TrxR2 overexpression alleviates inflammation-mediated neuronal death via reducing the oxidative stress and activating the Akt-Parkin pathway.

Neuronal death caused by inflammatory cytokine-mediated neuroinflammation is being extensively explored. Thioredoxin reductase (TrxR) 2 is a novel mediator of inflammation response. In the current study, we focus on the mechanisms of TrxR2 overexpression in inflammation-mediated neuronal death. LPS was used to induce neuroinflammation in N2a cells in vitro. Adenovirus-loaded TrxR2 was transfected into N2a cells to up-regulate TrxR2 expression. Then, cell viability was determined via MTT assay and TUNEL assay. Apoptosis was measured via western blotting and ELISA. Oxidative stress was detected via ELISA and flow cytometry. A pathway inhibitor was used to verify the role of the Akt–Parkin pathway in the LPS-mediated N2a cell death in the presence of TrxR2 overexpression. With the help of immunofluorescence assay and western blotting, we found that TrxR2 expression was significantly reduced in response to LPS treatment, and this effect was associated with N2a cell death via apoptosis. At the molecular level, TrxR2 overexpression elevated the activity of the Akt–Parkin pathway, as evidenced by the increased expression of p-Akt and Parkin. Interestingly, inhibition of the Akt–Parkin pathway abolished the regulatory effect of TrxR2 on LPS-treated N2a cells, as evidenced by the decreased cell viability and increased apoptotic ratio. Besides, TrxR2 overexpression also reduced oxidative stress, inflammation factor transcription and mitochondrial apoptosis. However, inhibition of Akt–Parkin axis abrogated the protective effects of TrxR2 on redox balance, mitochondrial performance and cell survival. LPS-mediated neuronal death was linked to a drop in TrxR2 overexpression and the inactivation of the Akt–Parkin pathway. Overexpression of TrxR2 sustained mitochondrial function, inhibited oxidative stress, repressed inflammation response, and blocked mitochondrial apoptosis, finally sending a pro-survival signal for the N2a cells in the setting of LPS-mediated inflammation environment.

Neurodegeneration is a chronic disease characterized by the progressive loss of structure or function of neurons. There are many neurodegenerative diseases, including amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, and Huntington's disease. Neurodegenerative diseases occur as a result of death of neurons due to excessive inflammation.1,2 Notably, many neurodegenerative diseases are incurable and debilitating conditions. There is an increasing recognition that inflammation plays a critical role in neurodegenerative diseases.3,4 Inflammatory responses that establish feed-forward loops may overwhelm normal resolution mechanisms. Mechanically, uncontrolled inflammation may lead to activation of NF-κB and production of reactive oxygen species (ROS) and proinflammatory mediators.5,6 These factors act directly on neurons that die in the progression of neurodegenerative diseases.7,8 Besides, these factors also activate microglia, which amplify the inflammatory response in a positive feedback loop, leading to further activation of microglia.9–11 Accordingly, understanding the prominent neuropathological hallmarks of neurodegenerative diseases may pave a new road to the treatment of chronic neurodegenerative diseases.12,13 Recently, thioredoxin reductase (TrxR) 2 has been found to be associated with inflammation injury. For example, in metabolic syndrome, TrxR2 overexpression reduces inflammation response via downregulating inflammatory factors’ transcription.14 Besides, the regulatory effect of TrxR2 on immunity has been verified, especially in nerve cells.15 Also, TrxR2 modulates the endothelial response to shear stress,16 affects lung innate immunity,17 controls antioxidant capacity,18 regulates mitochondrial homeostasis,19,20 and attenuates ER stress.21,22 These results thus highlight new functions of TrxR2 that they are the key mediators of inflammation response.23,24 However, there is no study to explore whether TrxR2 is implicated in the progression of inflammation-mediated neurodegenerative diseases.25 Therefore, in the present study, LPS treatment was used to induce a neuroinflammation model and then neural cell viability was measured in response to TrxR2 overexpression in the