Butyric acid alleviated chronic intermittent hypoxia-induced lipid formation and inflammation through up-regulating HuR expression and inactivating AMPK pathways

Abstract To investigate whether butyric acid could alleviate chronic intermittent hypoxia (CIH)-induced lipid formation in human preadipocytes-subcutaneous (HPA-s) through accumulation of human antigen R (HuR) and inactivation of AMP-activated protein kinase (AMPK) pathway, HPA-s were obtained and divided into three groups: Control group: cells were cultured under normal conditions; CIH group: cells were cultured in a three-gas incubator (10% O2); Butyric acid group: 10 mmol/l butyric acid added into cell culture medium. HuR-siRNA was futher transfected into CIH group for verification the function of HuR. Oil Red O was implemented for observation of lipid droplets within cells. Cell Counting Kit-8 (CCK8) assay was used for detecting cell viability. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labeling (TUNEL) assay as well as flow cytometry analysis was employed for determining cell apoptosis. Western blotting was used for measurement of protein expression levels. RT-qPCR analysis was used for detecting mRNA expression. CIH treatment increased adipocytes proliferation, while butyric acid inhibited cell proliferation and promoted cell apoptosis. The treatment of butyric acid in CIH group down-regulated expression of inflammatory factors and increased cell apoptotic rate. Butyric acid treatment increased HuR expression in both cytoplasm and nucleus and decreased the level of p-AMPK and p-ACC, while transfection of AMPK activator or HuR-siRNA would down-regulate HuR expression. Moreover, butyric acid alleviated CIH-induced cell proliferation, lipid formation and inflammatory status and promoted cell apoptosis through regulating related genes including p21, PPARγ, C/EBPa, IL-1β, IL-6, TLR4, caspase-8 and caspase-3. In conclusion, butyric acid could alleviate CIH-induced inflammation, cell proliferation and lipid formation through accumulation of HuR and inactivation of AMPK pathway.


Introduction
Butyric acid, as one of saturated short-chain fatty acids, which is the main components of fats in the foods and lipids, affords health benefits against lipid disorders [1]. In recent years, obesity, a systemic inflammatory disease, characterized by excessive lipid storage in adipose tissue, has been an increasingly significant public health problem [2,3]. People suffering from obesity have a higher prevalence of metabolic disorders such as cardiovascular disease, diabetes, and some types of cancer [4]. In addition, a clinical research indicated that children in obesity had shorter sleep time, and a higher incidence of obstructive sleep apnea [5], which leads to chronic intermittent hypoxia (CIH). Recent evidence suggests that butyric acid is beneficial to the lipid disorders [6] and exerts a dramatic hypotensive effect at a dose [7]. However, little attention has been paid to the mechanism of inhibiting lipogenesis by butyric acid.
As a major repressor during adipogenesis, human antigen R (HuR) plays a critical role in RNA metabolism [8]. And HuR protein is expressed in various cell types, such as adipose, intestine, spleen and testis [9]. Generally, HuR improves the efficiency of translation by positively regulating target mRNAs and their stability [10].
Researches show that HuR is intimately related to the inflammation, apoptosis, proliferation and polarization [11,12] and is a crucial repressor of adipogenesis and negatively regulate the inflammation [8]. Therefore, HuR has been identified to have However, mechanism insights are currently lacking. In consideration of the functions of HuR, it's vital and urgent to find how it affects lipogenesis and inflammation.
AMP-activated protein kinase (AMPK), as a regulator of metabolism, could inhibit cytoplasmic export of HuR by influencing the mRNA turnover [13], and exert a wide range of benefits on energy homeostasis [14]. Thus, the mechanism of adipogenesis and anti-inflammation is set out to investigate further. Recently investigators have examined the effect of CIH on inducing oxidative stress and cytokines production, which was commonly found in obese suffering from obstructive sleep apnea [15,16].
Surveys such as that conducted by Su et al. have shown that frequent episodes of CIH result in the increase of excitability in bladder receptors, leading to bladder dysfunction, indicating the destructive effects of CIH on physical functions [17].
In this study, we hypothesized that butyric acid could increase cytoplasm accumulation of HuR and regulate the expression of its downstream genes via inhibiting the activation of AMPK in CIH, thus, promotes apoptosis to inhibit the generation of adipocytes and plays an important role in anti-inflammatory property.

Cell Culture
Human preadipocytes-subcutaneous (HPA-s) were obtained from ScienCell Research Laboratories, Carlsbad, CA, USA. Cells were inoculated in a poly-L-lysine medium at Downloaded from http://portlandpress.com/bioscirep/article-pdf/doi/10.1042/BSR20203639/908561/bsr-2020-3639.pdf by guest on 21 April 2021 a density of 1×10 5 cells/cm 2 , and then cultured in an incubator with 5% CO2 at 37℃ for 2 d using PAM. When the cell fusion degree reached 80%, PAM was replaced by PADM which were refreshed every 3 d. After 7 days, human preadipocytes differentiated into mature adipocytes that were cultured in ADM.
Cells were divided into three groups: Control group: cells were cultured under normal conditions; Chronic intermittent hypoxia (CIH) group: cells were incubated in CIH chamber and exposed to 9h-deoxygenation-reoxygenation cycle of 5% oxygen for 60 min and 20% oxygen for 30 min [18]; Butyric acid group: 10 mmol/L butyric acid added into cell culture medium.

