Pomiferin Exerts Anti-Neuroinammatory Effects Through Activating Akt /Nrf2 Pathway and Inhibiting NF-κB pathway

Background:Neurodegenerative diseases are associated with neuroinammation. However, the proinammatory mediators produced during the occurrence of neuroinammation will damage neurons and aggravate the process of neurodegenerative diseases. Therefore, inhibiting neuroinammation may be an effective way to alleviate neurodegenerative diseases. The orange mulberry brass has a wide range of anti - oxidation and anti - inammatory effects in peripheral tissues. However, it is not clear whether it inhibits neuroinammation. Methods:In our experiment, we studied the effect of Pomiferin on BV2 cell inammation and its mechanism with Quantitative PCR, ELISA and western blot. Results:The results showed that Pomiferin inhibited the production of ROS, NO and proinammatory mediators (IL-6, TNF-α, iNOS, COX2) in BV2 cells. Further mechanism studies showed that Pomiferin activated the Akt /Nrf2 pathway and inhibited the NF-κB pathway. Conclusion: Our study demonstrated that Pomiferin exerts anti-neuroinammatory effects through activating Akt /Nrf2 pathway and inhibiting NF-κB pathway.


Introduction
Degenerative diseases are diseases in which the structure or function of neurons is gradually lost, including death, leading to dysfunction, such as Parkinson's disease (PD), Alzheimer's disease (Alzheimer's disease) and Huntington's disease (HD) etc. These diseases are mainly characterized by neurodegeneration in one or more parts of the central nervous system, with a long course and complex pathogenesis [1][2][3]. At present, it can only be alleviated in the early stage clinically, and there is no effective cure. Therefore, it is of great signi cance to study the pathogenesis of neurodegenerative diseases and search for effective therapeutic drugs.
Studies have shown that neuroimmunity and in ammatory responses are involved in the process of neurodegenerative diseases [4][5][6]. In the event of disease, immune cells are activated in large numbers and ock to the site of injury to remove harmful factors and repair damaged tissue. However, persistent in ammatory response leads to excessive activation of local immune cells in the central nervous system and accumulation of large amounts of toxic mediators such as IL-6, TNF-α, NO and PEG2, which further damages peripheral neurons and aggravates the progression of neurodegenerative disease [7,8].
Therefore, it is of great signi cance to suppress the overactivation of immune cells and the neuroin ammatory response caused by it for the remission of neurogenic diseases.
Pomiferin is a kind of avonoids extracted from the fruit of the orange mulberry. Studies show that Pomiferin s has anti -oxidation and anti -in ammatory effects. In the oxidative damage model, Pomiferin has a protective effect on the DNA damage caused by catalpa glucoside, showing a strong antioxidant activity [9,10]. In macrophages, Pomiferin activates the nuclear factor infrared 2-related factor 2 (Nrf2), triggers the transcription of antioxidant genes, exerts the antioxidant effect and the degradation of IκB α, and plays an anti-in ammatory role [11,12]. In human skin broblasts (NHDF) and normal human skin keratinocytes, Pomiferin can upregulate antioxidant genes such as ACLY, AQP3 and COX1 [13,14].Given its extensive antioxidant and anti-in ammatory activity, we hypothesized that Pomiferin might have an effect on neuroin ammation. Therefore, the purpose of this study was to explore the effect and mechanism of Pomiferin on neuroin ammation.

Cell culture
We incubated BV2 cells (purchased from shanghai Binsui Biological (Shanghai, China)) in complete medium (high glucose, 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin) in a 5% carbon dioxide cell incubator at 37℃. The cell culture medium is replaced daily. When the density reached about 80%, the cells were digested with 0.05% trypsin and subcultured into new cell culture ask.

CCK-8 assay
When the density of BV2 cells inoculated in 96-well plates reached about 50%, the cells were treated with different concentrations of Pomiferin (0.25 µM, 0.5 µM, 1 µM,) and LPS (500 ng/mL) for 24 h. Then we added CCK-8 diluent to each well and incubated it in a cell incubator for 2 h. After that, the survival rate of the cells was measured at the absorbance of 450 nm.

LDH assay
When the density of BV2 cells inoculated in 96-well plates reached about 80%, the complete culture medium was changed to incomplete culture medium. Then the cells were treated with different concentrations of Pomiferin (0.25 µM, 0.5 µM, 1 µM,) and LPS (500 ng/mL) for 24 h. After that, according to the instructions of the LDH detection kit, the LDH working uid was added to the 96-well plates, and the release of LDH was detected according to the LDH manufacturer's instructions.

ROS measurement
When the density of BV2 cells inoculated in 96-well plates reached about 80%, we added Pomiferin (1µM) and LPS (500 ng/mL) to treat the cells for 12 h. Then the ROS level was measured with a ROS detection kit according to the manufacturer's instructions.

NO measurement
When the density of BV2 cells inoculated in 96-well plates reached about 80%, we added Pomiferin (1µM) and LPS (500 ng/mL) to treat the cells for 12 h, and obtained the cell supernatant. Then the concentration of NO in the supernatant was measured using a NO detection kit according to the NO manufacturer's instructions.

