observed in five viewing fields, and was counted manually by a person unfamiliar with this study.
The number of autophagosomes and autolysosomes in each cell was calculated as the yellow dots and red dot, respectively.

| Oxidative stress induced by hydrogen peroxide (H 2 O 2 ) and cytotoxicity assay
Oxidative stress was induced by addition of H 2 O 2 (400 μmol/L) in lowglucose DMEM (Dulbecco's modified Eagle medium) supplemented with 0.1% FBS. We first performed LDH release assay to analyse H 2 O 2mediated cytotoxicity. The destruction of plasma structure caused by cell death results in the release of enzymes in cytoplasm into the culture medium, including LDH, which has relatively stable enzymatic activity. LDH release was analysed using a LDH Cytotoxicity Assay Kit (Beyotime, Shanghai, China). After H 2 O 2 treatment for 24 hours, the cell culture plates were centrifuged for 5 minutes at 400 g. Cell culture supernatant (120 μL/well) was transferred from each well to 96-well culture plates. Cells were washed once with PBS and incubated with 150 μL LDH release reagent (1:10 dilution) at 37°C for 1 hour; then, the supernatants were also transfer to a new culture plate. 60 μL LDH test solution was added to the transferred supernatant. The plate was incubated at room temperature (22-25°C) in the dark for 30 minutes.
Finally, the absorbances were read at 490 nm using a microplate spectrophotometer (Epoch, BioTek). The percentage of LDH release was calculated using the following formula:

| Cell apoptosis assay
After being incubated with H 2 O 2 or PBS for 24 hours, cells were fixed with 4% paraformaldehyde at room temperature for 30 minutes. In Situ Cell Death Detection Kit (Roche) was used for TUNEL staining. Briefly, cells were incubated in a 0.1% Triton-100 solution and then incubated with a TUNEL reaction mixture. Finally, nuclei were stained with 1 μg/mL DAPI. Besides, apoptosis was also detected using the Annexin-V-FITC Apoptosis Detection Kit (BD Pharmingen).
The protocol was performed in accordance with the manufacturer's instructions. Cells were digested into single cell suspensions with EDTA-free trypsin and then subjected to the Annexin V/PI. The stained cells were analysed by FCM in one hour after staining.

| Malondialdehyde (MDA) and glutathione (GSH) measurement
The contents of MDA and GSH were respectively measured using a Lipid Peroxidation MDA Assay Kit and a Total Glutathione Assay Kit (Beyotime), according to the manufacturer's instruction. Cells were homogenized with PBS, and the homogenate was centrifuged at 10 000 g for 10 minutes. The supernatant was taken for the subsequent measurement.
For MDA measurement, 0.1 mL homogenate or standards were added to a tube, and subsequently 0.2 mL MDA test working solution was added. The mixture was heated in boiling water bath for 15 minutes, then cooled to room temperature and centrifuged at 1,000 g for 10 minutes. 200 μL supernatant was added to a 96-well plate, and then, the absorbance at 532 nm was measured using a microplate spectrophotometer. For GSH measurement, 10 μL homogenate or standards, 10 μL protein removal reagent S solution and 150 μL total glutathione detection working solution were sequentially added to each well of a 96-well plate, then mixed and incubated at room temperature for 5 minutes. 50 μL NADPH solution (0.5 mg/ mL) was added to each well. After mixing, the absorbance at 412 nm was measured every 5 minutes for a total of 25 minutes. MDA or GSH content was calculated according to the standard curve.

| Cell migration assay
Transwell migration assay was performed using Transwell chambers    Collectively, these results demonstrate that SAL and hypoxia pre-conditionings synergistically attenuate H 2 O 2 -mediated cell death partly by protecting rASCs against apoptosis and ACD.  Figure 6D).

| SAL and hypoxia pre-conditionings synergistically inhibit H 2 O 2 -induced oxidative stress and NF-кB activation
These results suggest that SAL and hypoxia pre-conditionings synergistically protect rASCs against oxidative injury though anti-oxidant and anti-inflammation mechanisms.