setting of LPS-mediated neurodegradation. Recently, emerging evidence also suggests a role for Parkin in several major neurodegenerative diseases, including Alzheimer's disease (AD).26 Mechanistically, Parkin activity is downregulated in the development of neurodegenerative diseases, and this process is associated with mitochondrial damage, especially mitochondrial oxidative stress and mitochondrial pro-apoptotic factors’ leakage.27,28 Excessive oxidative stress exacerbates the brain inflammation response.29–31 Besides, the mitochondria-released pro-apoptotic factor is the primary trigger of neural death via activating the caspase-9 pathway.32,33 Interestingly, re-activation of Parkin has been shown to attenuate acute and chronic brain damage.34 For example, genetic activation of Parkin rescues TAF15-induced neurotoxicity in a drosophila model of amyotrophic lateral sclerosis. Brain reperfusion injury could be attenuated via Parkin-mediated mitophagy. Besides, the regulatory effects of Parkin on inflammation response have been widely explored. For example, in astrocyte endoplasmic reticulum stress, Parkin targets NOD2 and the latter attenuates inflammation injury.35,36 In addition, Parkin overexpression also reduces the chronic obstructive pulmonary disease via modulating inflammation response.37,38 Activation of Parkin also inhibits sepsis-induced cardiomyopathy. However, the role of Parkin in inflammation-mediated neurodegradation has not been identified. In the end, TrxR2 overexpression has been found to be associated with Parkin activation and mitochondrial protection in diabetic cardiomyopathy.39,40 A similar finding is also noted in endometriosis. Based on this finding, we asked whether TrxR2 overexpression has an ability to attenuate inflammation-mediated neural damage, especially neuron death, partly via modulating Parkin-modified mitochondrial protection.

and materials Cell experiments and reagent treatment Our study was performed in accordance with the Declaration of Helsinki. Mouse N2a cells were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA). The N2a cells were grown at a density of 5 × 106 in l-DMEM at 37 °C in a humidified atmosphere containing 5% CO2. The medium was supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic ATB (Pan Biotech, Aidenbach, Germany).41 The 10 μg ml–1 LPS incubation for 12 hours was used to mimic the inflammatory environment. To inhibit Akt activity, LY294002 (Selleck Chemicals) was applied for about 45 min42 Immunofluorescence Samples were first washed three times in PBS and then fixed in 3.7% paraformaldehyde. Then, the samples were incubated with primary antibodies at 4 °C overnight to label the targeted proteins. Subsequently, after washing three times with PBS, they were incubated with the Alexa Fluor® 594 (goat anti-mouse IgG, red) fluorescent-labeled secondary antibody at room temperature.43 After washing with PBS, they were loaded with DAPI to stain nuclei. Finally, the samples were observed using a laser confocal microscope (TCS SP5; Leica Microsystems, Inc., Buffalo Grove, IL, USA). The following primary antibodies were used: p-Akt (1 : 1000, Abcam, #ab81283) and Parkin (1 : 1000, Abcam, #ab77924).44,45 Quantitative PCR After treatment, total RNA was isolated using the TRIzol (Invitrogen, Carlsbad, CA) reagent. Subsequently, oligo-dT primers and PrimeScript RTase (TaKaRa, PrimeScript II 1st Strand cDNA Synthesis Kit) were used to generate cDNA based on a previous study. Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) was used to conduct the qPCR with the help of an ABI 7900HT Fast Real-Time PCR System.46 Immunoblotting After washing with PBS, cells were trypsinized and incubated with RIPA buffer. After incubation at 4 °C for 30 minutes, the cells were centrifuged at 10 000g for approximately 10 minutes. Then, the supernatant was collected and separated by SDS-PAGE followed by transfer to PVDF membranes. Primary antibodies were used to incubate the targeted proteins at 4 °C overnight with 5% non-fat milk in TBST. After washing three times with TBST, the samples were incubated with secondary antibodies at room temperature for approximately 45 minutes.47 The membranes were observed using an enhanced chemiluminescence detection kit (Santa Cruz Biotechnology, Nanterre, France). The primary antibodies used for immunoblotting were as