Quantitative PCR
When the density of BV2 cells inoculated in 96-well plates reached about 80%, the complete culture medium was changed to incomplete culture medium. We added Pomiferin (1µM) and LPS (100 ng/mL) to treat the cells for 12 h, and extracted RNA using the Trizol reagent (Sigma-Aldrich, St. Louis, MO, USA).
RNA is reverse-transcribed into cDNA using the Prime Script®1stStrand cDNA Synthesis Kit (Roche, South San Francisco, CA, USA). After that, the mRNA level of IL-6, TNF-α, iNOS and COX-2 were measured with Quanti Tect SYBR Green RT-PCR Kit (Takara Biotechnology, Ltd., Kyoto, Japan) according to the ELISA manufacturer's instructions. Primer sequences refer to previous studies [15,16].

ELISA
When the density of BV2 cells inoculated in 96-well plates reached about 80%, the complete culture medium was changed to incomplete culture medium. We added Pomiferin (1µM) and LPS (500 ng/mL) to treat the cells for 24 h, and obtained the cell supernatant. The protein concentration of IL-6 and TNF-α in the supernatant was measured according to the ELISA manufacturer's instructions.

Western blot
Total cell proteins were extracted using a protein extraction kit according to the manufacturer's instructions (Beyotime Biotechnology, Shanghai, China). A total of 50 µg of protein was added to the well for electrophoresis. After electrophoresis, the proteins were transferred to brous PVDF membranes (Millipore, Billerica, MA, USA). Then the membrane was placed in 5% skim milk powder and sealed for 4 h. After that, the membrane was incubated with the primary antibody at 4℃ for 12 h and the secondary antibody at room temperature for 2 h. After cleaning with a membrane wash solution, the membrane is colorized with an ECL luminescent solution and the proteins are imaged with a protein imager. All primary antibodies are purchased from Abcam (Cambridge, UK) and the secondary antibody is purchased from Biological Technology (Wuhan, China). Data analysis All data were presented in mean ± SD form and analyzed using SPSS 19.1 software. One-way analysis of variance was used to compare the different groups. P values < 0.05 were considered statistically

Effect of Pomiferin on the growth of BV2 cells
In order to study whether Pomiferin has toxic effects, BV2 cells were treated with LPS (100 ng/mL) and different concentrations of Pomiferin (0.25 µM, 0.5 µM, 1 µM), and then the effects of Pomiferin on the growth of BV2 cells were detected by CCK-8 ( Fig. 2A) and LDH (Fig. 2B) experiments. The results showed that LPS and Pomiferin had no toxic effects on BV2 cells.

Effect of Pomiferin on expression of ROS and NO in BV2 cells
In the process of oxidative stress, ROS and NO are produced, which accumulate in large amounts and damage the surrounding tissues. In order to study the effect of Pomiferin, we treated BV2 cells with LPS (100 ng/mL) and Pomiferin (1 µM) for 12 h, and then detected the effect of Pomiferin on the production of ROS (Fig. 3A) and NO (Fig. 3B) in BV2 cells. The results showed that Pomiferin inhibited the production of ROS and NO.

Effect of Pomiferin on Akt /Nrf2 pathway in BV2 cells
Akt signaling pathway is involved in basic cellular processes. Nuclear factor erythroid2-related factor 2(Nrf2) is an important protective transcription factor that generally exists in various organisms and cellules. Studies have shown that Akt/ Nrf2 is involved in the regulation of oxidative stress [17,18]. In order to investigate the mechanism of action of Pomiferin, we studied the effect of Pomiferin on Akt and Nrf2 pathways. The results showed that Pomiferin promoted the phosphorylation of Akt and the nuclear translocation of Nrf2.Then we treated BV2 cells with MK2206 (an Akt inhibitor, 10 µM) and BT (a Nrf2 inhibitor, 200 nM) for 6 h, and studied the effects of Pomiferin on ROS and NO after blocking Akt or Nrf2 pathway. The results showed that MK2206 or BT treatment weakened the inhibition of Pomiferin on ROS and NO. These results proved that Pomiferin inhibited the production of ROS and NO through the Akt/Nrf2 pathway.

