rhEPO Upregulates the PPARγ Pathway in Long-Term Cultured Primary Nerve Cells via PI3K/Akt to Delay Cell Senescence

doi:10.21203/rs.3.rs-739611/v1

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rhEPO Upregulates the PPARγ Pathway in Long-Term Cultured Primary Nerve Cells via PI3K/Akt to Delay Cell Senescence

https://doi.org/10.21203/rs.3.rs-739611/v1

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Previous studies have confirmed that both recombinant human erythropoietin(rhEPO) and peroxisome proliferatoractivated receptorsγ (PPARγ) activator pioglitazone can protect senescent nerve cells, and their mechanisms involve enhancing cell antioxidant capacity and reducing cell apoptosis. However, whether the PPARγ pathway is involved in the rhEPO anti-aging process in neuronal cells is still unclear. In this study, to explore the relationship between rhEPO and the PPARγ pathway at the cellular level, primary nerve cells cultured for 22 days were used to simulate the natural aging process of nerve cells. Starting on the 11th day of culture, rhEPO, LY294002 and GW9662 were added for treatment. Immunochemical methods and SA-β-gal staining were used to observe the changes in cellular antioxidant capacity and the fraction of senescent cells. The results showed that PPARγ blockade retarded the effect of rhEPO on the cellular antioxidant capacity and altered the fraction of senescent cells. It was confirmed that PPARγ was involved in rhEPO’s anti-aging process in neuronal cells. Real-time fluorescent quantitative RT-PCR, Western blotting and immunofluorescence staining were used to observe the changes in PPARγ pathway-related factors in nerve cells after rhEPO treatment. The results showed that rhEPO significantly upregulated the expression of PPARγ coactivator-1α(PGC-1α), PPARγ and nuclear PPARγ in cells but did not affect the level of phosphorylated PPARγ protein, confirming that rhEPO has the ability to upregulate the PPARγ pathway. PI3K/Akt and PPARγ pathway blockade experiments were used to explore the relationships among rhEPO, PI3K/Akt and PPARγ. The results showed that after PPARγ blockade, rhEPO had no significant effect on the PI3K/Akt pathway-related factor p-Akt, while after PI3K/Akt blockade, rhEPO’s effects on PPARγ-related factors (PGC-1α, PPARγ and nuclear PPARγ) were significantly decreased. It is suggested that rhEPO delays the PI3K/Akt pathway in the process of neuronal senescence, which is located upstream of PPARγ regulation. In conclusion, this study confirmed that rhEPO can upregulate the expression of PGC-1α and PPARγ in cells and the level of PPARγ protein in the nucleus to enhance the antioxidant capacity of cells and delay the senescence of nerve cells through the PI3K/Akt pathway. These findings will provide ideas for finding new targets for neuroprotection research and will also provide a theoretical basis and experimental evidence for rhEPO anti-aging research in neural cells.

Erythropoietin

Aging

Nerve cells

Peroxisome proliferatoractivated receptorsγ

PI3K/Akt pathway

Erythropoietin(EPO) is an important glycoprotein hormone in the body that is widely distributed in the central and peripheral nervous systems(Genc et al.,2004). Studies have shown that EPO has neuroprotective effects in various forms of nerve damage resulting from ischemia and hypoxia(Sirén et al.,2001;Simon et al.,2019), exogenous toxic substances(Mammis et al.,2009), primary or secondary inflammatory reactions(Nagańska et al.,2010), and nervous system aging(Wang et al.,2017). The PI3K/Akt signaling pathway exerts a wide range of biological effects in the nervous system(Feng et al.,2021;Tang et al.,2019). A number of studies have found that EPO can exert its biological effects through the PI3K/Akt pathway under both basic and stress conditions(Shen et al.,2010;Si et al.,2019).

Peroxisome proliferator-activated receptor γ (PPARγ) is a type of nuclear transcription factor activated by ligands and is a member of the nuclear receptor superfamily. Activated PPARγ combines with peroxisome response elements (PPAR response elements, PPREs) in the promoter region of the gene to initiate transcription of target genes and play a biological role(Berger and Moller,2002;Lehrke and Lazar,2005). Recent experiments have shown that the activation of the PPARγ pathway has a significant protective effect on various acute and slow forms of damage in the central nervous system and may be a new target for the treatment of various diseases, such as ischemic cerebrovascular disease(Li et al.,2020), Alzheimer’s disease(Khan et al.,2019),Parkinson’s disease(Corona and Duchen,2015)and Multiple sclerosis(Liu et al.,2020). Its mechanism involves regulating oxidative stress(Collino et al.,2006), inhibiting inflammation(Lecca et al.,2015) and reducing cell apoptosis(Kaundal and Sharma,2011). Our previous studies have shown that activation of the PPARγ pathway can enhance the antioxidant capacity of cells, reduce cell apoptosis, and protect senescent long-term cultured primary nerve cells(Wang et al.,2019).

The mechanism involved in the neuroprotection of EPO is similar to the neuroprotection mechanism activated by PPARγ, but it is still unclear whether there is a cross-connection between the two. Based on this, this study aimed to investigate and clarify the relationship between recombinant human erythropoietin(rhEPO) and the PPARγ pathway at the cellular level by observing the changes in PPARγ pathway-related factors and the fraction of senescent cells in the long-term culture of primary nerve cells after rhEPO treatment to find new targets for neural anti-aging research.