follows: t-Akt (1 : 1000, Abcam, #ab8805), p-Akt (1 : 1000, Abcam, #ab81283), Parkin (1 : 1000, Abcam, #ab77924), cleaved caspase3 (1 : 1000, Abcam, #ab49822), cleaved caspase3 (1 : 1000, Abcam, #ab49822), and TrxR2 (1 : 1000, Abcam, #ab180493).47 TUNEL assay The TUNEL assay was conducted using the one-step TUNEL kit (Beyotime Institute of Biotechnology, China) according to the manufacturer's instructions. After washing with PBS, the cells were fixed in 3.7% paraformaldehyde. The paraffin slices were then dewaxed with dimethyl benzene and incubated with proteinase K and TUNEL reaction reagents at 37 °C for approximately 2 hours in the dark. Then, the cells were loaded with DAPI to label nuclei.48 Finally, the images were captured under a laser confocal microscope (TCS SP5; Leica Microsystems, Inc., Buffalo Grove, IL, USA). At least five fields were recorded, and the number of TUNEL positive cells was measured.49 Flow cytometry analysis of calcium To observe cellular oxidative stress, flow cytometry analysis was used. In brief, the samples were washed with PBS and then the MitoSOX red mitochondrial superoxide indicator (Molecular Probes, USA) was incubated with the cells for approximately 30 minutes at 37 °C in the dark. Subsequently, the cells were washed with PBS to remove the probe and digested with 0.25% pancreatin.50 After resuspending in PBS, the cells were then immediately analyzed using a flow cytometer (Partec, Münster, Germany). Quantification of cellular calcium was performed per 10 000 cells in each group, and the data were analyzed with Flowmax software (Partec, Münster, Germany).51 MTT assay and caspase 3/9 activity MTT assay was determined according to a previous study. Caspase 3 and caspase 9 activities were measured spectrophotometrically according to previous studies. The caspase-3/9 activity kits (Beyotime Institute of Biotechnology, China) were used according to the manufacturer's protocols. To analyze caspase 3 activity, 5 μL of 4 mM DEVD-p-NA substrate (200 μM final concentration) were added to the samples for 2 hours at 37 °C. To measure caspase 9 activity,52 the samples were incubated with 5 μL of 4 mM LEHD-p-NA substrate (200 μM final concentration) for 1 hour at 37 °C. Then, the 400 nM wavelength was recorded using a microplate reader to reflect the caspase 3 and caspase 9 activities.53 ELISA Cellular oxidative stress was measured via ELISA. GPX Assay Kit (A003-1, NanJing JianCheng Bioengineering Institute, Nanjing, China), Glutathione Peroxidase Activity (GSH) Assay Kit (A005, NanJing JianCheng Bioengineering Institute, Nanjing, China), and Superoxide Dismutase (SOD) Assay Kit (A001-3, NanJing JianCheng Bioengineering Institute, Nanjing, China) were used according to the instructions of the manufacturer. Cellular LDH release and ATP production were determined via Lactate Assay Kit (A019-2, NanJing JianCheng Bioengineering Institute, Nanjing, China) and ATP Production Assay (S0026, Beyotime Biotechnology, Shanghai, China) according to the instructions of the manufacturer.54 Adenovirus-mediated TrxR2 overexpression assay In brief, adenovirus TrxR2 (ad-TrxR2) was obtained from Vigene Biosciences and then was transfected into N2a cells when the cells were grown to 80% to 90% confluency. After 72 hours, the cells were harvested and western blots were used to confirm the overexpression efficiency. The null vector transfection group was set as the control group (Ad-ctrl).36 Mitochondrial membrane potential detection JC-1 staining was performed to evaluate mitochondrial depolarization according to the manufacturer's protocol. Samples were washed with PBS and then incubated with 10 mg ml–1 of JC-1 for approximately 30 minutes at 37 °C in the dark.55 PBS was then used to wash the cells and subsequently, the cells were visualized using a laser confocal microscope (TCS SP5; Leica Microsystems, Inc., Buffalo Grove, IL, USA).56 Statistical analysis

were obtained from at least three independent experiments on different days with different cells. The data, a representation of one of the 3 independent experiments, are expressed as the mean ± SEM. Statistical significance was analyzed via ANOVA. Analysis was performed using SigmaStat version 4.0 software from Jandel Scientific (San Diego, CA, USA). A probability value less than 0.05 (p < 0.05) was considered statistically significant.

