Hsp22 pretreatment protects against LPS-induced hippocampal injury by alleviating neuroinflammation and apoptosis by regulating the NLRP3/Caspase1/IL-1β signaling pathway in mice

Animals and ethics statement

Adult male C57BL/6 mice (7–8 weeks old, 22–26 g) were purchased from Henan Skbeth Laboratory Animal Co., Ltd. (Henan, China). All mice are kept in the Animal Management Center of Nanchang University, the photoperiod was 12 hours, and food and water were freely supplied. The room temperature (RT) was controlled at 23°C ± 2°C, and the relative humidity was maintained at 50% ~ 60%. To reduce the pain of the mice, we obtained the approval of the Institutional Animal Care and Utilization Committee of Nanchang University (Animal Care and Utilization Committee Number: 2016020) before the experimental operation. All experimental procedures were performed in accordance with the guidelines of the International Association for Pain Research.

Intracerebroventricular injection (IVC)

Briefly, the mice were anesthetized by an intraperitoneal injection (i.p.) of 3% pentobarbital sodium and fixed in a stereotactic frame (RWD Life Sciences, Shenzhen, China). In a sterile surgical environment, skin approximately 1 cm along the midline was cut. Referring to the brain atlas the right ventricle was targeted as follows (millimeters from bregma): 0.8 mm behind the bregma, 1.3 mm on the right side of the median line, and 3.5 mm subdural. The compound (5 μl) was injected at a rate of 1 μl/min with a microsyringe, and the syringe was kept in place for 10 min after instillation to prevent reagent reflux. Gentamicin ointment was applied locally for disinfection. After the operation, the skin was sutured, and the mouse was resuscitated using a thermostatic heating blanket, and then returned to the breeding room for the next experiment.

Experimental design and establishment of the model

First, we pretreated the mice with the Hsp22 plasmid overexpression. Eighteen mice were randomly divided into the control group, LPS+Lv-Hsp22 group (the lentiviral vector was used to overexpress Hsp22) and LPS+Lv-Hsp22-NC group (the negative lentiviral vector of Hsp22) (n = 6 per group). According to the manufacturer’s instructions, the mice in the transfection groups were ICV-injected with Hsp22 (1 × 10⁶ TU). The Hsp22 lentiviral vector was constructed by China Shanghai GeneChem Co., Ltd. After the positioning navigation experiment, the mice in each group were administered saline/Hsp22 respectively. After 72 of hours pretreatment with Hsp22 (refer to Tai for the pretreatment time) [35]. The hippocampus was collected for enzyme-linked immunosorbent assay (ELISA) and Western blot analysis.

Next, mice in the LPS+Lv-Hsp22-NC and LPS+Lv-Hsp22 groups were subjected to LPS stimulation treatment to observe the changes of cognitive behavior and hippocampal neuroinflammation in mice. A total of 12 mice were pretreated with Hsp22 and divided into the LPS+Lv-Hsp22-NC and LPS+Lv-Hsp22 groups (n = 6 per group), and 6 mice were randomly selected for LPS stimulation. The administration concentration of LPS was 5 μg, which was dissolved in 5 ul of normal saline (the concentration is 0.9% of Nacl solution), because these doses have been reported to induce disease behavior in mice [36, 37]. As described in the previous protocol, 24 mice were subjected to behavioral tests 1 day before LPS injection. LPS (L2880, O55: B5) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

After the last behavioral experiment, the mice were sacrificed using sodium pentobarbitone (i.p., 45 mg/kg), and hippocampal tissues were collected for hematoxylin and eosin (H&E) staining, Nissl staining, immunohistochemistry, and TUNEL staining. All mice were sacrificed 24 h after the LPS injection, and the collected the hippocampus was collected for protein extraction and Western blot analysis.