1.1 Long-term culture of primary nerve cells (Wang et al.,2017;Wang et al.,2019)

One-day-old SD rats (Animal Experiment Center of Wuhan Buffal Biotechnology Co., Ltd., No. 42000600012249) were killed by cervical dislocation. The cerebral cortex was digested with 0.125% trypsin, filtered, and centrifuged for 5 min (800 n/min), and Neurobasal (Invitrogen Gibco, New York, USA) + B27 medium (Invitrogen Gibco, New York, USA) was used to prepare the cell suspension. The cells were inoculated in culture wells treated with polylysine and cultivated at 37°C at 5% CO₂ with a saturated humidity. Half of the medium was changed every 2 days for a total of 22 days of culture. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal use protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Xi’an Jiaotong University.

1.2 Experimental grouping

For the aging control group, the cells were cultured for a total of 22 days, representing aging nerve cells. For the EPO group, rhEPO (100 U/ml, PeproTech, Rocky Hill, USA) was added to the cells on the 11th day(Wang et al.,2017). For the EPO+ LY294002 group, LY294002 (5 µM, Selleck, Houston, USA) and rhEPO (100 U/ml, PeproTech, Rocky Hill, USA) were added starting on the 11th day. For the LY294002 group , LY294002 (5 µM, Selleck, Houston, USA) was added on the 11th day(Petrozziello et al.,2017). For the EPO+GW9662 group, GW9662 (1 µM, Selleck, Houston, USA) and rhEPO (100 U/ml, PeproTech, Rocky Hill, USA) were added starting on the 11th day. For the GW9662 group, GW9662 (1 µM, Selleck, Houston, USA) was added starting on the 11th day(Wang et al.,2019). For the pioglitazone group, pioglitazone (1 µM, Selleck, Houston, USA) was added starting on the 11th day(Wang et al.,2019).

1.3 Observations of the fraction of senescent cells by SA-β-gal staining and of the antioxidant capacity by immunochemical methods (Wang et al.,2017)

The fraction of senescent cells was observed by SA-β-gal staining (Nanjing KeyGen Biotech Co. Ltd., Nanjing, China). Superoxide dismutase (SOD) and Glutathione (GSH) are important intracellular antioxidant enzyme.Malondialdehyde (MDA) is the final product of intracellular oxidation reactions.they represent the intracellular antioxidant levels.SOD activity,GSHand MDA contents were determined respectively using a SOD assay kit (Jiancheng Bioengineering, Nanjing, China), a GSH detection kit (Jiancheng Bioengineering, Nanjing, China) and an MDA assay kit (Jiancheng Bioengineering, Nanjing, China).

1.4 Real-time fluorescent quantitative RT-PCR detection of PPARγ and PGC-1α mRNA

Total mRNA was extracted from the hippocampus of rats using TRIzol reagent (Aidlab, Beijing, China), and complementary DNA was then reverse-transcribed from 1 μg of total RNA with a reverse transcription kit (Vazyme Biotech Co., Ltd., Nanjing, China) according to the manufacturer’s instructions. PCR was performed using SYBR Green Master Mix (Vazyme Biotech Co., Ltd., Nanjing, China), and β-actin served as the internal control. The relative expression levels of PPARγ and PGC-1α were determined using the 2−ΔΔCt method and are shown as fold changes compared to the controls. The primers were 5'-AGGGCGATCTTGACAGGAAA-3' (forward) and 5'-CGAAACTGGCACCCTTGAAA-3' (reverse) for PPARγ, 5'-CACTACAGACACCGCACACA-3' (forward) and 5'-CAGACT CCCGCTTCTCA TAC-3' (reverse) for PGC-1α, and 5'- CACGATGGAGGGGCCGGACTCATC-3' (forward) and 5'- TAAAGACCTCTATGCCAACACAGT-3' (reverse) for β-actin. All primers were designed and synthesized by Tianyi Biotech (Wuhan, China). The cycling conditions included heat treatment at 95°C for 10 min, one cycle of 95°C for 1 min, and 40 cycles of 95°C for 30 seconds, 60°C for 1 min, and 72°C for 1 min, followed by one cycle at 72°C for 10 min. The specificity of amplification was assessed by melting curve analysis and gel electrophoresis.

1.6 Western blot detection of phosphorylated Akt(p-Akt), PPARγ, PGC-1α and phosphorylated PPARγ(p-PPARγ)

The cells were lysed with RIPA lysate, the protein was extracted, and the protein concentfractionn was measured with a DG-3022A microplate reader. After the electrophoresis gel was prepared, the prepared protein samples and marker were loaded into the wells with a micropipette, with the total protein content of each sample of 40 µg. After loading the samples, the gels were electrophoresed at a constant voltage of 80 V until the bromophenol blue indicator dye formed a line at the junction of the concentrating gel and the sepafractionn gel, after which the voltage was changed to 120 V until the bromophenol blue reached the bottom of the gel. The gel was removed, and the target band was excised according to the marker, followed by rinsing with distilled water and transfer to a membrane. For Western blot hybridization after transfer, the following primary antibodies were used: rabbit anti-rat p-Akt antibody (1:1000, species: rabbit, CST, Boston, USA), rabbit anti-rat PGC-1α antibody (1:1000, species: rabbit, Thermo Fisher Scientific, Waltham, USA), rabbit anti-rat PPARγ antibody (1:1000, species: rabbit, Sanyin Biological Technology Co. Ltd., Wuhan, China), rabbit anti-rat p-PPARγ (1:300, species: rabbit, Santa Cruz Biotechnology, Santa Cruz, USA), mouse anti-rat β-actin (1:200, species: Mouse, Boster Biological Technology Co. Ltd., Wuhan, China), and rabbit anti-rat Lamin B antibody (1:200, species: rabbit, Boster Biological Technology Co. Ltd, Wuhan, China). The primary antibodies were incubated with the membrane overnight at 4°C. After washing, HRP goat anti-rabbit (1:5000, species: goat, Boster Biological Technology Co. Ltd., Wuhan, China) and HRP goat anti-mouse (1:5000, species: goat, Boster Biological Technology Co. Ltd., Wuhan, China) secondary antibodies were added. The ECL chemiluminescence method was used to detect protein bands, and the image was recorded by a gel imager. The BandScan system was used to analyze the film gray value.

