Shikimic acid (SA) was obtained from Shanghai yuan ye Biotech Co., Ltd (Shanghai, China). The purity is ≥98%. Dimethyl sulfoxide (DMSO) and Lipopoly-saccharide (LPS) were obtained from Sigma Aldrich (St Louis, MO, United States). Serum (FBS), Trypsin (0.05%), Penicillin-Streptomycin Solution (PS) and Dulbecco’s modified eagle’s medium (DMEM) were purchased from Gibco (Grand Island, NY, United States). RA (nrf2 inhibitor), JSH-23 (NF-κB inhibitor) and MK2206 (AKT inhibitor) were purchased from Selleck Chemicals Aldrich (Shanghai, China). In our experiments we used MK2206、RA or JSH treatments to inhibit the activation of the AKT、Nrf2 and NF-κB pathways in cells, with pretreatment concentrations and times of MK2206 (10 μM) for 4 h、RA (5 μM) for 4 h and JSH (1 μM) for 3 h. The concentration and duration of pretreatment were referred to previous studies (Peng et al., 2019; Gao et al., 2020; He et al., 2021a).

A mouse microglia (BV2 cells, from Jin Yuan Biotechnology Co. (Shanghai, China)) was cultured with DMEM medium containing 10% FBS and 1% PS (the condition: air atmosphere, 95%; carbon dioxide (CO₂), 5% and temperature, 37°C). When density reaches 90%, cells were processed and passaged with 0.05% trypsin.

BV2 cells growing in good condition were inoculated into 96-well plates at a density of 2–3 × 10⁵. After 12 h, different concentrations of SA (2.5 μM、5 μM、10 μM、20 μM、40 μM) were added into well and cultured for another 20 h. After that, the medium was replaced with fresh DMEM without FBS and cultured for another 2 h. Subsequently CCK8 (Shang Bao Biological, Shanghai, China) was added and incubated for another 3 h. Finally, absorbance was measured with a microplate reader (BioTek, Winooski, VT, United States) at 450 nm.

NO level in the culture media was assayed with the Griess reagent (Beijing Solarbio Science and Technology Co., Beijing, China) according to the manufacturer’s instructions. Briefly, BV-2 cells were inoculated in the 12-well plates at a density of 2–3 × 10⁴. After 12 h, cells were treated with SA and/or LPS for 24 h. The supernatant (100 μL) was mixed with an equal volume of Griess reagent (parts 1 and 2) and incubated in the dark for 30 min. After that, the absorbance were measured with a microplate reader (BioTek, Winooski, VT, United States) at 530 nm and NO concentrations were calculated based on the standard curve of sodium nitrite.

Release of ROS in BV2 cells is detected using ROS detection kits (Thermo Fisher Scientific Inc., Shanghai, China). Briefly, BV-2 cells were inoculated in the 96-well plates at a density of 2–3×10⁵. After 12 h, cells were treated with SA and/or LPS for 24 h. The medium was replaced with DMEM without FBS and DCFH-DA (10 μM) was added to each well, and then incubated for 30 min at 37°C. After that, the absorbance were measured with a microplate reader (BioTek, Winooski, VT, United States) under the condition: excitation wavelength 488 nm and emission wavelength 525 nm.

Expression of associated proteins in tissues and cells is detected using the Western blot method. Firstly, proteins are cleaved using NP40 reagents (Beyotime Biotechnology, Shanghai, China) and concentrations are quantified using BCA kits (Beyotime Biotechnology, Shanghai, China). Subsequently, Electrophoresis (120 V, 1.5 h) was performed with 50 μg of protein. After electrophoresis, proteins were transferred to PVDF membranes (Millipore, Billerica, MA, United States) (70 V, 2 h). After closed for 3 h in 5% skim milk, the membrane was incubated with the primary antibody (iNOS (1:1,000), p-NF-κB p65 (1:1,000), p-IκB (1:1,000),β-actin (1:10,000), TH (1:1,000), p-AKT (1:2000), Nrf2 (1:5,000), iba-1 (1:500), PCNA (1:5,000), COX2 (1:1,000), IκB (1:1,000), NF-κB p65 (1:2000), β-actin (1:10,000)) at room temperature for 4 h and the secondary antibody (1:4,000) for 2 h at room temperature. The primary antibodies are from Proteintech (Wuhan, China) and Cell Signaling Technology (Massachusetts, United States). The secondary antibody is from Invitrogen (California, America). After that, the membranes were developed using ECL ultrasensitive luminescent solution (Yeasen Biotechnology Co., Shanghai, China) and imaged using Kodak X-ray film (Ruike Medical Equipment Co., Xiamen, China). Results are analyzed using ImageJ software.