Behavioral tests

The Morris water maze (MWM) was used for cognitive behavioral testing [38]. Testing included one continuous daily of the positioning sailing trial (6 days of training) and one space exploration trial (1 day of training). The MWM device (XR-XM101, Shanghai Xinruan, China) consisted of a circular pool with a diameter of 150 cm and a height of 60 cm. The wall had four water inlet points marked on the top to divide the pool into four quadrants (upper left, upper right, lower right, and lower left), which were filled with tap water (30 cm in-depth, 24–26°C). At the center of the right quadrant, a circular white platform (12 cm in diameter) was placed 3 cm below the water surface. During training, white food additive white pigment (GB25577-2010) was used to dye the pool white and hide the white platform. First, the experimental mouse was placed on the platform for 30 seconds to familiarize it with the surrounding environment. The MWM method includes a continuous daily positioning navigation test and a space exploration test. In the positioning and navigation tests, one of the quadrants was randomly selected and the mouse could travel back to the platform, which was randomly placed in the water, and the mouse was allowed to explore the hidden platform for 60 seconds and stay on it for 15 seconds. If the mouse could not approach the platform within 60 seconds, it was guided to the platform and allowed to stay for another 30 seconds. This process was repeated in the four quadrants, and record the escape latency. At the end of the positioning navigation test, the platform was removed, and the space exploration test was carried out. The number of platform crossings in 60 s, the escape latency and the time spent in the target quadrant were recorded. The MWM test was carried out from 8:00–12:00 in the morning.

Tissue and serum collection

Twenty-four hours after LPS injection, the mice were deeply anesthetized by an intraperitoneal injection of 1.5% pentobarbital sodium (45 mg/kg), and blood was taken after eyeball removal, and centrifuged at 4000 rpm in a low-temperature centrifuge (Thermo Fisher Scientific, Sorvall Legend Micro 17R). After centrifugation for 15 minutes, the supernatant was used for ELISA analysis, and then the mice were perfused through the heart with cold 0.9% NaCl injection to remove blood cells and proteins in the blood circulation. Next, the brain was removed by craniotomy, and the hippocampal tissue was separated, placed in a cryopreservation tube, and then transferred to a −80°C freezer for Western blot analysis. Finally, PBS (150 mL) was perfused, and 4% polymer was perfused again. Formaldehyde fixation (pH = 7.3, 150 mL), H&E staining, Nissl staining, immunohistochemistry and TUNEL staining were performed on mice.

Cell culture and groupings

The mouse microglial (BV2) cell line was purchased from Shanghai Tongpai Biotechnology Co., Ltd., China. The culture conditions were 37°C in 95% and 5% CO2, and DMEM high-glucose medium (Cell max, +4500 mg/L glucose) supplemented with 10% fetal bovine serum. During the whole experiment, the cells were randomly divided into the control group and LPS group. Our previous studies showed that 5 ug of LPS at a concentration of 1 μg/ul could induce a significant inflammatory response in microglia [39]. Therefore, we used 5 μg of LPS to stimulate BV2 microglia in this study.

In the second step of the cell transfection experiment, the cells were randomly divided into 2 groups: LPS+OE-Control (Hsp22 empty vector plasmid) and LPS+OE-Hsp22 (Hsp22 overexpression plasmid). Simply put, the BV2 cells were seeded in a 6-well plate and grown to 30–40% confluence before being transfected. According to the manufacturer’s instructions (GenePharma, Suzhou, China), the Hspb8 (65064-1) plasmid empty vector and Hspb8 (65064-1) overexpression plasmid (5 ul dissolved in 195 ul of serum-free medium), and GP-transfect-mate (Lot NO: 200703) reagents (dissolve 25 ul of transfection reagent in 175 ul of serum-free medium) were mixed to prepare 400 μl of working solution, and added to a 6-well plate. The medium was replaced with complete medium 8 hours after transfection. After 72 h of transfection, BV2 cells were stimulated with 5 ug of LPS (1 μg/ul) for 24 h, and BV2 cells were collected for protein separation.

Enzyme-linked immunosorbent assay (ELISA)

The concentrations of proinflammatory cytokines (TNF-α, IL-1β and IL-6) in the mouse hippocampal tissues were detected by ELISA assays using ELISA kits (CUSABIO, Wuhan, China) according to the manufacture’s protocols.