1.7 PPARγ immunofluorescence staining

After fixation with 4% paraformaldehyde for 15 min, immersion and washing were performed, and 0.5% Triton X-100 was used for permeabilization at room temperature for 20 min. Goat serum was used for blocking for 30 min. The primary antibody against PPARγ (1:50, species: rabbit, Sanyin Biological Technology Co. Ltd., Wuhan, China) was added and incubated for 24 hours at 4°C in a humid box. Fluorescence (Cy3)-labeled goat anti-rabbit IgG (1:100, species: goat, Boster Biological Technology Co. Ltd., Wuhan, China) was added, incubated at 37°C for 1 hour in a humid box, and then blocked with goat serum for 30 min. DAPI was added and incubated in the dark for 5 min, followed by mounting with mounting solution containing an anti-fluorescence quencher. The cells were observed under a laser scanning confocal microscope, and images were collected. CY3 exhibits red fluorescence with an excitation wavelength of 550 nm and an emission wavelength of 570 nm, and DAPI exhibits blue fluorescence with an excitation wavelength of 340 nm and an emission wavelength of 488 nm. IPP6.0 software was used to analyze the optical density of the immunofluorescence photos.

1.8 Statistical analysis

All data were analyzed using SPSS (version 23.0) software. All experiments were performed in triplicate, and the data are presented as the mean ± standard deviation (SD). Data were analyzed using one-way analysis of variance (ANOVA). The level of statistical significance was set at P < 0.05.

2.1 PPARγ participates in the process by which rhEPO enhances the antioxidant capacity of cells and delays the aging process of nerve cells

Superoxide dismutase (SOD) and Glutathione (GSH) are important intracellular antioxidant enzyme.Malondialdehyde (MDA) is the final product of intracellular oxidation reactions.They represent the intracellular antioxidant levels.To clarify whether PPARγ is involved in the process by which rhEPO enhances the antioxidant capacity of long-term cultured primary nerve cells, immunochemical methods to were used determine the changes in cellular antioxidant enzyme SOD activity and GSH and MDA content after rhEPO and GW9662 treatment (Figure 1). The results showed that the SOD activity (P =0.0015 VS. aging control group) and GSH content (P =0.0003 VS. aging control group) of the cells in the EPO group were significantly higher than those in the aging control group, while the MDA content was significantly reduced (P =0.0005 VS. aging control group). SOD activity (P =0.0311 VS. EPO group) and GSH content (P =0.0036 VS. EPO group) of the cells in the EPO+GW9662 group were significantly lower than those in the EPO group, while the MDA content increased significantly (P =0.0103 VS. EPO group), but each index still did not reach the corresponding index level of aging control group (SOD: P =0.0110 VS. aging control group; GSH: P =0.0033 VS. aging control group; MDA: P =0.0044 VS. aging control group). SOD activity in GW9662 group(P =0.0029 VS. aging control group, P =0.0003 VS. EPO group, P =0.0009 VS. EPO+GW9662 group) and GSH content (P =0.0160 VS. aging control group, P =0.0002 VS. EPO group, P =0.0009 VS. EPO+GW9662 group) were significantly lower than those of the other threEPO groups, while the MDA content was significantly increased (P =0.0051 VS. aging control group, P =0.0005 VS. EPO group, P =0.0011 VS. EPO+GW9662 group). All indicators of pioglitazone group were better than those of aging control group (SOD: P =0.0193 VS. aging control group; GSH: P =0.0066 VS. aging control group; MDA: P =0.0013 VS. aging control group), but worse than EPO group (SOD: P =0.0100 VS. EPO group; GSH: P =0.0013 VS. EPO group; MDA: P =0.0321 VS. EPO group).

To observe whether PPARγ is involved in the process by which rhEPO reduces the fraction of senescent cells in long-term cultured primary neural cells, SA-β-gal staining was used to detect the changes in the fraction of senescent cells in each group after rhEPO and GW9662 treatment (Figure 2). The results showed that the fraction of senescent cells in the EPO group was significantly lower than that in aging control group (P =0.0001 VS. aging control group). The fraction of senescent cells in the EPO+GW9662 group was significantly higher than that in the EPO group (P =0.0131 VS. EPO group) , but still lower than that in the aging control group (P =0.0001 VS. aging control group). The fraction of senescent cells in the GW9662 group was significantly higher than that in EPO group (P <0.0001 VS. EPO group) and EPO+GW9662 group(P =0.0001 VS.EPO+GW9662 group). The fraction of senescent cells in pioglitazone group was lower than that in aging control group (P =0.0001 VS. aging control group), but higher than that in EPO group (P =0.0058 VS. EPO group).