| SAL and hypoxia pre-conditionings show synergistical effects on migration but not proliferation of rASCs under H 2 O 2 -induced oxidative stress
As shown in Figure  We also determined the expression of Akt, p-Akt, Erk1/2 and p-Erk1/2 using Western blot. The levels of p-Akt and p-Erk1/2 were obviously decreased after the NC pre-treatment followed by the H 2 O 2 treatment ( Figure 7E,F) F I G U R E 6 Effects of SAL and hypoxia pre-treatments on H 2 O 2 -induced oxidative stress and NF-кB activation. The contents of MDA (A) and GSH (B) and the enzymatic activity of CAT (C) were quantitated in the extracts of rASCs using the colorimetric method. NF-кB-p65 phosphorylation (D) of rASCs was determined by Western blot. *P < .05, ***P < .001

F I G U R E 7
Effects of SAL and hypoxia pre-treatments on cell proliferation, migration and activation of Akt and Erk1/2 after the H 2 O 2 treatment. The cell proliferation was analysed by BrdU incorporation assay (A and B), and the cell migration was measured by Transwell migration assay (C and D). The phosphorylation levels of Akt (E) and Erk1/2 (F) were analysed by Western blot. *P < .05, **P < .01, ***P < .001 In summary, these results indicate that SAL and hypoxia pre-conditionings enhance the proliferation and migration of rASCs under oxidative stress in association with Akt and Erk1/2 activation, and SAL pre-conditioning has a enhanced potential in improving the migration but not proliferation of hypoxia-pre-conditioned rASCs.

| D ISCUSS I ON
This study demonstrated that SAL and hypoxia pre-conditionings Recent studies have revealed that low oxygen tension or hypoxia promotes the survival and function of MSCs. 14,[22][23][24] In addition, pre-conditioning with the components of the traditional Chinese herbs is also a promising strategy. [25][26][27][28][29] Using the cell number counting assay and BrdU incorporation assay, we found that the treatment Oxidative stress-induced ROS have been shown to cause cell death and cellular ageing in different cell types. 35,36 In response to the infarct microenvironment, the stressed stem cells release various immunomodulatory signalling factors, including both pro-inflammatory and anti-inflammatory cytokines, thereby displaying their immunomodulatory roles. 35,37 As one of the common ROS, H 2 O 2 can easily cross the plasma membrane and stimulate consecutive reactions leading to cell apoptosis and inflammatory responses. 36 Our present results show that the H 2 O 2 treatment led to increased cytotoxicity, apoptosis, late-stage autophagy, oxidative stress and NF-κB activation in rASCs, which were alleviated by the SAL or hypoxia pre-treatment; moreover, the combined pre-treatment with SAL and hypoxia has synergetic effects (Figures 4-6). The results imply that SAL pre-conditioning combined with hypoxia pre-conditioning can Autophagy, an intracellular degradation and recycling of cytoplasmic contents, plays a double-edged sword effect on cell fate. 13 Several studies have reported that autophagy is involved in the survival and proliferation of MSCs. [44][45][46] However, recent reports have indicated that chronic stress and ROS can induce ACD of stem cells. [16][17][18][19][20][21] In the present study, rASCs were transfected with LV which mediated stable expression of stubRFP-sens-GFP-LC3 in rASCs. During autophagy, LC3-I is converted to an autophagosome-associating form called LC3-II. 47 Sens-GFP is an acid-sensitive protein, while stub-RFP is a stable fluorescent protein unaffected by the internal environment of lysosome. Thus, the yellow dots (overlays of red and green channels) and red dots respectively represent autophagosomes and autolysosomes.
Here, we found the SAL, hypoxia and combined treatments dis- Hence, further investigations will need to focus on the underlying mechanisms of how SAL and hypoxia pre-conditionings counterbalance the autophagic activity of stem cells.

| CON CLUS IONS
In conclusion, our results shed light on a better understanding of the effects and mechanisms of SAL pre-conditioning combined with hypoxia pre-conditioning on rASC function (Figure 8). The present study clearly demonstrates for the first time that SAL pre-conditioning further improves the function of hypoxia-pre-conditioned rASCs by enhancing the proliferation, migration and tolerance against oxidative stress. Importantly, we partly identified the mechanisms underlying the multi-target effects of SAL and hypoxia preconditionings on rASC function. This study also suggests that SAL pre-conditioning combined with hypoxia may facilitate a higher therapeutic capacity of ASCs in post-ischaemic repair.

ACK N OWLED G EM ENTS
We

CO N FLI C T S O F I NTE R E S T
The authors declare that there is no conflict of interests regarding the publication of the paper.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.