BV2 microglia and HT22 hippocampal neurons were obtained from the Chinese Academy of Sciences (Shanghai, China). All cell lines were tested for mycoplasma and no mycoplasma contamination was observed. All cell lines were detected by Short Tandem Repeat (STR) test and all were detected correctly. Cells were maintained in DMEM with 10% FBS and 1% penicillin/streptomycin. The cell viability was examined periodically, and the medium was replaced every three days. The logarithmic growth phase cells were selected. BV2 microglia (5 $\times$ 10${}^4$) and HT22 hippocampal neurons (5 $\times$ 10${}^4$) were inoculated into 96-well plates and treated with LPS (1 ug/mL) for 24 hours with or without the treatment of 40 $\mu$M Gas.

The animal test was authorized by the Experimental Animal Ethics Committee of Zhejiang University School of Medicine and implemented under strict supervision (EAECZUSM20210013). Forty adult male Sprague-Dawley (SD) rats (220–270 g) were purchased from the Institute of Experimental Animals of Wuhan University. Rats were placed in a temperature (23 $\pm$ 2 °C) and light controlled room.

Rats were divided into sham, Gas, TBI, and TBI+Gas groups (10 rats in each group). The stereotactic impacter Impact One™ (Leica Biosystems, Deer Park, IL, USA) was applied to cause controlled cortical impingement (CCI) damage in rats [13]. The rats were immobilized in a stereotactic instrument and anesthetized with isoflurane through a nasal mask. The induction rate was 3%, and the maintenance rate was 2%. A midline incision was made in the skull. Unilateral craniectomy was carried out between Bregma and Lambda with a hand drill (5 mm in diameter). The rats were hit with a 4.0 mm stainless steel flat impacter tip. The conditions were as follows: stereotactic coordinates (AP: –2.26 mm and ML: +2.0 mm), depth (0.65 mm), speed (4.0 m/s), residence time (200 ms), and angle (0.4°). After the injury, the bleeding was cleaned up, the incision was sutured, and the anesthesia was immediately removed. After the surgery, the rats were placed in cages on heating pads. The recovery of each rat was observed until they were standing on their four paws. The sham-operated rats underwent the same process as those with CCI injury, without craniotomy and later hits. Rats in the Gas group and TBI+Gas group were intraperitoneally injected with Gas (100 mg/kg; Sigma, USA) one hour before CCI, once daily for seven days. In contrast, the sham and TBI groups were not treated.

As previously stated [14], the rats were decapitated after the nerve injury was assessed. Their brains were harvested immediately at 3 and 7 days after the surgery. For craniotomy, a portion of the cerebral cortex was isolated (about 2 mm; 200 $\pm$ 20 mg). Blood and cerebrospinal fluid were cleaned with filter paper. After the wet weight was measured with a digital scale, the sample was dried at 100 °C for one day. Subsequently, the weight of each sample was determined. The brain water content = 100% $\times$ (wet weight – dry weight)/dry weight.

The Rotarod test was utilized to test motor coordination and balance [15, 16]. Before CCI induction, train rats to hold on to the rotarod for 120 s at 10 rpm (adaptation phase). After the rat was accustomed to running on the stick, the speed was accelerated to 40 rpm for 300 s. The mean time to fall of the last three trials was recorded as the baseline latency. After CCI, rats were examined three times on days 1, 3, 7, 14 and 21, and the mean fall latency was recorded.

TUNEL staining and Nissl staining were performed with the method of Wang H [17]. First, antigen-retrieved brain slices were incubated with a TUNEL reaction mix (Beyotime, China) for one hour at room temperature. After washing with phosphate buffered saline (PBS) for 20 min, the slides were incubated with DAPI for 5 min. Then, the sections were rinsed twice and observed with an inverted fluorescence microscope. ImageJ software was used to count TUNEL-positive cells. The number of apoptotic cells relative to DAPI was considered the apoptotic index. For Nissl staining, sections were stained in Nissl staining solution (Beyotime, China) for 5 min, washed with double distilled water, and mounted with Permount. This photo was taken under an optical microscope. Neurons with visible nuclei and relatively intact cell morphology were counted by ImageJ (version 2, LOCI, University of Wisconsin, Madison, WI, USA) and Cellcounter software 1.0 (Ningbo Shunyu Technology, China).