2.2 Inhibition of PPARγ does not affect rhEPO activation of the PI3K/Akt pathway

Akt phosphorylation is a sign of PI3K/Akt pathway activation(He et al.,2021). To clarify whether PPARγ affects rhEPO to delay the activation of the PI3K/Akt pathway in the process of nerve cell aging, Western blotting was used to detect the expression of phosphorylated Akt (p-Akt) protein in nerve cells after PPARγ inhibition (Figure 3). The results showed that the p-Akt protein expression levels of the EPO group and EPO+GW9662 group were significantly higher than that of the aging control group (P = 0.0015 VS. EPO group, P = 0.0011 VS. EPO+GW9662 group). However, there was no significant difference between the EPO group and the EPO+GW9662 group (P >0.05).

2.3 rhEPO upregulates the expression of PPARγ and PGC-1α mRNA in long-term cultured primary neural cells through the PI3K/Akt pathway

PGC-1α is an important transcriptional cofactor of PPARγ and plays an important role in promoting the transcriptional activity of PPARγ(Rowe et al.,2010). PGC-1α and PPARγ are key factors in the PPARγ pathway. To clarify whether rhEPO upregulates the expression of PPARγ and PGC-1α mRNA in long-term cultured primary neural cells through the PI3K/Akt pathway, real-time fluorescent RT-PCR was used to observe the expression of PPARγ and PGC-1α mRNA in nerve cells after rhEPO and LY294002 treatment (Figure 4). The results showed that the mRNA expression of PPARγ and PGC-1α in the EPO group was significantly higher than that in the aging control group (PPARγ: P = 0.0002 VS. aging control group; PGC-1α: P = 0.0003 VS. aging control group), while the expression of PPARγ and PGC-1α mRNA in the EPO+ LY294002 group was significantly lower than that in the EPO group (PPARγ: P = 0.0007 VS. EPO group; PGC-1α: P = 0.0020 VS. EPO group). The mRNA expression of PPARγ and PGC-1α in the LY294002 group was significantly lower than that in the other groups (PPARγ: P = 0.008 VS. aging control group, P = 0.0001 VS. EPO group, P = 0.0004 VS.EPO+ LY294002 group; PGC-1α: P = 0.0011 VS. aging control group, P = 0.0001 VS. EPO group, P = 0.0006 VS.EPO+ LY294002 group).

2.4 rhEPO upregulates the protein expression of PGC-1α and PPARγ in long-term cultured primary neural cells through the PI3K/Akt pathway but does not affect the phosphorylation level of the PPARγ protein

PGC-1α protein plays an important role in promoting the transcriptional activity of PPARγ. To determine whether rhEPO upregulates the expression of PGC-1α in senescent long-term cultured primary neurons through the PI3K/Akt pathway, Western blotting was used to detect changes in the PGC-1α protein (Figure 5a) in nerve cells after rhEPO and LY294002 treatment. The results showed that the PGC-1α protein level in the EPO group was significantly higher than that in the aging control group (P = 0.0409 VS. aging control group), while the PGC-1α protein level in the EPO+ LY294002 group was significantly lower than that in the EPO group (P = 0.0213 VS. EPO group). However, there was no significant difference compared with the aging control group. The protein level of PGC-1α in the LY294002 group was significantly lower than that of the other groups (P = 0.0006 VS. aging control group, P = 0.0003 VS. EPO group, P = 0.0025 VS. aging control group).

The expression level of PPARγ protein is an important indicator of PPARγ pathway activation in cells(Berger and Moller,2002). To clarify whether rhEPO upregulates the expression of PPARγ in long-term cultured primary neurons through the PI3K/Akt pathway, Western blotting was used to detect the expression of whole PPARγ protein (w-PPARγ) in nerve cells after rhEPO and LY294002 treatment (Figure 5b). The results showed that the w-PPARγ protein level of the EPO group was significantly higher than that of the aging control group (P = 0.0062 VS. aging control group). The w-PPARγ protein level of the EPO+ LY294002 group was significantly lower than that of the EPO group (P = 0.0292 VS. EPO group), but there was no significant difference with aging control group. The w-PPARγ protein level in LY294002 groupwas significantly lower than that of the other groups (P = 0.0080 VS. aging control group, P = 0.0008 VS. EPO group, P = 0.0034 VS. EPO+ LY294002 group).

Phosphorylation of PPARγ is an important modification pathway of the PPARγ protein(Montanari et al.,2020) and is one of the main factors affecting the transcriptional activity of the PPARγ pathway. The results of Western blotting in this study showed that the levels of phosphorylated PPARγ (p-PPARγ) protein in nerve cells in each group were not significantly different (P >0.05) (Figure 5b).

2.6 rhEPO increases the level of PPARγ protein in the nucleus of long-term cultured primary nerve cells through the PI3K/Akt pathway