The mRNA expression of inflammatory mediators in tissues and cells is detected using quantitative PCR methods. Briefly, RNA was extracted using trizol reagent (Sigma Aldrich, Shanghai, China). Then, 500 ng of RNA was reverse transcribed into cDNA using a reverse transcription kit (Yeasen Biotechnology, Shanghai, China). Next, cDNA is amplified using the 2×M5 Hi per PCR Super mix (Yeasen Biotechnology, Shanghai, China) and Cq values are recorded with Bio Rad system. Finally, the mRNA level of the mediators was assessed relative to β-actin according to the 2^−ΔΔCT. Primer sequences of the mediators refer to previous studies (Hou et al., 2018).

BV2 cells growing in good condition were seeded into 24-well plates at a density of 1–2×10⁴. After 12 h, the culture medium was replaced and SA and/or LPS were added. After continuing incubation for 24 h, the culture medium was collected and the expression of IL-6 and TNF-α was tested using ELISA kits (Bio Legend, San Diego, CA, United States) according to the manufacturer’s protocol.

Eight weeks-old mice (C57/BL6) were purchased from Chang sheng Biological Company (Changchun, China). Mice were allowed 2 weeks to adapt after being purchased. The mice were housed in a normal temperature environment (temperature of 22°C ± 2°C and humidity of 40%–60%) with free intake of food and water. The animal experimental process is presented in Figure 5A. Briefly, mice were randomly divided into four groups: Saline group (Injection of saline in the SN), SA group (Intraperitoneal drug delivery SA) single-injection LPS group (Injection of LPS in the SN) and SA + LPS (Injection of LPS in the SN and Intraperitoneal drug delivery SA). LPS (3 μL) dissolved in saline (concentration for 3 mg/mL) was injected into the SN ((anteroposterior (AP) = 5.2 mm, lateral (LAT) = 2.1 mm and dorsoventral (DV) = 7.8 mm)). The injection rate is 0.3 μL/min. SA (100 mg/kg) dissolved in saline is administered by intraperitoneal injection as once a day from LPS injection. Concentrations of SA used are referenced to previous studies (Rabelo et al., 2016; Lu et al., 2019). Four weeks later, behavioral tests and other experiments are performed. All animal experiments were approved by the Ethics Committee of the First Hospital of Jilin University and performed in strict accordance with the relevant provisions of the Guidelines for the Care and Use of Animals in Animal Laboratories (Declaration of Helsinki 2013).

After 4 weeks of administration, the mental state and motor activity of mice were evaluated by mental state scoring, rod climbing, rod turning and open field test. Behavioral evaluation methods refer to previous articles (He et al., 2021b; Snyder et al., 2021). The brief methodology is as follows:

Mental state scoring: After modeling, the mice were scored by observing their mental state. Scoring criteria: 0 points for no adverse performance; 1 point for mild reduction of autonomous activity and slightly slow reaction; On top of the symptoms of 1 point, a significant decrease in voluntary activity, loss of balance, unsteady gait, and bowed back are recorded as 2 points; On top of the symptoms of 2 points, 3 points were recorded for symptoms such as weakness of both hind limbs, splitting of hind limbs after lifting the tail, and involuntary trembling of the body; On top of the symptoms of 3 points, 4 points were recorded for symptoms such as paralysis of both hind limbs, inability to stand, increased muscle tone in the extremities, and cessation of eating; Rod climbing: Mice were placed at the top of a wooden pole about 60 cm long and 1–2 cm in diameter so that they could move freely. The timing started when the hind limbs of the mice left the top of the pole and stopped when the head touched the bottom. The time taken for the mice to climb from the top to the bottom was recorded. Each mouse was repeated 3 times and the average was calculated.

Rod turning: Mice were placed on a rotor bar fatigue apparatus. The speed of the rotor bar was adjusted to approximately 40 revolutions per minute. The time that the mice remained on the rotor bar fatigue apparatus without falling was recorded. Each mouse was repeated 3 times and the average was calculated.

Open field test: Mice were placed in a 50 cm × 50 cm × 30 cm open field box and allowed to move freely. A camera was used to record the trajectory of the mice running in the open field area within 5 min. The total distance run by the mice in 5 min and the time of entry into the central region were analyzed using Top Scan software (Any-maze, Stoelting Co.).