Rats were sacrificed under chloral hydrate anesthesia. The injured side brain tissue was taken, stored in 100 g/L paraformaldehyde solution, and fixed at room temperature for 24 hours. Cut the brain tissue into 3–4 $\mu$m coronal sections and place in the oven for 4–6 h. Then, the sections were cleaned with PBS 3 times (2 min each time) and incubated with the rabbit anti-IBA1 antibody (1:500) in a 4 ℃ refrigerator overnight. After being washed, the sections were maintained with the horseradish peroxidase-labeled goat anti-rabbit IgG (1:800) at 37 ℃ for 2 hours. After the sections were washed, 3,3’-Diaminobenzidine (DAB) was utilized for color development. The control sections were incubated with PBS. Five fields of view on the damaged side of the section were taken without overlapping each other, and the IBA1-positive cells were calculated. The mean $\pm$ standard deviation of the number of all positive cells in the sections of each group was taken as the immunohistochemical positive cell value of IBA1 in the damaged side brain tissue area of the group. The pathological alterations of brain tissues were reviewed under a microscope to confirm the degree of recovery.

Total RNA in tissues was extracted using the TRIzol reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. The RNA concentration and purity were determined with Nanodrop-spectrophotometer (Invitrogen, Waltham, MA, USA). Complementary DNA (cDNA) was synthesized from 1 $\mu$g of total RNA with the PrimeScript-RT Kit (Takara, Japan). The qRT-PCR was performed on the ABI7300 Realtime PCR system (ABI, USA) using SYBR®Premix-Ex-Taq™ (Takara, Japan). The total volume of the PCR reaction was 30 $\mu$L containing 300 ng of cDNA. The amplification was carried out with 10 min of initial denaturation at 95 °C, followed by 45 cycles of 95 °C for 10 seconds, 60 °C for 30 seconds, and 85 °C for 20 seconds. The GAPDH was set as the endogenous control of IL-1$\beta$, IL-6 and TNF-$\alpha$. qRT-PCR was implemented three times. The primers were listed in Table 1.

WB was carried out using the methods of predecessors [18].

The membranes were incubated with the primary antibodies (1:1000; Abcam, Waltham, MA, USA) overnight at 4 °C as follows: Bad (ab32445, Abcam, Waltham, MA, USA), Bax (ab32503, Abcam, Waltham, MA, USA), Bcl-2 (ab32124, Abcam, Waltham, MA, USA), iNOS (ab178945, Abcam, Waltham, MA, USA), TLR4 (ab13556, Abcam, Waltham, MA, USA), NF-$\kappa$B (ab32536, Abcam, Waltham, MA, USA), p-NF-$\kappa$B (ab76302, Abcam, Waltham, MA, USA), PKA (ab32514, Abcam, Waltham, MA, USA), CREB1 (ab32515, Abcam, Waltham, MA, USA), p-CREB1 (ab32096, Abcam, Waltham, MA, USA), BDFN (ab203573, Abcam, Waltham, MA, USA), Bcl-2 (ab32124, Abcam, Waltham, MA, USA), and GAPDH (ab9485, Abcam, Waltham, MA, USA). Then, the membranes were rinsed with tris buffered saline with Tween 20 (TBST) twice and maintained with a fluorescein-labeled secondary antibody for 1 hour at RT. After being cleaned three times, the membranes were exposed to the enhanced chemiluminescence (ECL) chronotropic agent and imaged with a membrane scanner.

The interleukin (IL)-1$\beta$, IL-6, tumor necrosis factor-$\alpha$ (TNF-$\alpha$) and other cytokines were quantified by commercial ELISA kits (MultiSciences, Hangzhou, China) following the manufacturer’s instructions [19].

Cell viability was determined by CCK-8 assay. The cells were plated into 96-well plates at a density of 1 $\times$ 10${}^3$ cells per well and cultured for 24 hours. Each well was added 10 $\mu$L of CCK-8 reagent (Dojindo Molecular Technologies, Kumamoto, Japan). After 1 hour of incubation at 37 ℃, the optical density (OD) at 450 nm was determined with a spectrophotometer (Bio-Rad, CA, USA).