PPARγ enters the nucleus to bind to PPREs, and thus the level of PPARγ protein in the nucleus represents the ability of PPARγ to initiate gene transcription(Orzechowska-Jastrząb et al.,2019). To determine whether rhEPO can increase the PPARγ protein level in the nucleus of long-term cultured primary nerve cells through the PI3K/Akt pathway, immunofluorescence staining was used to observe the distribution of PPARγ in long-term cultured primary nerve cells after rhEPO and LY294002 treatment. The results showed that PPARγ was distributed in the cytoplasm and nucleus of primary nerve cells in each group (Figure 6a). Statistical analysis demonstrated that the PPARγ expression intensity and nuclear-positive cell fraction of the EPO group were significantly higher than those of the aging control group (cellular PPARγ expression intensity: P = 0.0257 VS. aging control group; nuclear-positive cell fraction: P = 0.0080 VS. aging control group), while the expression intensity of PPARγ and the fraction of nuclear-positive cells in the EPO +LY294002 group were significantly lower than those in EPO group (PPARγ expression intensity of cells: P = 0.0184 VS. EPO group; fraction of nuclear-positive cells: P = 0.0312 VS. EPO group), but there was no significant difference with aging control group. The expression intensity of PPARγ and the fraction of nuclear-positive cells in the LY294002 group were significantly lower than those of the other groups (the expression intensity of PPARγ in cells: P = 0.0424 VS. aging control group, P = 0.0007 VS. EPO group, P = 0.0022 VS.EPO+ LY294002 group; the fraction of nuclear-positive cells: P = 0.0443 VS. aging control group, P = 0.0003 VS. EPO group, P = 0.0041 VS. EPO+ LY294002 group) (Figure 6b).

To further observe the effect of rhEPO on the PPARγ protein level in the nucleus of long-term cultured primary nerve cells through PI3K/Akt, Western blotting was used to detect the PPARγ protein level in the nerve cell nucleus (n-PPARγ) after rhEPO and LY294002 treatment (Figure 6c). The results showed that the n-PPARγ protein level of the EPO group was significantly higher than that of the aging control group (P = 0.0044 VS. aging control group), while the n-PPARγ level of the EPO+ LY294002 group was significantly lower than that of the EPO group (P = 0.0022 VS. EPO group), but there was no significant difference with aging control group. The level of n-PPARγ in LY294002 groupwas significantly lower than that of the other groups (P = 0.0007 VS. aging control group, P = 0.0003 VS. EPO group, P = 0.0022 VS. EPO+ LY294002 group).

PPARγ is a nuclear transcription factor activated by ligands. Activated PPARγ enters the nucleus and binds to PPREs under the combined action of coactivators, initiates target gene transcription, and participates in various physiological activities of the body(Harada etal.,2015;Zhu et al.,2021;Shang et al.,2020;Álvarez-Almazán et al.,2017). Existing studies have shown that PPARγ pathway activation has an important neuroprotective effect on a variety of central nervous system pathological changes and have proposed that PPARγ may be a therapeutic target for neurodegenerative diseases(Li et al.,2020;Khan et al.,2019;Corona and Duchen,2015;Liu et al.,2020;Katsouri et al.,2012). Our previous studies have shown that after treatment with the PPARγ inhibitor GW9662, the antioxidant capacity of long-term cultured primary nerve cells decreases and the apoptotic cell fraction increases, while after treatment with the PPARγ agonist pioglitazone, the cell antioxidant capacity significantly increases and the apoptotic cell fraction decreases significantly. It is suggested that the PPARγ pathway is involved in the protection from senescence and the long-term culture of primary nerve cells and that it is one of the protective pathways of aging nerve cells(Wang et al.,2019). Another study of ours showed that the mechanism by which rhEPO protects senescent nerve cells is the same as that of the PPARγ pathway activator pioglitazone, which also involves the enhancement of intracellular antioxidant capacity(Wang et al.,2017). However, whether the PPARγ pathway is involved in the anti-senescence process of rhEPO in neuronal cells has not yet been reported. This study showed that the antioxidant capacity of cells in the EPO+GW9662 group was significantly lower than that of the EPO group and that the fraction of senescent cells was significantly increased. It is suggested that the PPARγ pathway is involved in the physiological process of rhEPO's enhancement of the antioxidant capacity of cells and protection against senescence in the long-term culture of primary nerve cells.

The transcriptional activity of PPARγ is affected by the expression level of PPARγ(Berger and Moller,2002), protein phosphorylation(Montanari et al.,2020), and the intensity of ligand stimulation(Santos et al.,2021). The level of PPARγ in the nucleus(Orzechowska-Jastrząb et al.,2019) and transcription cofactors such as PGC-1α(Zhang et al.,2018) are also critical. To further explore the effect of rhEPO on the PPARγ pathway in long-term cultured primary nerve cells, this study compared the expression of the PPARγ pathway-related factors PPARγ and PGC-1α after rhEPO treatment. Both RT-PCR and western blotting results showed that rhEPO significantly enhanced the expression of the PPARγ pathway-related factors PPARγ and PGC-1α in nerve cells and increased the level of PPARγ in the nucleus but did not affect the phosphorylation of PPARγ. This proves that rhEPO can significantly increase PPARγ pathway activity in long-term cultured nerve cells, but the mechanism is still unclear.

The PI3K/Akt pathway is one of the key pathways by which EPO protects nerve cells(Shen et al.,2010;Si et al.,2019;Ma et al.,2018), but it is not clear whether there is a connection between the PI3K/Akt pathway and the PPARγ pathway in the process by which rhEPO protects senescent nerve cells. Previous studies have shown that the PI3K/Akt pathway is the downstream pathway of PPARγ in most cases and that it can affect the activity of the PI3K/Akt pathway by regulating the phosphorylation level of Akt protein(Li et al.,2014;Li et al.,2018;Lv et al.,2019). Based on this, we speculate that the PI3K/Akt pathway may also be a downstream pathway of PPARγ in the process by which EPO protects senescent nerve cells. To verify this hypothesis, in this study, we first compared and observed the changes in the PI3K/Akt pathway-related protein p-Akt after rhEPO treatment while inhibiting the PPARγ pathway. The results showed that there was no significant change in the protein expression of p-Akt between the EPO+GW9662 group and the EPO group. This suggests that the PI3K/Akt pathway is not located downstream of the PPARγ pathway in the process of rhEPO's protection from senescence in the long-term culture of primary nerve cells, and its relationship with the PPARγ pathway is different from what has been demonstrated in previous studies.

