Mouse microglial cells, BV2, were purchased (The Institute of Cell Biology, Chinese Academy of Sciences, China) and cultured with DMEM, a high-glucose medium containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), at a constant temperature of 37˚C in 5% CO₂. The microglia were used in the experiment when the cells reached the logarithmic growth stage.

BV2 cell suspension was placed in a 6-well plate (2x10⁵ cells/ml). The cells were stimulated with LPS at different concentrations (100, 200 and 500 ng/ml; Sigma-Aldrich; Merck KGaA) for 24 h. For LMTK2 overexpression, the cells were transfected with plasmids overexpressing LMTK2. After 24 h, the cells were stimulated with LPS (500 ng/ml) for 24 h. Subsequently, cells were collected to extract total protein using RIPA lysis solution (cat. no. R0278; Sigma-Aldrich; Merck KGaA). The protein concentrations were detected by BCA method. Then, 10% SDS-PAGE electrophoresis was performed to separate the proteins (40 µg protein in each well). Skim milk powder (5%) was utilized to block the PVDF membrane for 40 min at room temperature. Primary antibodies (LMTK2 (1:500; cat. no. DF3344; Affinity Biosciences), inducible nitric oxide synthase (1:1,000; iNOS; cat. no. ab178945; Abcam), cyclooxygenase 2 (1:1,000; COX2; cat. no. ab179800), nuclear factor erythroid 2-related factor 2 (1:1,000; NRF2; ab137550; Abcam), heme oxygenase-1 (1:2,000; HO-1; cat. no. ab189491; Abcam), NAD(P)H dehydrogenase quinone 1 (1:20,000; NQO1; cat. no. ab28947; Abcam), Histone H3 (1:2,000; cat. no. ab1791; Abcam), GAPDH (1:5,000; cat. no. ab8245; Abcam) were incubated with the membrane at 4˚C overnight. This was followed by incubation with the secondary antibodies goat anti-rabbit IgG (1:10,000; cat. no. ab6721; Abcam) and rabbit anti-mouse IgG (1:10,000; cat. no. ab6728; Abcam) at room temperature for 1 h. ECL was used to visualize the protein bands which then was quantified with ImageJ 1.52v software (National Institutes of Health).

The plasmids overexpressing LMTK2 were constructed by Shanghai GenePharma Co., Ltd. BV2 cells were seeded into 6-well plates (2x10⁵ cells/ml). LMTK2 overexpression plasmids (Oe-LM; 4 µg) and empty plasmids (Oe-NC; 4 µg) were respectively transfected into BV2 cells using Lipofectamine™3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to manufacturer's protocol. After transfection of 24 h at 37˚C, cells were activated by LPS (500 ng/ml).

BV2 cell suspension was placed in a 6-well plate (2x10⁵ cells/ml). After experimental treatment, total RNA was extracted using TRIzol method and reverse transcribed into cDNA 42˚C for 1 h (RevertaidTM First Strand cDNA Synthesis kit, Fermentas; Thermo Fisher Scientific, Inc.). Relative determination of LMTK2 and GADPH mRNA was performed by SYBR-Green dye method (Clontech Laboratories, Inc.) and calculated using the 2^-ΔΔCq method (22). The primers of LMTK2 mRNA were as follows: Forward, 5'-TTGCCCGCCACAGTCTAAAC-3' and reverse, 5'-GATGACTCTTGCTACGCTAGT-3'; The primers of GADPH mRNA were as follows: Forward 5'-GCCTTCCGTGTTCCTACCC -3' and reverse 5'-TGCCTG CTTCACCACCTTC-3'. The target mRNA was amplified in the following thermocycling conditions: Predenaturation at 95˚C for 3 min; 30 cycles of 95˚C 30 sec, 58˚C for 30 sec and 72˚C for 1 min. The total extension at 72˚C for 10 min.