Effect of Pomiferin on the expression of proin ammatory factors in BV2 cells
Continued oxidative stress usually leads to an in ammatory response, and abnormal in ammatory responses produce large amounts of proin ammatory factors (IL-6 and TNF-α), causing damage to neurons. Therefore, we treated BV2 cells with LPS (100 ng/mL) and Pomiferin (1 µM)for 12 h (mRNA) and 24 h (protein), and then detected the effect of Pomiferin on the production of proin ammatory factors (IL-6 and TNF-α) in BV2 cells via quantitative PCR and ELISA methods. The results showed that Pomiferin inhibited the mRNA (Fig. 5A, B) and protein (Fig. 5C, D) expression of proin ammatory factors (IL-6 and TNF-α).
Effect of Pomiferin on the expression of proin ammatory enzymes in BV2 cells In order to further study the effect of Pomiferin on in ammation, we treated BV2 cells with LPS (100 ng/mL) and Pomiferin (1 µM) for 12 h (mRNA) and 24 h (protein), and then detected the effect of Pomiferin on the production of proin ammatory proteinase (iNOS and COX2) in BV2 cells via quantitative PCR and western blot methods. The results showed that Pomiferin inhibited the mRNA (Fig. 6A, B) and protein (Fig. 5C, D, E) expression of proin ammatory proteinase (iNOS and COX2).
Effect of Pomiferin on NF-κB and MAPK pathways in BV2 cells NF-κB and MAPK pathways are classical in ammatory pathways, whose activation regulates transcription of in ammatory mediators [19,20]. To investigate the anti-in ammatory mechanism of Pomiferin, we treated BV2 cells with Pomiferin (1 µM) and LPS (100 ng/mL), and then examined the effects of Pomiferin on activation of NF-κB and MAPK pathways. The results showed that orange mulberry brass inhibited the activation of NF-κB (Fig. 7A, C, D, E) and MAPK (Fig. 7B, F, G, H) pathways in BV2 cells.
As a avonoid extracted from the fruit of Moraceae, Pomiferin has been reported to regulate oxidative stress [21][22][23]. In our experiment, we examined the effects of Pomiferin on the production of ROS and NO in BV2 cells. The results showed that Pomiferin inhibited the production of ROS and NO. The Akt pathway or PI3K-Akt pathway is the transduction pathway of signals corresponding to extracellular survival and growth. Activated Akt mediates downstream reactions by phosphorylation of a series of intracellular proteins that regulate cell survival, growth, proliferation, cell migration, and angiogenesis [24][25][26]. In our study, we examined the effect of Pomiferin on Akt phosphorylation, and the results showed that Pomiferin treatment promoted the phosphorylation of Akt. Nrf2 is a key factor in the regulation of oxidative stress, and its activation can regulate a variety of genes related to oxidative stress. Under normal circumstances, Nrf2 is located in the cytoplasm and does not play a role. After activation, Nrf2 translocates into the nucleus and binds with the antioxidant reaction elements to form the Nrf2-ARE signaling pathway, thereby regulating the expression of a series of downstream antioxidant genes and proteins and playing the antioxidant role [27][28][29]. In our experiment, we investigated the effect of Pomiferin on Nrf2 activation and found that Pomiferin promoted the nuclear translocation of Nrf2. Further studies showed that the inhibitors of Akt and Nrf2 could weaken the inhibition of ROS and NO by Pomiferin. It is suggested that Pomiferin inhibits the production of ROS and NO in BV2 cells by activating Akt and Nrf2 pathways.
The imbalance of oxidative and antioxidant reactions in the body will lead to oxidative stress. Continued and complex oxidative stress can trigger an in ammatory response in the body. In ammation can lead to the imbalance of oxidation and antioxidant reactions in the body [30][31][32]. Therefore, regulation of oxidative stress plays an important role in inhibiting in ammatory response. The main pathological feature of neurodegenerative diseases is the degenerative loss of neurons. In ammation plays an important role in this process [33][34][35]. Therefore, the inhibition of in ammatory response is of great signi cance in the treatment of neurodegenerative diseases. In our experiment, we examined the effect of Pomiferin on the production of pro-in ammatory mediators in BV2 cells and found that Pomiferin inhibited the production of pro-in ammatory mediators in BV2 cells.
NF-κB (enhanced κ-light chain of nuclear factor B) is a protein complex that plays a key role in regulating the immune response to infection. Its target genes are involved in immune response, in ammatory response, cell proliferation, anti-apoptosis, etc. When the cells were stimulated, IκBα kinase in the cytoplasm was activated, and part of IκBα was phosphorylated, part of IκBα was degraded by ubiquitination, and the p65 subunit associated with it was dissociated and phosphorylated [36][37][38]. In our experiment, we investigated the effect of Pomiferin on NF-κB activation and found that Pomiferin inhibited the phosphorylation of IκBα and p65 and the ubiquitination degradation of IκBα. MAPK (Mitogen-activated protein kinase) is a group of important transmitters that can be activated by different extracellular stimuli and make signals from the cell surface to the nucleus. It consists of ERK, JNK and P38 [39,40].In our experiment, we detected the effect of Pomiferin on the activation of MAPK pathway, and the results showed that Pomiferin had no effect on the activation of MAPK pathway.

Conclusion
Our study found that Pomiferin inhibited microglial in ammation by activating the Akt /Nrf2 pathway and inhibiting the NF-κB pathway. In addition, the unique pharmacological properties and easy availability of Pomiferin suggest that it may be a candidate drug for anti-neuroin ammation. Our experiment has laid a certain foundation for further study of the effect of Pomiferin. Availability of data and materials The datasets used or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
All authors claim that there is no con ict of interest.