Western blot analysis

RIPA lysis buffer, phenylmethylsulfonyl fluoride (PMSF), and protein phosphatase inhibitor were mixed in a ratio of 100:1 and added to hippocampal tissue and BV2 microglia to homogenize and incubated for 30 minutes to lyse the samples. Then, the samples were centrifuged at 4°C for 30 min (15 min/time), with a relative centrifugal force at 12,000 relative centrifugal force (rcf), and the supernatant was collected. The protein concentration was determined by a bicinchoninic acid protein assay kit (TIANGEN, Beijing, China). Protein samples (35–50 μg) were separated protein samples by SDS-polyacrylamide gel electrophoresis (10%), and then transferred them to PVDF membranes (0.22 μm). The membranes were blocked in 5% skim milk at RT for 1 h and incubated with the following primary main antibodies overnight at 4°C: rabbit polyclonal anti-Hsp22 (Abcam, 1:1000, ab151552, USA), rabbit monoclonal anti-NLRP3 (Cell Signaling Technology 1:1000, D4D8T, USA), rabbit monoclonal anti-Caspase-1 (Cell Signaling Technology 1:500, E2Z1C, USA), rabbit monoclonal anti Cleaved caspase-1 (Cell Signaling Technology 1:500, E2G2I, USA), rabbit monoclonal anti-IL-1β (Cell Signaling Technology 1:1000, D4T2D, USA), rabbit monoclonal Cleaved IL-1β (Cell Signaling Technology 1:1000, D3A3Z, USA) and rabbit monoclonal anti-Iba1 (Cell Signaling Technology 1:1000, E404W, USA), rabbit polyclonal anti-Bcl2 (ABclonal 1:1000, A0208, China), rabbit monoclonal anti-Bax (ABclonal 1:1000, A19684, China), rabbit polyclonal anti-TNF-α (Proteintech 1:1000, 17590-1-AP, China), rabbit monoclonal anti-IL-6 (Proteintech 1:1000, 66146-1-Ig, China), and mouse polyclonal β-actin (Proteintech 1:5000, 66009-1-Ig, China). Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies respectively (HRP-conjugated Affinipure goat anti-Mouse IgG, SA00001-1, Goat Anti-Rabbit IgG, SA00001-2, 1:6000, Proteintech, China) for 1 h at RT. Bands were visualized with a Bio-Rad Gel Doc EZ imager (Bio-Rad, USA), and the intensity was analyzed by ImageJ software (NIH Image analysis website: http://rsb.info.nih.gov/ij/). β-Actin served as an internal control. Each experiment was repeated at least three times.

Cell apoptosis analysis

Apoptosis analysis was performed using a TUNEL kit (Roche, 11684817910, Wuhan, China). Hippocampal tissue was collected after the different treatments and add TdT, dUTP and DAPI staining solutions were added. After being incubated in the dark at 4°C for 20 minutes, the samples were washed once with PBS. Cell apoptosis was observed with a Nikon inverted fluorescence microscope (Nikon Eclipse TI-SR, Tokyo, Japan) and analyzed with ImageJ software.

H&E staining, Nissl staining and immunohistochemistry

H&E and Nissl staining were used to observe neuronal morphology and damage, and immunohistochemistry was used to detect the activation of Hsp22 and microglia in the hippocampus of mice. The brain tissue was embedded in paraffin, and a 4 μm thick brain tissue sections were prepared using a microtome (RM2016, Leica, Germany). After the sections were deparaffinized and rehydrated with xylene and ethanol, H&E staining, Nissl staining or immunohistochemical staining were performed. Using standard H&E and Nissl staining methods, H&E and toluidine blue staining were performed on the 4 μm sections, respectively. For immunohistochemistry, the sections were placed in 100°C (0.01 M, pH = 6.0) citrate buffer to induce antigen retrieval and then soaked them in 3% hydrogen peroxide solution for 10 minutes at RT to quench the endogenous peroxidase activity. After being blocked with 5% bovine serum albumin, the tissue was incubated with anti-Hsp22 antibodies (Abcam, 1:1000, ab151552, USA) or anti-Iba1 (Cell Signaling Technology 1:1000, E404W, USA) at 4°C overnight. Then, the sections were rinsed 3 times with PBS, incubated with anti-rabbit IgG secondary antibodies (1:2000, Boster, China, Wuhan) at RT for 1 h, dyed in 3,3′-diaminobenzidine solution, and reverse stained with hematoxylin. The slices were visualized and analyzed by an optical microscope (NIKON ECLIPSE CI, Tokyo, Japan) equipped with an imaging system and ImageJ software.