The mice’s dopaminergic neurons and microglia in the SN were detected using IHC staining. These experimental procedures refer to previous study. Briefly, after being euthanized, the midbrain tissue of the mice was acquired and fixed. The IHC process is then performed using Ultrasensitive TMS-P kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s protocols. The dopaminergic neurons were labeled with the anti-tyrosine hydroxylase (TH) antibody (1:200, Abcam, Cambridge, United Kingdom) and microglia were labeled with the anti-ionized calcium binding adapter molecule 1(IBA-1) antibody (1:200, Abcam, Cambridge, United Kingdom). The numbers of TH and IBA-1 positive cells were counted and averaged separately by three investigators unrelated to this experiment were recorded.

All data are presented as mean ± SEM and analyzed using Graph Pad Prism 9. The normality of data distribution was verified by Shapiro-Wilk test. Homocedasticity of variance was verified by Brown-Forsythe test (for multiple groups). Differences between treatment groups were compared using a combination of one-way ANOVA and Tukey’s multiple comparisons test. p < 0.05 were considered statistically significant.

To determine the effect of SA on neuroinflammation, we cultured microglial cell lines (BV2) in vitro. Firstly, we investigated the changes of BV2 cell survival rate after different concentrations of SA treatment (2.5 μM、5 μM、10 μM、20 μM、40 μM) via CCK8 assay. Results showed that SA not exceeding 10 μM did not have a significant effect on the viability of BV2 (Figures 1B, C ). Therefore, we chose 5 μM and 10 μM SA for the follow-up study.

Next, we treated BV2 with SA (5 μM and 10 μM) and LPS (1 μg/mL) for 24 h, and investigated the effect of SA on NO release and ROS production in LPS-exposed BV2 cells via NO and ROS detection assay. Results showed that SA inhibited the release of NO (Figure 1D) and the production of ROS (Figure 1E) in BV2 cells.

It was shown that NO release and ROS production were regulated by AKT and Nrf2 pathways. To further elucidate the role of SA, we investigated the effect of SA on AKT and Nrf2 pathways in BV2 cells. Results displayed that SA promoted the activation of AKT and Nrf2 pathways in BV2 (Figures 2A–C).

Subsequently, we treated BV2 with AKT inhibitor MK2206 (10 μM) for 4 h and then examined the effect of SA on the activation of AKT pathway and Nrf2 pathway in BV2 cells. Results displayed that MK2206 inhibited the activation of Nrf2 pathway by SA (Figures 2D–F). These results demonstrate that SA promotes the activation of the Nrf2 pathway through the AKT pathway. After that, we treated BV2 with Nrf2 inhibitor RA (5 μM) for 4 h, and then examined the activation of Nrf2 by SA. Results showed that RA inhibited the activation of Nrf2 by SA (Figures 2G, H).

Finally, we also investigated the effect of SA on NO release and ROS production in BV2 cells after pretreatment of BV2 cells with MK2206 or RA. Results displayed that MK2206 or RA pretreatment reversed the effect of SA on NO and ROS in BV2 cells to some extent (Figures 2I, J). These results demonstrate that SA inhibits NO release and ROS production through activation of the AKT/Nrf2 pathway.

To further elucidate the role of SA, we investigated the effect of SA on the production of inflammatory mediators in BV2 cells. First, BV2 cells were treated with SA (5 μM and 10 μM) and LPS (1 μg/mL) for 12 h, and the mRNA expression of inflammatory mediators were detected using a quantitative PCR method. Results displayed that SA inhibited mRNA expression of inflammatory mediators (IL-6 (Figure 3A), TNF-α (Figure 3B), iNOS (Figure 3C) and COX-2 (Figure 3D)) in LPS-stimulated BV2 cells. Subsequently, BV2 cells were treated with SA (5 μM and 10 μM) and LPS (1 μg/mL) for 24 h, and the protein expression of inflammatory mediators were detected using ELISA and Western blot methods. Results displayed that SA inhibited protein expression of inflammatory mediators (iNOS (Figures 3E, F), COX-2 (Figures 3E, G), IL-6 (Figure 3H) and TNF-α (Figure 3I)). These results demonstrate that SA inhibits the production of pro-inflammatory mediators in LPS-stimulated BV2 cells.

NF-κB pathway is a key pathway of inflammation, and to elucidate potential mechanism by which SA regulates inflammatory mediators, we further examined the effect of SA on NF-κB activation. First, BV2 cells were treated with SA (10 μM) and LPS (1 μg/mL) for 1 h, and the changes in p65, IκB and its phosphorylated proteins were detected using Western blot method. Results showed that SA inhibited the phosphorylation of IκB (Figures 4A, B), p65 (Figures 4A, C) and the degradation of IκB (Figures 4A, D).