The PI3K/Akt pathway affects the expression of a variety of proteins in cells(Xie et al.,2019;Xu et al.,2020), and PPARγ pathway-related factors are regulated by a variety of proteins(Villapol,2018;Vallée and Lecarpentier,2018;Chen et al.,2019). Since the PI3K/Akt pathway is not downstream of rhEPO's activation of the PPARγ pathway for protection from senescence in long-term cultured primary nerve cells, is it upstream of the factors that regulate the PPARγ pathway? To solve this problem, we observed changes in the protein and mRNA levels of the PPARγ pathway-related factors PPARγ and PGC-1α in long-term cultured primary nerve cells while inhibiting the PI3K/Akt pathway after rhEPO treatment. The results showed that the PGC-1α, w-PPARγ, and n-PPARγ protein and mRNA levels in the EPO+ LY294002 group were significantly lower than those in the EPO group, while the p-PPARγ protein level did not change significantly. This result confirms that the PI3K/Akt pathway is upstream of the PPARγ pathway in the process by which rhEPO protects nerve cells from senescence. This shows that rhEPO can upregulate the expression of PGC-1α and PPARγ in cells and the level of PPARγ protein in the nucleus by activating the PI3K/Akt pathway, and play a role in protecting senescent nerve cells. However, its specific regulatory molecular mechanism is still unclear and needs further study.

At the same time, we also found an interesting phenomenon in the study: the PGC-1 α and w-PPARγ proteins in the EPO+ LY294002 group were significantly lower than those in the EPO group, and there was no significant difference compared with the aging control group, but in the EPO+ LY294002 group, the mRNA levels of PGC-1 α and PPARγ were significantly lower than those of EPO group, although they were still higher than those of aging control group. This difference in protein and mRNA levels suggests that PI3K/Akt pathway inhibition at the protein level can almost completely block the effect of rhEPO on the PPARγ pathway of nerve cells, but it does not achieve a complete blocking effect at the mRNA level. Analysis suggests that the reason for this phenomenon may be that PI3K/Akt pathway regulation of PPARγ-related factors not affects not only gene transcription but may also the translation and modification of related proteins by regulating the activity of certain modified enzymes. In addition, this study also found that the protein and mRNA levels of w-PPARγ, n-PPARγ, and PGC-1α in the LY294002 group of nerve cells in which the PI3K/Akt pathway was inhibited were significantly lower than those in the aging control group. It is suggested that even without rhEPO treatment, the PI3K/Akt pathway may be one of the important upstream pathways that regulates the activity of the PPARγ pathway in the process of neuronal senescence. This is the direction of our further research in the future.

In this study, by first observing the effects of rhEPO treatment in combination with a PPARγ inhibitor to hinder changes in cell antioxidant capacity and the fraction of senescent cells, it was confirmed that PPARγ participates in rhEPO's anti-aging process in neuronal cells. Furthermore, by observing the changes in PPARγ pathway-related factors in long-term cultured primary nerve cells after rhEPO treatment, it was confirmed that rhEPO has the ability to upregulate the cellular PPARγ pathway. Finally, PPARγ and PI3K/Akt pathway blockade experiments confirmed that rhEPO delays the process of neuronal senescence and that the PI3K/Akt pathway is located upstream of PPARγ regulation. In conclusion, this study confirms that rhEPO can upregulate the expression of PGC-1α and PPARγ in cells and the level of PPARγ protein in the nucleus through the PI3K/Akt pathway, enhance cell antioxidant capacity, and delay the senescence of primary neurons in long-term culture, but its specific regulatory molecules are unclear, and further research is needed.

Disclosures

None of the authors have any disclosures.

Acknowledgments

This research was supported by the National Natural Science Foundation of China (No.81170330).

Conflicts of interest

All of the authors declare that they have no conflicts of interest regarding this paper.

ETHICAL STATEMENT:
Ethics approval and consent to participate: This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal use protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Xi’an Jiaotong University.
Consent for publication: No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication.
Availability of data and materials: No applicable
Competing interests: The authors declare that they have no competing financial interests.
Funding: This research was supported by the National Natural Science Foundation of China (No.81170330).
Authors' contributions: Huqing Wang, Xiaorui Yu and Haiqin Wu designed the study. Ming Chen and Tao Zhang participated in several steps of the experiment: long-term culture of primary nerve cells and observations of the antioxidant capacity by immunochemical methods. Zhen gao and Yu Gong participated in the SA-β-gal staining, immunofluorescence staining, RT-PCR and Western blot. Huqing Wang participated in the whole process of the experiment and wrote the manuscript. Xiaorui Yu and Haiqin Wu revised the manuscript.