TrxR2 is downregulated in LPS-treated N2a cells and contributes to the LPS-mediated N2a cell death In the present study, LPS was used to mimic the inflammatory microenvironment. Then, TrxR2 expression was determined via western blotting. As shown in Fig. 1A and B, compared to the control group, LPS reduced the expression of TrxR2 in N2a cells, indicative of the inactivation of TrxR2 in the setting of inflammatory microenvironment. Subsequently, adenovirus-mediated TrxR2 (Ad-TrxR2) overexpression assay was performed in order to reverse the TrxR2 expression in LPS-treated N2a cells. The overexpression efficiency was confirmed via western blotting (Fig. 1A and B) and immunofluorescence (Fig. 1C and D). As shown in Fig. 1C and D, compared to the control group, LPS reduced the fluorescence intensity of TrxR2 in N2a cells, and this alteration could be reversed by Ad-TrxR2 transfection. To verify the role of TrxR2 in LPS-mediated neural dysfunction, cell viability was determined via LDH release assay. As shown in Fig. 1E, compared to the control group, LPS increased the content of LDH in the medium, indicative of cell death in response to LPS treatment. Interestingly, TrxR2 overexpression reduced the levels of LDH in the medium, suggesting that TrxR2 overexpression protected N2a cell viability in the setting of LPS-mediated inflammation environment. Previous studies have reported that mitochondria apoptosis is the primary pathogenesis responsible for the N2a cell death in the context of LPS-mediated inflammation environment. The molecular feature of mitochondrial apoptosis is the pro-apoptotic factor (such as HtrA2/Omi) release from cytoplasm into the nucleus. With the help of immunofluorescence, we found that LPS treatment promoted the translocation of HtrA2/Omi leakage from cytoplasm into the nucleus (Fig. 1F and G). Interestingly, TrxR2 overexpression prevented the translocation of HtrA2/Omi (Fig. 1F and G). As a consequence of HtrA2/Omi release, the activity of caspase-9 was rapidly increased in response to the LPS treatment (Fig. 1H). Notably, TrxR2 overexpression prevented the LPS-mediated caspase-9 activation. These data indicated that TrxR2 downregulation was associated with the activation of mitochondrial apoptosis in N2a cells in the setting of LPS-mediated inflammatory microenvironment. TrxR2 overexpression activates the Akt–Parkin pathway Previous studies have found that mitochondrial protection could be achieved via the activation of the Akt–Parkin pathway. In the present study, we asked whether TrxR2 overexpression had an ability to modulate the Akt–Parkin pathway. First, immunofluorescence assay was used to observe the alterations of p-Akt and Parkin in response to TrxR2 overexpression. As shown in Fig. 2A–C, compared to the control group, LPS treatment reduced the fluorescence intensity of p-Akt and Parkin in N2a cells, indicative of the inactivation of the Akt–Parkin pathway in response to inflammation microenvironment. Interestingly, TrxR2 overexpression reversed the activity of the Akt–Parkin pathway as evidenced by increased p-Akt expression and Parkin levels. This information indicated that TrxR2 overexpression prevented the decrease in the activity of the Akt–Parkin pathway. Similar results were obtained in N2a cells via western blotting. Compared to the control group, the protein expression of p-Akt and Parkin was rapidly downregulated in N2a cells in response to LPS treatment (Fig. 2D–F). However, TrxR2 overexpression reversed the expression of p-Akt and Parkin in the setting of inflammation injury (Fig. 2D–F). Therefore, these data supported the necessary role of TrxR2 in sustaining the activity of the Akt–Parkin pathway in N2a cells. Inhibition of the Akt–Parkin pathway abolished the anti-apoptotic effect of TrxR2 overexpression on N2a cells To verify the influence of the Akt–Parkin pathway in N2a cell viability, pathway blocker LY294002 was added into the medium of N2a cells in the presence of LPS-mediated inflammatory microenvironment. Then, cell viability was determined via MTT assay. As shown in Fig. 3A, compared to the control group, the cell viability was markedly reduced in response to LPS treatment. However, TrxR2 overexpression reversed cell viability in N2a cells, and this effect was abolished by Akt inhibition because application of LY294002 significantly reduced cell viability despite overexpression of TrxR2 (Fig. 3A). This finding was also validated via LDH release assay (Fig. 3B). Subsequently, cell death was determined via TUNEL assay. As shown in Fig. 