Subsequently, BV2 cells were treated with the NF-κB inhibitor JSH (100 μM) and then the effect of SA on inflammatory mediator production were investigated. Results showed that SA inhibited the production of inflammatory mediators in BV2 cells in the same way as JSH (Figures 4E–H). These results demonstrate that SA inhibits the production of inflammatory mediators in BV2 cells in the same way as JSH.

In clinic, PD mainly manifests in the loss of motor ability, some with depressed, depression and other mental symptoms. In order to further study the effect of SA, we injected LPS to SN of mice and administered SA, and studied the effect of SA on clinical state and motor behavior in mice with LPS injection (Figure 5A). First, we tested the weight changes in mice before and after the experiment and showed that the SA treatment ameliorated the weight loss caused by the LPS injection (Figure 5B). Subsequently, we focused on the mice’s mental status, motor level and muscle tremor to score the mice for clinic pathology and found that SA treatment reduced the clinic pathology scores of LPS-injected mice (Figure 5C).

Next, to further elucidate the effect of SA on the locomotor behaviour of mice, we investigated the effect of SA on the locomotor ability of LPS injected mice through behavioural experiments. The results of the pole climbing experiment displayed that SA treatment ameliorated the reduction in balance in mice caused by LPS injection (Figure 5D). The results of the rotating bar experiment displayed that SA treatment ameliorated the reduction in fatigue tolerance in mice caused by LPS injection (Figure 5E). The results of the open-field experiments displayed that SA treatment improved the reduction in locomotion and the ability to explore unknown areas in mice caused by LPS injection (Figures 5F, G).

In summary, the above results illustrate that SA administration ameliorates clinical status and dyskinesia in mice exposed to LPS.

The main pathological change in PD is the degenerative absence of dopaminergic (DA) neurons in the SN. To further elucidate the neuroprotective effects of SA in vivo, we investigated the effects of SA on DA neurons in the SN of LPS-injected mice. The results of immunohistochemistry showed that SA treatment alleviated the reduction of TH-positive cells in the SN of mice caused by LPS injection (Figures 6A, B). We then examined the changes of TH protein in the mice midbrain by Western blot. Results displayed that SA restrained the reduction of TH protein in the mice midbrain caused by LPS injection (Figures 6C, D). These results prove that SA administration ameliorates dopaminergic neuron loss in mice exposed to LPS.

PD patients have large numbers of activated microglia in the SN of the midbrain, which are also the main effector cells of neuro-inflammation. To further elucidate the role of SA in vivo, we also investigated the effect of SA on microglia activation in the SN of LPS-injected mice. First, we examined the number of IBA-1-positive cells in the SN of the mouse midbrain using immunohistochemistry. The results showed that SA treatment reduced the number of IBA-1-positive cells in the SN of the brain in LPS-injected mice (Figures 7A, B). We then examined changes in OX-42 protein in the midbrain of mice by Western blot. The results showed that SA treatment reduced the expression of OX-42 protein in the midbrain of mice (Figures 7C, D). These results prove that SA administration inhibits microglia over-activation in mice exposed to LPS.

LPS is typically an inflammation-inducing factor and neuro-inflammation is related in the process of PD. Therefore, we next investigated the effect of SA on the inflammatory response in the brain of LPS-injected mice. Firstly, we measured the protein levels of pro-inflammatory factors (IL-6, IL-1β and TNF-α) in the mouse midbrain by ELISA. Results displayed that SA inhibited the protein expression of pro-inflammatory factors (Figures 8A–C) in the midbrain of LPS-injected mice. Afterwards, we examined the changes of pro-inflammatory mediator (IL-6, TNF-α, iNOS and COX2) mRNA in the midbrain of mice by qPCR. Results displayed that SA inhibited the mRNA expression of pro-inflammatory mediator (Figures 8D–G) in the midbrain of LPS-injected mice. Afterwards, we examined the effect of SA on ROS in the brain of LPS-injected mice. Results displayed that SA treatment inhibited the expression of ROS in the brain of LPS-injected mice (Figure 8H). These results prove that SA administration inhibits inflammatory responses in the midbrain region in mice exposed to LPS.

To further clarify the mechanism of SA’s inhibitory neuroinflammatory effects. We next examined the activation of AKT and NF-κB pathways in the midbrain of mice by Western blot. The results showed that the phosphorylation level of AKT in the midbrain of mice in the SA + LPS group was increased and the phosphorylation level of NF-κB p65 was decreased compared with that in the LPS-injected group (Figure 9). These results demonstrate that SA administration activates the AKT pathway and inhibits the NF-κB pathway in the midbrain region in mice exposed to LPS.