Cells viability was detected as per the manufacturer's protocol. BV2 cells were seeded into 96-well plates (1x10⁵ cells/ml). After cells were treated, MTT solution of 5 g/l (APExBIO Technology LLC) was added and placed in an incubator at 37˚C and 5% CO₂ for 4 h. The dimethyl sulfoxide (DMSO) solution of 100 µl was added to each well. After shaking the mixture, the absorbance (A) value at the wavelength of 490 nm was detected by a microplate reader and the cell survival rate was calculated.

NO2 was formed from NO in aqueous and reacted with Griess reagent (Beyotime Institute of Biotechnology). Therefore, NO levels in the supernatant were indirectly detected. Cells were seeded into 24-well plates (2x10⁵ cells/ml). After LPS treatment (500 ng/ml) for 24 h, the supernatant in the medium was collected and then 50 µl of Griess reagent was supplemented into the supernatant. After 10 min, the absorbance at 540 nm was detected.

BV2 cells were cultured in 6-well plates (2x10⁵ cells/ml). After treatment, the supernatant in medium was collected. Interleukin (IL)-1β, IL-6 and IL-10 levels were analyzed through ELISA kits (Mouse IL-1β ELISA kit; cat. no. PI301; IL-6, Mouse IL-6 ELISA kit; cat. no. PI326; and IL-10; Mouse IL-10 ELISA kit, cat. no. PI522; all from Beyotime Institute of Biotechnology), along with the detection of tumor necrosis factor (Mouse TNF-α ELISA Standard Recombinant Protein; cat. no. 29-8321-65; Invitrogen; Thermo Fisher Scientific, Inc.) and prostaglandin E2 (PGE2) levels (Prostaglandin E2 ELISA; ab133021; Abcam).

GraphPad Prism 8.0 software (GraphPad Software, Inc.) was used for statistical analysis of data, and one-way ANOVA was used to perform the comparison among groups, followed by Turkey's test. P<0.05 was considered to indicate a statistically significant difference.

LPS of different concentrations (100, 200 and 500 ng/ml) was used to stimulate BV2 cells. The LMTK2 protein and mRNA levels presented a gradual decrease with the increasing dose of LPS (Fig. 1A and B). Therefore, 500 ng/ml LPS was utilized to perform the follow-up experiments. Subsequently, the plasmids Oe-LM and Oe-NC were utilized to pre-treat BV2 cells. A significant upregulation of LMTK2 mRNA levels was observed in cells transfected with Oe-LM. (Fig. S1). Following plasmid transfection for 24 h, LPS was used to activate BV2 cells. A marked upregulation of LMTK2 protein and mRNA levels was observed in BV2 cells transfected with Oe-LM compared with Oe-NC (Fig. 1C and D).

In subsequent experiments, cell viability was evaluated through MTT assay in BV2 cells stimulated with LPS. Compared with the control group, LPS stimulation increased the cell viability of BV2 cells (Fig. 2A). As shown in a previous study (23), the levels of cytoskeletal protein α-tubulin and Iba1 were significantly increased in BV2 cells following LPS induction. Moreover, succinic acid dehydrogenase decreased exogenous MTT into water-insoluble blue-purple crystal formazan, contributing to the increase in OD value. In the present study, LMTK2 overexpression significantly decreased cell viability compared with cells treated with LPS alone (Fig. 2A). Subsequently, it was observed that the levels of proinflammatory mediators, consisting of NO generated by iNOS, PGE2 generated by COX-2), iNOS and COX-2, showed significant decreases in the presence of LMTK2 overexpression in LPS-activated BV2 cells (Fig. 2B-E).

The levels of proinflammatory and anti-inflammatory factors were analyzed in LPS-treated BV2 cells with or without transfection of Oe-LM plasmids. As the result displayed, the proinflammatory mediators, TNF-α, IL-1β and IL-6, were markedly decreased in response to LMTK2 overexpression compared with LPS treatment alone (Fig. 3A). Subsequently, Nrf2 signals were analyzed by detecting the expression of Nrf2 and its downstream genes (HO-1 and NQO1). Nrf2 was upregulated in the cytoplasm and nucleus, as well as HO-1 and NQO1, following overexpression of LMTK2 in BV cells (Fig. 3B), implying that the activation of Nrf2 is dependent on LMTK2.