Álvarez-Almazán S, Bello M, Tamay-Cach F, Martínez-Archundia M, Alemán-González-Duhart D, Correa-Basurto J, Mendieta-Wejebe JE,2017. Study of new interactions of glitazone's stereoisomers and the endogenous ligand 15d-PGJ2 on six different PPAR gamma proteins[J]. Biochem Pharmacol,142:168-193.
Berger J, Moller DE,2002. The mechanisms of action of PPARs[J]. Annu Rev Med,53:409-35.
Chen T, Zhang Y, Liu Y, Zhu D, Yu J, Li G, Sun Z, Wang W, Jiang H, Hong Z,2019. MiR-27a promotes insulin resistance and mediates glucose metabolism by targeting PPAR-γ-mediated PI3K/AKT signaling[J]. Aging (Albany NY),11:7510-7524.
Collino M, Aragno M, Mastrocola R, Gallicchio M, Rosa AC, Dianzani C, Danni O, Thiemermann C, Fantozzi R,2006. Modulation of the oxidative stress and inflammatory response by PPAR-gamma agonists in the hippocampus of rats exposed to cerebral ischemia/reperfusion[J]. Eur J Pharmacol,530:70-80.
Corona JC, Duchen MR,2015. PPARγ and PGC-1α as therapeutic targets in Parkinson's[J]. Neurochem Res,,40:308-16.
Feng H, Gui Q, Wu G, Zhu W, Dong X, Shen M, Fu X, Shi G, Luo H, Yang X, Xue S, Cheng Q,2021. Long noncoding RNA nespas inhibits apoptosis of epileptiform hippocampal neurons by inhibiting the PI3K/Akt/mTOR pathway[J]. Exp Cell Res,398:112384.
Genc S, Koroglu TF, Genc K,2004. Erythropoietin and the nervous system[J]. Brain Res,1000:19-31.
Harada S, Hiromori Y, Nakamura S, Kawahara K, Fukakusa S, Maruno T, Noda M, Uchiyama S, Fukui K, Nishikawa J, Nagase H, Kobayashi Y, Yoshida T, Ohkubo T, Nakanishi T,2015. Structural basis for PPARγ transactivation by endocrine-disrupting organotin compounds[J]. Sci Rep,5:8520.
He Z, Yang C, He Y, Gong B, Yin C, Feng J, Chen L, Tang J, Chen Y. CAMTA1,2021. a novel antitumor gene, regulates prolifefractionn and the cell cycle in glioma by inhibiting AKT phosphorylation[J]. Cell Signal,79:109882.
Katsouri L, Blondrath K, Sastre M,2012. Peroxisome proliferator-activated receptor-γ cofactors in neurodegenefractionn[J]. IUBMB Life,64:958-64.
Kaundal RK, Sharma SS,2011. Ameliorative effects of GW1929, a nonthiazolidinedione PPARγ agonist, on inflammation and apoptosis in focal cerebral ischemic-reperfusion injury[J]. Curr Neurovasc Res,8:236-45.
Khan MA, Alam Q, Haque A, Ashafaq M, Khan MJ, Ashraf GM, Ahmad M，2019. CurrentProgress on Peroxisome Proliferator-activated Receptor Gamma agonist as an emerging therapeutic spproach for the treatment of Alzheimer's disease: an update[J]. Curr Neuropharmacol,17:232-246.
Lecca D, Nevin DK, Mulas G, Casu MA, Diana A, Rossi D, Sacchetti G, Fayne D, Carta AR,2015. Neuroprotective and anti-inflammatory properties of a novel non-thiazolidinedione PPARγ agonist in vitro and in MPTP-treated mice[J]. Neuroscience,302:23-35.
Lehrke M, Lazar MA,2005. The many faces of PPARgamma[J]. Cell,123:993-9.
Li H, Lu W, Cai WW, Wang PJ, Zhang N, Yu CP, Wang DL, Liu BC, Sun W,2014. Telmisartan attenuates monocrotaline-induced pulmonary artery endothelial dysfunction through a PPAR gamma-dependent PI3K/Akt/eNOS pathway[J]. Pulm Pharmacol Ther,28:17-24.
Li J, Zhang K, Zhang Q, Zhou X, Wen L, Ma J, Niu L, Li C,2020. PPAR-γ Mediates Ta-VNS-induced angiogenesis and subsequent functional recovery after experimental stroke in rats[J]. Biomed Res Int,2020:8163789.
Li X, Bilali A, Qiao R, Paerhati T, Yang Y,2018. Association of the PPARγ/PI3K/Akt pathway with the cardioprotective effects of tacrolimus in myocardial ischemic/reperfusion injury[J]. Mol Med Rep,17:6759-6767.
Lv S, Wang W, Wang H, Zhu Y, Lei C,2019. PPARγ activation serves as therapeutic strategy against bladder cancer via inhibiting PI3K-Akt signaling pathway[J]. BMC Cancer,19:204. Liu Y, Wang J, Luo S, Zhan Y, Lu Q.,2020 The roles of PPARγ and its agonists in autoimmune diseases: A comprehensive review[J]. J Autoimmun,113:102510.
Ma S, Chen J, Chen C, Wei N, Xu J, Yang G, Wang N, Meng Y, Ren J, Xu Z,2018. Erythropoietin rescues memory impairment in a rat model of chronic cerebral hypoperfusion via the EPO-R/JAK2/STAT5/PI3K/Akt/GSK-3β pathway[J]. Mol Neurobiol,55:3290-3299.
Mammis A, McIntosh TK, Maniker AH,2009. Erythropoietin as a neuroprotective agent in traumatic brain injury Review[J]. Surg Neurol,71:527-31.
Montanari R, Capelli D, Yamamoto K, Awaishima H, Nishikata K, Barendregt A, Heck AJR, Loiodice F, Altieri F, Paiardini A, Grottesi A, Pirone L, Pedone E, Peiretti F, Brunel JM, Itoh T, Pochetti G,2020. Insights into PPARγ phosphorylation and its inhibition mechanism[J]. J Med Chem,63:4811-4823.