3C and D, compared to the control group, LPS treatment increased the ratio of TUNEL-positive cells, and this effect could be repressed by TrxR2 overexpression. Interestingly, inhibition of the Akt pathway using LY294002 could abrogate the anti-apoptotic effects of TrxR2 overexpression on N2a cells (Fig. 3C and D). Furthermore, we explored whether apoptosis was responsible for LPS-mediated cell death. Western blotting was used to observe the alterations of caspase-3 and its substrate PARP. As shown in Fig. 3E–G, compared to the control group, LPS treatment elevated the expression of caspase-3 and its substrate PARP, suggesting that LPS activated apoptosis in N2a cells. Notably, TrxR2 overexpression reduced caspase-3 and PARP expression, indicative of the anti-apoptotic action of TrxR2 overexpression on N2a cells. Interestingly, LY294002 treatment abolished the anti-apoptotic effects of TrxR2 overexpression on N2a cells, as evidenced by the increased expression of caspase-3 and PARP (Fig. 3F and G), suggesting that TrxR2 overexpression inhibited LPS-mediated apoptosis via the Akt–Parkin pathway. Taken together, these results indicated that LPS activated apoptosis in N2a cells and that TrxR2 overexpression inhibited N2a cell apoptosis via augmenting the activity of the Akt–Parkin pathway. TrxR2 overexpression attenuates LPS-mediated oxidative stress in N2a cells via the Akt–Parkin pathway Oxidative stress has been acknowledged as a primary mediator of cell death. Although TrxR2 is the anti-oxidative factor via reducing ROS production, it is unknown whether TrxR2 modulated oxidative stress via the Akt–Parkin pathway. In the present study, ELISA was used to assess the changes in cellular antioxidants. Compared to the control group, LPS treatment reduced the content of SOD, GSH and GPx, indicative of the downregulation of cell antioxidants (Fig. 4A–C). Interestingly, TrxR2 overexpression reversed the levels of SOD, GSH, and GPx; these effects could be negated by LY294002 treatment (Fig. 4A–C). This information indicated that TrxR2 attenuated LPS-mediated oxidative stress in a manner dependent on the Akt–Parkin pathway. Subsequently, flow cytometry was used to analyze ROS overproduction. As shown in Fig. 4D and E, compared to the control group, LPS treatment elevated ROS production, indicative of ROS overloading in the presence of LPS stress. Interestingly, TrxR2 overexpression attenuated ROS overproduction, and this effect seemed to be dependent on the Akt–Parkin pathway (Fig. 4D and E). To provide more evidence to support the oxidative stress in LPS-treated N2a cells, mitochondrial membrane potential was evaluated. At the molecular level, mitochondrial membrane potential reduction is the primary reason for ROS production. Subsequently, JC-1 probe was used observe the mitochondrial membrane potential. As shown in Fig. 4F and G, compared to the control group, LPS treatment reduced the mitochondrial membrane potential, as shown in decreased red fluorescence intensity and increased green fluorescence intensity. Interestingly, TrxR2 overexpression stabilized the mitochondrial membrane potential, and this effect was abolished by LY294002 (Fig. 4F and G), suggesting that the Akt–Parkin pathway was involved in TrxR2-mediated mitochondrial membrane potential stabilization. Altogether, our results indicated that TrxR2 modulated oxidative injury in LPS-treated N2a cells via the Akt–Parkin pathway. LPS-mediated inflammation would be improved by TrxR2 via the Akt–Parkin pathway In addition to oxidative stress, inflammation is also involved in LPS-mediated neural dysfunction and cell death. With the help of ELISA, we found that the levels of inflammatory factors were rapidly increased in response to LPS treatment (Fig. 5A–D). Notably, TrxR2 overexpression repressed the upregulation of inflammation factors in the presence of LPS stress (Fig. 5A–D). Interestingly, inhibition of the Akt–Parkin pathway could abolish the regulatory effects of TrxR2-mediated inflammatory factors’ downregulation (Fig. 5A–D). These findings were further supported via qPCR. As shown in Fig. 5E–H, compared to the control group, the transcription of MMP9, TNFa, IL-2 and IL-12 was rapidly increased in response to LPS treatment.57 However, TrxR2 overexpression inhibited the LPS-mediated inflammation factors’ upregulation, and this effect was dependent on the Akt–Parkin pathway (Fig. 5E–H). Altogether, our results indicated that LPS-mediated inflammation response could be inhibited by TrxR2 in a manner dependent on the Akt–Parkin pathway.