The cells were trypsinized and harvested by centrifugation at 1500 rpm for 3 min. The cell apoptosis was determined by the apoptosis detection kit (Shanghai Aladdin biological technology Co., Ltd). Briefly, the cells were washed with PBS twice and then incubated with 400 $\mu$L of pre-cooled PBS. Subsequently, cells were added 10 $\mu$L of AnnexinV-FITC and 5 $\mu$L of PI. After incubation in the dark for 30 min at 4 ℃, the cell apoptosis was determined by flow cytometry (BD, USA). The cells were gated with FSC-A vs. SSC-A, and four populations including Annexin V${}^{-}$/PI${}^{-}$, Annexin V${}^{+}$/PI${}^{-}$, Annexin V${}^{+}$/PI${}^{+}$ and Annexin V${}^{-}$/PI${}^{+}$ were gated with Annexin V FITC-A vs. PI-A. The proportion of apoptotic cells was determined.

Statistical analysis was performed by SPSS17.0 statistical software (IBM SPSS, Chicago, IL, USA). One-way analysis of variance (ANOVA) was used to compare between groups. The measurement data were presented as mean $\pm$ standard deviation (SD). p 0.05 indicated statistical significance.

To understand the effect of Gas (100 mg/kg) on the neurological function of TBI rats, we implemented a modified Neurological Severity Score (mNSS), rotarod test and the wet/dry method to test the neurological function, motor function and brain water content of TBI rats 1, 3, 7, 14 and 21 days after TBI. The findings demonstrated that mNSS in the sham and Gas group did not change at the corresponding time points (with a score of 1–3). Nevertheless, neurological function was seriously damaged by TBI. After the treatment of Gas, the mNSS of rats in the TBI+Gas group was significantly lower than that of the TBI group (p 0.05, Fig. 1A). Meanwhile, the rotation performance of rats in the TBI+Gas group was better than that of the TBI group (p 0.05, Fig. 1B). Additionally, compared with the sham group, the water content of brain tissues in the TBI group was dramatically higher (82.32 $\pm$ 0.83%, p 0.05). Moreover, Gas treatment in the TBI model attenuated the water content (80.32 $\pm$ 0.62%, p 0.05, Fig. 1C).

Fig. 1.

Gas reduced the neurological deficits and brain water content of TBI rats. (A,B) The recovery of the nervous system was analyzed by mNSS (A), and Rota-rod (B) tests at 1, 3, 5, 7, 14 and 21 days after TBI. (C) The water content in the brain of TBI rats was assessed by the dry and wet method at 3, 7 and 21 days after TBI. *p 0.05, ***p 0.001 (vs. sham), NS p $\mathrm{>}$ 0.05, & p 0.05, && p 0.01, &&& p 0.001 (vs. TBI).

Nissl and TUNEL staining were performed to verify neuronal apoptosis in the damaged side of the cerebral cortex of TBI rats. The results illustrated that the neurons in the sham group were clear and complete. In contrast, damaged neurons in the TBI group exhibited irregular cell bodies, enhanced crinkling and nuclear pigmentation, and increased TUNEL-positive cells. After the treatment of Gas, the neuronal loss and TUNEL-positive neurons decreased in the TBI group (p 0.05, Fig. 2A–D). In addition, the levels of apoptosis-related proteins in the damaged side of brain tissues of TBI rats were determined. The results revealed that the Bax and Bad expression was increased, and the Bcl-2 level was decreased in the TBI group compared with the sham group. After Gas treatment, the results were the opposite (p 0.05, Fig. 2E). There was no significant difference between the TBI and the Gas groups. These results demonstrated that Gas reduced the apoptosis and degeneration of neurons in the brain of TBI rats.

Fig. 2.