Oil red O staining
Oil red O and distilled water were mixed for 10 min at room temperature in the ratio of 3:2, after which the mixture was filtered. Human preadipocytes and adipocytes which have been induced for 7 days were removed and washed with PBS for 3 times.
The cells were fixed with 4% paraformaldehyde at room temperature for 15min, and washed with PBS for 3 times. The cells were stained with oil red O for 30 min at room temperature and washed with PBS again. After drying, a microscope was implemented for observation and recording.

CCK8 Assay
Mature adipocytes were collected to prepare cell suspension, after which cells were inoculated into 96 well plates (2 × 10 4 cells per well), and then cultured in an incubator with 5% CO2 at 37℃ for 12 h. After implementation of different treatments in each group and incubated for another 48 h, 10 μL CCK-8 solution was added into each well and incubated for 2 h. The absorbance of each well was detected at 450 nm wavelength.

TUNEL assay
TUNEL assay was obtained for detecting the effect of different treatments on apoptosis of adipocytes. Cells in each group were fixed with 4% paraformaldehyde at room temperature for 15 min and washed with PBS for 3 times, after which cells were treated with 1% Triton X-100 solution for 3 min and then washed again with PBS. used to re-stain the nucleus and incubated in dark 37℃ for 10 minutes. A microscope was implemented for observation and recording.

Flow cytometry analysis
The cell culture medium was digested with trypsin at room temperature until the adherent cells were blown down, after which the cells were transferred into a centrifuge tube and centrifuged at 1000g for 5 minutes. The supernatant was discarded, and the cells were collected and washed with PBS. Then 195 μL annexin V-FITC solution was added, after which 10 μL propidium iodide solution was added and incubated for 20 minutes at room temperature (20-25℃). After washing with PBS, cells were resuspended with 300 μL PBS and detected by flow cytometry.

Western Blotting
The cells were placed into a 1.5 mL EP tube and washed twice with PBS. After centrifugation, protein lysate was added and the cells were lysed at 4℃ for 1 h. The lysed protein solution was centrifuged at 12000 g for 10 min, and the supernatant was collected and the concentration of protein was measured using a BCA protein assay kit. Total protein was separated with SDS-PAGE and transferred onto a PVDF membrane. The membrane was then incubated in blocking liquid for 1 h at 25°C.

RT-qPCR analysis
The total RNA was extracted from cells using Trizol reagent. cDNA was synthesized using the TaqMan MicroRNA reverse transcription kit according to the manufacturer's   instructions with the following condition: 16˚C for 30 min, 37˚C for

HuR-siRNA transfection
Cells were diluted using 10% FBS without antibiotics and then distributed into a 6-well plate at a density of 5×10 4 /cell, after which cells were incubated in a 5% CO2 incubator at 37℃ for 24 h. The transfection of HuR-siRNA was performed according to the manufacturer's instructions. Briefly, 5 μL siRNA and 5μL lipo2000 was dissolved into 50 μL serum-free medium and incubated at room temperature for 5 min, respectively. Then the solution was mixed and incubated at room temperature for 20 min and added into cells. Cells were further incubated in a 5% CO2 incubator at 37℃ for 24 h.

Statistical analysis
All experiments were performed in triplicates and all statistical data were expressed as ± standard deviation (SD). The comparisons among multiple groups were performed using one-way analysis of variance (ANOVA). If the variance was homogeneous, LSD test was used for further pairwise comparison, else nonparametric test was used and Kruskal Wallis h test was used for further pairwise comparison via SPSS 24.0. A value of P < 0.05 was considered statistically significant.

Butyric acid treatment increased the apoptotic rate of adipocytes
HPA-s were cultured in PADM and observed under microscope. After 4 d of culturing, lipid droplets were observed, which were growing into larger droplets with the extension of induction time. After 7 d, the lipid droplets in the cytoplasm were stained using oil red O staining (Fig. 1A), whose results indicated that HPA-s were successfully differentiated into adipocytes. For determining the appropriate concentration of butyric acid, 1, 2.5, 5 and 10 mmol/L of butyric acid were added into adipocytes, after which cells were cultured at 37 ℃ in a 5% CO2 incubator for 24 h.
Then cell viability of each group was detected. As presented in Fig. 1B, compared with the control group, the cell viability was significantly decreased in butyric acid group when the concentration was 5 and 10 mmol/L (P < 0.001). Therefore, 2.5 mmol/L of butyric acid was selected for subsequent experiments. After 48 hours of hypoxia or butyric acid treatment (Fig. 1C and 1D), no significantly difference in the apoptotic rate were observed in CIH group compared with control group (P > 0.05).
However, the apoptotic rate in butyric acid group was significantly increased (P < 0.001). The OD value in the CIH group was dramatically increased (Fig. 1E), while it was decreased in butyric acid group compared with control group (P < 0.001). These results suggested that hypoxia treatment had no significant effect on apoptosis and could promote the proliferation of adipocyte, while butyric acid treatment would induce adipocyte apoptosis and suppress cell proliferation.