Statistical analysis

GraphPad Prism 8 software was used for statistical analysis. The data are expressed as the mean ± standard error (SEM). Two groups were compared using Student’s t test, and multiple groups were compared using repeated-measures one-way analysis of variance followed by post hoc of Bonferroni test or Dunnett’s test.

Availability of data

The datasets supporting the conclusions of this study are included in this article.

Hsp22 overexpression pretreatment ameliorates LPS-induced cognitive impairment of in mice in the MWM test

To study the effect of LPS injection and lentiviral vector-mediated was utilized to Hsp22 overexpression on the learning and memory in mice, the mice were subjected to a MWM experiment after 7 days of adaptive feeding, After MWM training, mice in the LPS+Lv-Hsp22-NC and LPS+Lv-Hsp22 groups were pretreated with the corresponding lentiviral vector for 72 hours, and then stimulated with LPS drugs. Twenty-four hours after LPS injection, the water maze space exploration experiment was performed to observe the changes in the spatial learning and memory abilities of mice before and after the drug was administered. After the experiment, the mice were sacrificed, and serum and the hippocampus were collected for subsequent experiments. No mice in each group died during the experiment. The experiment flow chart is shown in Figure 1A. The water maze activity track was recorded for each group of mice after modeling (Figure 1B). The results of the MWM test showed that compared with the control group, the LPS group and the LPS+Lv-Hsp22-NC group significantly prolonged the escape latencies of mice on day (p < 0.05–0.005, Figure 1C), In addition, there was a significant difference in the time spent in the target quadrant between the control group and the LPS-treated group mice (p < 0.005, Figure 1D). To further evaluate the effect of Hsp22 overexpression on LPS-induced memory impairment, a lentiviral vector was used to overexpress Hsp22 before LPS injection. The results showed that compared with the LPS group and the LPS+Lv-Hsp22-NC group, the LPS+Lv-Hsp22 group had a shortened escape latency of LPS-treated mice and increased the number of crossing platforms (p < 0.01, Figure 1D). These findings indicate that LPS can impair the memory and learning abilities of mice, indicating that the animal model of cognitive impairment was successfully constructed. Hsp22 overpression pretreatment can improve the cognitive decline in mice induced by LPS.

Figure 1. Hsp22 overexpression preconditioning improved LPS induced learning and memory impairment in mice. (A) Flowchart figure of animal experiment. (B) Representative diagram of Morris water maze of mice in each group. Hsp22 overexpression preconditioning reduces escape latency in mouse MWM test (C) and Hsp22 overexpression pretreatment increases the number of mice crossing the platform (D). (^***p < 0.005, ^***p < 0.005 vs. Control; ns: p > 0.05, LPS vs. LPS+Lv-Hsp22-NC; ^#p < 0.05, ^##p < 0.01 vs. LPS+Lv-Hsp22-NC).

LPS increases the expression of NLRP3/Caspase-1/IL-1β and proinflammatory cytokines in the hippocampus of mice

An increasing number of studies have shown that neuroinflammation is an important factor leading to cognitive impairment [40, 41]. Our previous research showed that the levels of proinflammatory cytokines (IL-6 and TNF-α) in the hippocampus increased within 24 hours of LPS injection [39]. Therefore, we collected blood from mouse eyeballs 24 hours after LPS administration, which was centrifuged, and the obtained serum was used for ELISA analysis. The results showed that compared with that in the control group, the expression of the proinflammatory cytokines IL-6, IL-1β and TNF-α was upregulated in the LPS group and the LPS+Lv-Hsp22-NC group after 24 h. (p < 0.01–0.005, Figure 2A–2C). In contrast, the LPS+Lv-Hsp22 group exhibited significantly reduced the expression of these inflammatory factors (p < 0.01–0.05, Figure 2A–2C).