Gas inhibited neuronal degeneration and apoptosis after brain injury in rats. (A,B) Representative photomicrograph (A) (scale = 100 $\mu$m) and quantification (B) of Nissl staining of the damaged side of the cortex of TBI rats on day 7. The red arrow indicates neuronal damage. (C,D) Representative micrograph (C) (scale = 50 $\mu$m) and quantification (D) of TUNEL staining in the damaged side of the cortex of TBI rats on day 7. (E) WB was carried out to test the profiles of apoptosis-related proteins in the damaged cortex of TBI rats on day 7. Data were expressed as mean $\pm$ SD. n = 5 for each group. *** p 0.001 (vs. sham), &&& p 0.001 (vs. TBI).

First, IBA1-labeled microglia in the brain tissues of TBI rats were determined by IHC. The results indicated that IBA1 microglia were significantly increased in the TBI group compared with the sham group. However, the IBA1 microglia were reduced after Gas intervention (p 0.05, Fig. 3A). The levels of IL-1$\beta$, IL-6 and TNF$\alpha$ in the brain tissues of TBI rats were measured by ELISA and qRT-PCR. Our results indicated that IL-1$\beta$, IL-6 and TNF$\alpha$ were upregulated in the TBI group. However, they were downregulated after Gas treatment (p 0.05, Fig. 3B,C). WB results demonstrated that iNOS and TLR4 were upregulated, and the NF-$\kappa$B phosphorylation was enhanced in the TBI group. After the Gas intervention, iNOS and TLR4 were downregulated, and the NF-$\kappa$B phosphorylation was decreased (p 0.05, Fig. 3D). These results indicated that Gas attenuated microglial activation and inflammatory responses in TBI rats.

Fig. 3.

Gas repressed microglia-mediated inflammations in TBI rats. (A) IHC was utilized to check the representative images and quantification of IBA1-positive cells in the damaged side of the cortex of TBI rats on day 7, with a scale of 100 $\mu$m. (B,C) qRT-PCR (B) and ELISA (C) were applied to measure the expression of inflammatory factors in the damaged side of the cortex of TBI rats on day 7. (D) WB was conducted to monitor the levels of inflammatory proteins in the damaged side of the cortex of TBI rats on day 7. Data were expressed as mean $\pm$ SD. n = 5 for each group. NS p $>$ 0.05, *** p 0.001 (vs. sham), && p 0.01, &&& p 0.001 (vs. TBI).

To explore the molecular mechanism of Gas in vivo, the effects of Gas on the PKA/CREB/BDNF pathway were determined. Our results indicated that the PKA level was decreased, the phosphorylation of CREB was inhibited, and Bcl2 was significantly downregulated in the TBI group compared with the sham group. However, Gas intervention yielded opposite results (p 0.05, Fig. 4A–D). These results confirmed that the mechanism of Gas might be related to the activation of the PKA/CREB/BDNF pathway.

Fig. 4.

Gas activated the expression of PKA/CREB1/Bcl2. (A–D) WB was utilized to test the PKA expression, the CREB phosphorylation, and the BDNF profile in the damaged side of the cortex of TBI rats on day 7. NS p $>$ 0.05, *** p 0.001 (vs. sham), &&& p 0.001 (vs. TBI).

Inflammatory responses are the main pathological mechanism of brain injury. Therefore, the influence of Gas (40 $\mu$M) on inflammatory cytokines in LPS-treated BV2 microglia was determined. The IL-1$\beta$, IL-6 and TNF-$\alpha$ were augmented in LPS-treated BV2 microglia. However, levels of IL-1$\beta$, IL-6 and TNF-$\alpha$ in BV2 microglia were dramatically reduced after Gas intervention (p 0.05, Fig. 5A,B). WB results further confirmed that the levels of iNOS and TLR4 were strengthened, and the phosphorylation of NF-$\kappa$B was elevated in the LPS group compared with the control group. However, after Gas intervention, the expression of iNOS and TLR4 was impeded, and the phosphorylation of NF-$\kappa$B declined (p 0.05, Fig. 5C). Additionally, the effects of Gas on neurons were evaluated. CCK-8 and FCM were utilized to determine cell viability and apoptosis. The results demonstrated that LPS treatment attenuated cell viability and increased cell apoptosis, while Gas treatment increased cell viability (p 0.05, Fig. 5D) and reduced cell apoptosis (p 0.05, Fig. 5E). Furthermore, Gas treatment suppressed the expression of apoptotic proteins (p 0.05, Fig. 5F). These findings confirmed that Gas alleviated LPS-induced microglial inflammation and hippocampal neuronal damage.