Butyric acid alleviated CIH-induced lipid formation, cell proliferation and inflammatory status in adipocytes
For investigating the effects of butyric acid on CIH-treated adipocytes, oil red O staining was used and results showed that the lipid droplets were increased in the CIH group, while addition of butyric acid could reverse the effects ( Fig. 2A). Flow cytometry analysis was further implemented to determine the cell apoptosis. As shown in Fig. 2B and 2C, the apoptotic rate was significantly increased in CIH + butyric acid group compared with control group and CIH group (P < 0.001). Cell viability results (Fig. 2D) demonstrated that though cell viability was increased in CIH group compared with control group (P < 0.001), the treatment of butyric acid could decrease the cell viability but could not recover to the level in control group (P < 0.001). Moreover, inflammation status was evaluated via detection the level of inflammatory related proteins using western blotting ( Fig. 2E and 2F). Data revealed that the expressions of IL-1β, IL-6, IFN-r and TLR4 were upregulated in CIH group compared with control group, however, these expressions were downregulated in CIH + butyric acid group compared with CIH group (P < 0.01). RT-qPCR analysis was employed for determining relative mRNA expression. Fig. 2G illustrated that the expression of TLR4 was upregulated in CIH group compared with control group, while treatment of butyric acid had reverse effect but could not recover to the normal

Butyric acid inactivated AMPK pathway and upregualted HuR expression
In our pre-experiments, it was showed that the level of HuR was significantly decreased under CIH treatment, while butyric acid could upregulate HuR expression.
Therefore, we hypothesized that HuR might be an important regulatory factor in CIH treatment, which was further knockdown using HuR-siRNA to investigate its molecular mechanism. After transfection of HuR-siRNA into CIH group, the mRNA level of HuR was detected using RT-qPCR for efficacy verification. Results presented in Fig. 3A indicated that the expression of HuR was downregulated in siRNA1, siRNA2 and siRNA3 group compared with control group and siRNA-NC group (P < 0.001). Consistently, the implementation of western blotting also demonstrated that transfection of HuR-siRNA could downregulate HuR expression (Fig. 3B). Then AICAR, a kind of AMPK pathway activator, was also added into adipocytes. Western blotting results presented in Fig. 3C showed that the treatment of butyric acid in CIH group could upregulate the expression of HuR in both cytoplasm and nucleus and downregulate the expression of p-AMPK and p-ACC (P < 0.05). However, the treatment of AICAR and HuR-siRNA could suppress HuR expression, demonstrating that activation of AMPK might lead to the decrease of HuR level.
As shown in Fig. 4A-4D, the expression of p21, PPARγ, C/EBPa, IL-1β, IL-6 and TLR4 were downregulated, while caspase-8 and caspase-3 were upregulated in CIH + butyric acid group compared with CIH group. However, the activation of AMPK using AICAR or suppressing of HuR using HuR-siRNA had the reverse effect, while no significant change on the relative expression of cleaved caspase-8 and cleaved caspase-3 was observed. Consistently, RT-qPCR results in Fig. 5A and 5B also demonstrated the phenomenon. These evidences suggested that butyric acid could inhibit the formation of lipid and alleviate inflammatory status via regulating the expression of HuR. The present study was designed to determine the effect of HuR, which might be regulated by AMPK. Compared with CIH treatment, butyric acid increased the apoptotic rate of adipocytes. To investigate the effects of butyric acid on CIH-treated adipocytes, oil red O staining was used, and inflammation status was evaluated via detection the level of inflammatory-related proteins by western blotting. It was suggested that butyric acid alleviated CIH-induced lipid formation, cell proliferation and inflammatory status in adipocytes. According to the result of western blotting, the treatment of AICAR and HuR-siRNA could suppress HuR expression, indicating that activation of AMPK might lead to the decrease of HuR level. Next, expression of inflammation-related proteins, HuR, and AMPK were determined by qPCR. It was interesting to note that in this study butyric acid could inhibit the formation of lipid and alleviate inflammatory status via regulating the expression of HuR. However, the Therefore, a further study with more focus on the limitation above is therefore suggested.

Competing interests
The authors declare that they have no competing interests.

Funding
This study was supported by National Natural Science Foundation of China