Figure 2. Effect of Hsp22 on LPS-induced activation of NLRP3/Caspase-1/IL-1β pathways in the mice hippocampus tissue. The serum levels of IL-6, TNF-α and IL-1β. (A–C) (^**p < 0.01, ^***p < 0.005, ^***p < 0.005 vs. Control; ns: p > 0.05, LPS vs. LPS+Lv-Hsp22-NC; ^##p < 0.01, ^##p < 0.01, ^#p < 0.05 vs. LPS). The protein band of NLRP3, Caspase-1, IL-1β and its expression in the mice Hippocampus tissue (D–F). (^**p < 0.01, ^*p < 0.05, ^**p < 0.01 vs. Control; ns: p > 0.05, LPS vs. LPS+Lv-Hsp22-NC; ^#p < 0.05, ^###p < 0.005, ^#p < 0.05 vs. LPS+Lv-Hsp22-NC).

The western bolt results (Figure 2D–2F) showed that compared with those in the control group, the protein levels of NLRP3, IL-1β and Caspase-1 in the LPS group and LPS+Lv-Hsp22-NC group were significantly increased (p < 0.01–0.05), while, the mice of the LPS+Lv-Hsp22 group had significantly reduced the expression of these proteins in the hippocampus of mice (p < 0.05–0.005).

Hsp22 overexpression decreases pretreatment the expression of NLRP3/Caspase-1/IL-1β and proinflammatory cytokines in the hippocampus of LPS-treated mice

H&E staining was used to observe the neuronal morphology in the hippocampal (Figure 3A, 3B). In the control group, the cells in the hippocampus CA1 area were arranged tightly, with clear nuclei and rich cytoplasms, and there was complete brain parenchymal structure, and no neuronal degeneration or edema. In the LPS group and the LPS+Lv-Hsp22-NC group, a large amount of necrosis in nerve cells and deep staining of nuclear pyknosis were observed in the hippocampal CA1 area, as shown by the yellow arrow; The hippocampal neurons in the LPS+Lv-Hsp22 pretreatment group were arranged more densely and neatly than in the other groups.

Figure 3. Hsp22 overexpression pretreatment improves hippocampus tissue damage induced by LPS. (A) Histological analysis of Hippocampus tissue via HE staining (×200). (B) The degeneration rate of neurons in LPS group was higher than that in control group, and the difference was statistically significant (^***p < 0.005). The degeneration rate of neurons in LPS+Lv-Hsp22 group was lower than that in LPS+Lv-Hsp22-NC group, and the difference was statistically significant (^###p < 0.005). (C) Histological analysis of Hippocampus tissue via Nissl Staining (×200).

Some studies have shown that the structure and function of pyramidal cells in the hippocampus and cortex are related to learning and memory functions [42, 43]. Our histopathological observations on the mouse brain by Nissl-stained mouse brains showed that compared with those in the control group, the pyramidal cells in the hippocampus and cortex in the LPS group and the LPS+Lv-Hsp22-NC group were damaged, as shown by the yellow arrow, which were characterized by Nissl bodies and extensive loss of normal neurons (Figure 3C), However, the number of Nissl bodies in the LPS+Lv-Hsp22 group was decreased. Our Nissl staining results showed that the number of normal neurons in the hippocampus and cortex increased significantly after Hsp22 overexpression was given, suggesting that Hsp22 overexpression has a neuroprotective effect.

Immunohistochemistry showed that the number of Hsp22 positive cells in the hippocampus of mice in the LPS group and the LPS+Lv-Hsp22-NC group was higher than that in the control group. The LPS+Lv-Hsp22 group shown a reduction in these cells (p < 0.05, Figure 4A, 4B). Moreover, the Hsp22 protein levels in of the LPS+Lv-Hsp22 group was higher than that in the other groups, indicating that Hsp22 overexpression was successfully established (Figure 4C). These results indicated that Hsp22 overexpression pretreatment can decrease the expression of NLRP3/Caspase-1/IL-1β and proinflammatory cytokines in the hippocampus of LPS-treated mice.

Figure 4. Hsp22 levels were increased in hippocampus tissue after LPS stimulation and increased expression after Hsp22 pretreatment. (A, B) Immunohistochemical staining images of Hsp22(^*p < 0.05 vs. Control; ns: p > 0.05, LPS vs. LPS+Lv-Hsp22-NC; ^#p < 0.05 vs. LPS). (C) The protein band of Hsp22 and its expression (^*p < 0.05 vs. Control; ns: p > 0.05, LPS vs. LPS+Lv-Hsp22-NC; ^#p < 0.05 vs. LPS+Lv-Hsp22-NC). (D) The protein band of TNF-α and its expression (^*p < 0.05 vs. Control; ns: p > 0.05, LPS vs. LPS+Lv-Hsp22-NC; ^#p < 0.05 vs. LPS+Lv-Hsp22-NC).