Fig. 5.

Gas attenuated LPS-mediated inflammatory responses and neuronal apoptosis in BV2 microglia (suggestion: in row E, it would be better to use a bold or larger font to make the labels easy to read). BV2 microglia and HT22 hippocampal neurons were treated with LPS (1 g/mL) for 24 hours with or without Gas (40 $\mu$M). (A,B) qRT-PCR (A) and ELISA (B) were employed to assay the expression of inflammatory cytokines in BV2 microglia. (C) WB was conducted to monitor the profiles of inflammatory proteins in BV2 microglia. (D) The effect of Gas on the viability of HT22 hippocampal neurons was analyzed by CCK-8. (E,F) FCM (E) and WB (F) were utilized to assess the impact of Gas on HT22 hippocampal neuronal apoptosis. Data were expressed as mean $\pm$ SD. n = 5 for each group. NS p $>$ 0.05, * p 0.05, ** p 0.01, *** p 0.001 (vs. Con), & p 0.05, && p 0.01, &&& p 0.001 (vs. LPS).

The effects of Gas in vitro have been determined. First, the PKA/CREB/BDNF pathway expression in LPS-treated BV2 microglia and HT22 hippocampal neurons was evaluated. The results confirmed that PKA was downregulated, the phosphorylation of CREB was reduced, and the expression of BDNF was significantly attenuated in the LPS group compared with the control group (p 0.05, Fig. 6A,B). Therefore, the PKA/CREB/BDNF pathway was abnormally expressed in LPS-treated cells. Accordingly, BV2 microglia and HT22 hippocampal neurons were treated with the PKA inhibitors H89 (10 $\mu$M) and Gas (40 $\mu$M) and exposed them to LPS for 24 hours. The results confirmed that H89 treatment weakened the effects of Gas and enhanced inflammatory cytokines and inflammatory proteins in BV2 microglia (p 0.05, Fig. 6C–E), declined the viability of HT22 hippocampal neurons (p 0.05, Fig. 6F), and enhanced the apoptosis (p 0.05, Fig. 6G,H). Furthermore, the LPS+H89+Gas treatment resulted in reduced PKA expression and downregulated phosphorylation of CREB and BDNF expression (p 0.05, Fig. 6I). These findings demonstrated that inhibiting the PKA/CREB/BDNF pathway weakened the anti-inflammatory and anti-apoptotic effects of Gas in vitro.

Fig. 6.

Inhibiting the PKA/CREB/BDNF pathway weakened the anti-inflammatory and anti-apoptotic effects of Gas on cells (suggestion: for row G, please refer to the suggestion for Fig. 5 row E). (A,B) WB was employed to measure the PKA expression, CREB phosphorylation, and the BDNF profile in BV2 microglia and HT22 hippocampal neurons. (C,D) qRT-PCR (C) and ELISA (D) were applied to define the expression of inflammatory cytokines in BV2 microglia treated with H89 (10 $\mu$M) and Gas. (E) The profiles of inflammatory proteins in BV2 microglia treated with H89 (10 $\mu$M) and Gas were determined by WB. (F) CCK-8 was utilized to measure the effect of H89 (10 $\mu$M) and Gas on the viability of HT22 hippocampal neurons. (G,H) FCM (G) and WB (H) were employed to verify the effect of H89 (10 $\mu$M) and Gas on the apoptosis of HT22 hippocampal neurons. (I) The PKA/CREB/BDNF pathway expression in BV2 microglia treated with H89 (10 $\mu$M) and Gas (40 $\mu$M) was monitored by WB. Data were expressed as mean $\pm$ SD. n = 5 for each group. NS p $>$ 0.05, ** p 0.01, *** p 0.001 (vs. Con), & p 0.05, && p 0.01, &&& p 0.001 (vs. LPS), # p 0.05, ## p 0.01, ### p 0.001 (vs. LPS+Gas).