Nagańska E, Taraszewska A, Matyja E, Grieb P, Rafałowska J,2010. Neuroprotective effect of erythropoietin in amyotrophic lateral sclerosis (ALS) model in vitro. Ultrastructural study[J]. Folia Neuropathol,48:35-44.
Orzechowska-Jastrząb A, Ptak-Belowska A, Targosz A,2019. Effect of TLRs and nuclear PPARγ receptors on the mechanisms of intestinal carcinogenesis[J]. Folia Med Cracov,59:23-33. DOI: 10.24425/fmc.2019.128451.PMID: 31659346.
Petrozziello T, Secondo A, Tedeschi V, Esposito A, Sisalli M, Scorziello A, Di Renzo G, Annunziato L,2017. ApoSOD1 lacking dismutase activity neuroprotects motor neurons exposed to beta-methylamino-L-alanine through the Ca2+/Akt/ERK1/2 prosurvival pathway[J]. Cell Death Differ,24:511-522.
Rowe GC, Jiang A, Arany Z,2010. PGC-1 coactivators in cardiac development and disease[J]. Circ Res,,107:825-38.
Santos DFS, Melo-Aquino B, Jorge CO, Clemente-Napimoga JT, Taylor BK, Oliveira-Fusaro MCG,2021. Prostaglandin 15d-PGJ2 targets PPARγ and opioid receptors to prevent muscle hyperalgesia in rats[J]. Neuroreport,32:238-243.
Shen J, Wu Y, Xu JY, Zhang J, Sinclair SH, Yanoff M, Xu G, Li W, Xu GT,2010. ERK and Akt-dependent neuroprotection by erythropoietin (EPO) against glyoxal-AGEs via modulation of Bcl-xL, Bax, and BAD[J].Invest Ophthalmol Vis Sci,51:35-46.Simon F, Floros N, Ibing W, Schelzig H, Knapsis A,2019. Neurotherapeutic potential of erythropoietin after ischemic injury of the central nervous system[J]. Neural Regen Res,14:1309-1312.
Shang J, Mosure SA, Zheng J, Brust R, Bass J, Nichols A, Solt LA, Griffin PR, Kojetin DJ,2020. A molecular switch regulating transcriptional repression and activation of PPARγ[J]. Nat Commun,11:956.
Si W, Wang J, Li M, Qu H, Gu R, Liu R, Wang L, Li S, Hu X,2019. Erythropoietin protects neurons from apoptosis via activating PI3K/AKT and inhibiting Erk1/2 signaling pathway[J]. 3 Biotech,9:131.
Sirén AL, Fratelli M, Brines M, Goemans C, Casagrande S, Lewczuk P, Keenan S, Gleiter C, Pasquali C, Capobianco A, Mennini T, Heumann R, Cerami A, Ehrenreich H, Ghezzi P,2001. Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress[J]. Proc Natl Acad Sci U S A, 98:4044-9.
Tang T, Gao D, Yang X, Hua X, Li S, Sun H,2019. Exogenous netrin-1 inhibits autophagy of ischemic brain tissues and hypoxic neurons via PI3K/mTOR pathway in ischemic stroke[J]. J Stroke Cerebrovasc Dis,28:1338-1345.
Vallée A, Lecarpentier Y,2018. Crosstalk between peroxisome proliferator-activated receptor gamma and the canonical WNT/β-Catenin pathway in chronic inflammation and oxidative stress during carcinogenesis[J]. Front Immunol,9:745.
Villapol S,2018. Roles of peroxisome proliferator-activated receptor gamma on brain and peripheral inflammation[J]. Cell Mol Neurobiol,38:121-132.
Wang H, Fan J, Chen M, Yao Q, Gao Z, Zhang G, Wu H, Yu X,2017. rhEPO enhances cellular anti-oxidant capacity to protect long-term cultured aging primary nerve cells[J]. J Mol Neurosci,62:291-303.
Wang H, Fan J, Chen W, Gao Z, Zhang G, Wu H, Yu X,2019. Activation of PPARγ pathway enhances cellular anti-oxidant capacity to protect long-term cultured primary rat neural cells from apoptosis[J]. Nan Fang Yi Ke Da Xue Xue Bao,39:23-29.
Xie Y, Shi X, Sheng K, Han G, Li W, Zhao Q, Jiang B, Feng J, Li J, Gu Y,2019. PI3K/Akt signaling transduction pathway, erythropoiesis and glycolysis in hypoxia (Review)[J]. Mol Med Rep, 19:783-791.
Xu F, Na L, Li Y, Chen L,2020. Roles of the PI3K/AKT/mTOR signalling pathways in neurodegenerative diseases and tumours[J]. Cell Biosci,10:54.
Zhang X, Ji R, Sun H, Peng J, Ma X, Wang C, Fu Y, Bao L, Jin Y,2018. Scutellarin ameliorates nonalcoholic fatty liver disease through the PPARγ/PGC-1α-Nrf2 pathway. Free Radic Res,52:198-211.
Zhu Y, Jing L, Li X, Zheng D, Zhou G, Zhang Y, Sang Y, Shi Z, Sun Z, Zhou X,2021. Decabromodiphenyl ether disturbs hepatic glycolipid metabolism by regulating the PI3K/AKT/GLUT4 and mTOR/PPARγ/RXRα pathway in mice and L02 cells[J]. Sci Total Environ,763:142936.

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26 Jul, 2021
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rhEPO Upregulates the PPARγ Pathway in Long-Term Cultured Primary Nerve Cells via PI3K/Akt to Delay Cell Senescence

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