Furthermore, our western bolt results showed that the expression of TNF-α in the LPS group and the LPS+Lv-Hsp22-NC group was higher than that in the other groups, and the LPS+Lv-Hsp22 group had decreased TNF-α expression (p < 0.05, Figure 4D).

Hsp22 overexpression pretreatment decrease the expression of NLRP3/Caspase-1/IL-1β and proinflammatory cytokines in LPS-treated BV2 microglial cells

Next, we used LPS stimulation to study the effect of Hsp22 overexpression preconditioning on BV2 microglia in neuroinflammation in vitro. The cell modeling process is shown in Figure 5A. We analysed the morphological change of microglia (Supplementary Figure 1). Western blot analysis showed that the expression of Hsp22 was higher than that in the other groups, indicating that the OE-Hsp22 was successfully transfected into BV2 microglial cells (p < 0.05, Figure 5B). Western blotting was used to measure the expression of NLRP3, Caspase-1, and IL-1β. The results showed that the protein expression levels in the LPS group and the LPS+Lv-Hsp22-NC group were higher than those in the control group (p < 0.01–0.05, Figure 5C–5E), The protein expression level of cleaved form of caspase-1 and IL-1 β has a significant difference respectively (p < 0.05–0.005, Supplementary Figures 2, 3). After overexpressing Hsp22 in BV2 microglia that were treated with LPS, the expression of NLRP3, caspase-1, and IL-1β was significantly reduced compared with LPS group (p < 0.01–0.05, Figure 5C–5E). Compared with that in the LPS+Lv-Hsp22-NC group, the difference was not statistically significant (p > 0.05, Figure 5C–5E), and the protein expression level of cleaved form of caspase-1 and IL-1 β has a significant difference respectively (p < 0.05–0.005, Supplementary Figures 4, 5). In summary, Hsp22 overexpression pretreatment can decrease the expression of NLRP3, Caspase-1, and IL-1β.

Figure 5. Effect of Hsp22 on LPS-induced activation of NLRP3/Caspase-1/IL-1β pathways in BV2 Microglial Cells. (A) Cell Modeling Flowchart. (B) The protein band of Hsp22 and its expression (^*p < 0.05 vs. Control; ns: p > 0.05, LPS vs. LPS+OE-Control; ^#p < 0.05 vs. LPS+OE-Control). (C–E) The protein band of NLRP3, Caspase-1, IL-1β and its expression. (^**p < 0.01, ^*p < 0.05, ^**p < 0.01 vs. Control; ns: p > 0.05, LPS vs. LPS+OE-Control; ^##p < 0.01, ^#p < 0.05, ^##p < 0.01 vs. LPS+OE-Control).

Hsp22 overexpression pretreatment attenuates inflammation in the hippocampus and apoptosis in BV2 microglial cells

Western blotting was used to measure the expression of IL-6 and TNF-α, the results showed that the protein expression levels in the LPS group and the LPS+OE- Control group were higher than those in the control group (p < 0.01, Figure 6A, 6B). After Hsp22 overexpression, IL-6 and TNF-α were significantly reduced compared with those in the LPS group (p < 0.05, Figure 6A, 6B). We had carried out quantitative real-time polymerase chain reaction (qRT-PCR) experiment and analyzed the mRNA expression level of TNF-α, NLRP3, Caspase-1, IL-1β (Supplementary Figures 6–9), and obtained the results which are similar to Western blotting, which made our results more reliable.

Figure 6. Hsp22 overexpression pretreatment alleviates BV2 microglial cells inflammation and apoptosis induced by LPS. The protein band of IL-6 (A), TNF-α (B) and its expression (^**p < 0.01, ^**p < 0.01 vs. Control; ns: p > 0.05, LPS vs. LPS+OE-Control; ^#p < 0.05, ^#p < 0.05 vs. LPS+OE-Control). (C) The protein band of Bax and its expression (D); The protein band of Bcl2 and its expression (***p < 0.005, ^*p < 0.01 vs. Control; ns: p > 0.05, LPS vs. LPS+OE-Control; ^#p < 0.05, ^#p < 0.05 vs. LPS+OE-Control).