In the present study, we found that TrxR2 downregulation could be considered as a primary pathogenesis for the development of neuroinflammation. Overexpression of TrxR2 attenuated inflammation-mediated cell death and sustained mitochondrial dysfunction (Fig. 6). Therefore, activation of TrxR2 may be more beneficial than single anti-inflammation treatment in suppressing the inflammatory response to alleviate the progression of PD.58 It is well known that LPS is a component of the outer membrane of the most Gram-negative bacteria, which can directly activate glial cells in the brain and then release pro-inflammatory factors. The pro-apoptotic effects of LPS have been widely explored.59 For example, in sepsis-related myocardial injury, LPS promotes cardiomyocyte death and mitochondrial dysfunction via the Drp1/F-actin signaling pathway.60 Besides, LPS also modulates the metabolism and viability of SH-SY5Y cells in the presence of BV-2 microglia. Moreover, pro-inflammatory macrophages are activated by LPS via the PARP1–LSD1 axis under oxidative conditions. Interestingly, LPS uptake is associated with pyroptotic cell death. In acute lung injury model rats, LPS promotes the apoptosis of alveolar epithelium. In the present study, we found that LPS-mediated cell death was associated with TrxR2 downregulation. Enhancement of TrxR2 could attenuate LPS-mediated N2a cell death via inhibiting apoptosis.61 Besides, TrxR2 overexpression also sustained mitochondrial function, alleviated oxidative stress and blocked mitochondrial apoptosis. As far as we know, this is the first study to explore the influence of TrxR2 in the LPS-mediated neural dysfunction and mitochondrial apoptosis (Fig. 6). However, more clinical data are required to support our notion. The Akt pathway and Parkin protein have been acknowledged as the defenders to sustain CNS function in several diseases. In cerebral ischemia reperfusion injury, the Akt pathway is inactivated and promotes the neuronal autophagic injury in a manner dependent on the PI3K/mTOR signaling pathway.61 In seizure-induced brain injury, dynorphin activation protects against neuronal death via activating the Akt/HO-1 pathway. Besides, blood brain barrier function is also sustained by KY226 via the Akt pathway.62 Loureirin B promotes axon regeneration by inhibiting ER stress-induced mitochondrial dysfunction and regulating the Akt/GSK-3β pathway after spinal cord injury.63,64 With respect to Parkin, Parkinson's disease could be retarded via activating Parkin-dependent mitophagy.65 Besides, Parkin targets NOD2 to regulate astrocyte endoplasmic reticulum stress and inflammation. Genetic activation of Parkin rescues TAF15-induced neurotoxicity in a Drosophila model of amyotrophic lateral sclerosis. In rotenone-induced neurotoxicity, mutual antagonism of Parkin and PGC-1α contributes to maintenance of mitochondrial homeostasis. In the present study, we reported that Akt activity and Parkin expression were rapidly downregulated in response to inflammation response.66 Interestingly, overexpression of TrxR2 could reverse the activity of the Akt–Parkin pathway. These data support the functional importance of the redox balance in regulating the Akt–Parkin pathway.67 This result also illustrates the complex interactive effects between inflammation and oxidative stress and also defines the oxidative stress as the upstream mediator of cell viability and mitochondrial homeostasis in the presence of inflammation microenvironment. There are several limitations in the present study, and they require further work. First, our observation is limited to the short time treatment of LPS. More studies of inflammation-mediated neural dysfunction with various concentrations and treatment times are warranted to verify the protective effects of TrxR2 on inflammation-mediated neural dysfunction. Besides, our study primarily focused on the role played by oxidative stress, mitochondrial damage and caspase-related apoptosis in inflammation-mediated neuronal injury. However, the detailed mechanism by which TrxR2 modulates the mitochondrial damage and oxidative injury has not been fully addressed.

In conclusion, our report identified TrxR2 downregulation as an early event of inflammation-mediated neural dysfunction via affecting the activity of Akt–Parkin pathway. Overexpression of TrxR2 sustained neuron viability, stabilized mitochondrial function, and inhibited inflammation-induced apoptosis in N2a cells. Given this, our data support future studies targeting intervention of the TrxR2–Akt–Parkin pathway as a potential therapeutic strategy for the treatment of neuroinflammation. Author contributions JBG and YJL conceived the research; WDL and HJW performed the experiments; and all authors participated in discussing and revising the manuscript. Funding This work was supported by the National Natural Science Foundation of China (Grant/Award Numbers: 81771347) and Key Military Research Projects On Equipment. Conflicts of interest The authors have declared that they have no conflicts of interest.

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