Apoptosis is a form of the process of inducing programmed cell death that is induced by gene expression to maintain homeostasis under certain physiological and pathological conditions [44]. Previous studies have shown that LPS-induced cognitive impairment is related to apoptosis [17]. The ratio of proapoptotic molecules to antiapoptotic molecules is increased (Bax/Bcl-2). To confirm the influence of inflammation model on apoptosis, we performed TUNEL staining and Western blotting. Western blot analysis of BV2 microglial cells showed that, compared with the control group, Bax was upregulated in the hippocampus in the LPS and LPS+OE-control group, and Bcl2 was downregulated (p < 0.05–0.005, Figure 6C, 6D). We analyzed the Bax/Bcl-2 ratio in vivo and in vitro experiments and the results are the same as reliable (p < 0.05–0.005, Supplementary Figures 10, 11). The TUNEL assay and hippocampal tissue protein analysis yielded similar results (p < 0.05–0.005, Figure 7A–7D). Hsp22 overexpression in the pretreatment group reversed this outcome. Our research shows that overexpression of Hsp22 may exert a neuroprotective effect by reducing cell apoptosis.

Figure 7. Hsp22 overexpression pretreatment alleviates hippocampus tissue apoptosis induced by LPS. (A, B) Representative images of Tunel-stained brain sections (×200) (^***p < 0.005 vs. Control; ns: p > 0.05, LPS vs. LPS+Lv-Hsp22-NC; ^###p < 0.005 vs. LPS+Lv-Hsp22-NC). (C) The protein band of Bax and its expression. (D) The protein band of Bcl2 and its expression. (^*p < 0.05, ^***p < 0.005 vs. Control; ns: p > 0.05, LPS vs. LPS+Lv-Hsp22-NC; ^#p < 0.05, ^#p < 0.05 vs. LPS+Lv-Hsp22-NC).

Hsp22 overexpress pretreatment attenuates microglial activation in the hippocampus of LPS-treated mice and BV2 microglial cells

Iba1 is considered to be an activation marker of microglia [45, 46]. This factor plays an essential role in inflammatory diseases of the central nervous system [47]. In this study, we examined the number of Iba1-positive (microglia marker) cells by immunohistochemistry. We observed that compared with that in the control group, the number of Iba1 positive cells in the LPS group and the LPS+Lv-Hsp22-NC group was significantly increased (p < 0.005, Figure 8A, 8B). The Iba1-positive cells in the Hsp22 overexpression pretreatment group were significantly reduced compared with those in the LPS group (p < 0.005, Figure 8A, 8B). Next, we also detected the expression of Iba1 protein in the hippocampus and BV2 cells in each group. The expression of Iba1 protein in the hippocampus in the LPS group and the LPS+Lv-Hsp22-NC group was higher than that in the control group. The expression of Iba1 protein in the LPS+Lv-Hsp22 group was lower than that in the LPS group (p < 0.01–0.05, Figure 8C). Similar results were obtained in the LPS-induced microglial BV2 cell model in vitro (p < 0.05–0.005, Figure 8D). These results indicate that Hsp22 overexpression preconditioning negatively regulates microglia activation in a mouse model of cognitive dysfunction.

Figure 8. Hsp22 overexpression pretreatment alleviates Iba1 activation induced by LPS. (A, B) Immunohistochemical staining images of Iba1(^***p < 0.005 vs. Control; ns: p > 0.05, LPS vs. LPS+Lv-Hsp22-NC; ^###p < 0.005 vs. LPS+Lv-Hsp22-NC). The arrow points to Iba1 positive cell. (C) The protein band of Iba1 and its expression in Hippocampus tissue (^**p < 0.01 vs. Control; ns: p > 0.05, LPS vs. LPS+Lv-Hsp22-NC; ^#p < 0.05 vs. LPS+Lv-Hsp22-NC). (D) The protein band of Iba1 and its expression in BV2 Microglial Cells. (^***p < 0.005 vs. Control; ns: p > 0.05, LPS vs. LPS+OE-Control; ^#p < 0.05 vs. LPS+OE-Control).