Guizhi Fuling capsule relieves memory deficits by inhibition of microglial neuroinflammation through blocking JAK2/STAT3 pathway in presenilin1/2 conditional double knockout mice

Abstract

Chronic neuroinflammation has been regarded as an important part of the pathological initiation of Alzheimer's disease (AD), which is associated with the regulation of microglial activation. Preventing microglial activation to inhibit neuroinflammation may become a potential target for the treatment of neurodegenerative diseases. Guizhi Fuling capsule (GZFL) has a strong repression on inflammatory responses. Here, the presenilin1/2 conditional double knockout (PS cDKO) mice, a well-established mouse model of AD, were divided into: WT mice (WT), WT mice+GZFL (WT+GZFL), PS cDKO mice (cDKO), and PS cDKO mice+GZFL (cDKO+GZFL). Mice in the WT+GZFL and cDKO+GZFL group were fed standard chow containing 2000 ppm GZFL for 90 days. After 60 days of GZFL treatment, mice were given to behavioral tests for 30 days in order to explore the effects of GZFL on cognitive and motor function. Then, mice were sacrificed for examining the effects of GZFL on inflammation. Furthermore, primary microglia were obtained from neonatal Sprague-Dawley rats and pretreated with or without GZFL (50 μg/ml) for 1 h in the absence or presence of lipopolysaccharide (LPS) (100 ng/ml) stimulation to speculate whether the underlying mechanism of GZFL's anti-inflammatory potential was closely associated with Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Our findings indicated that GZFL has the ability to alleviate memory deficits in PS cDKO mice, which attributes to the improvement of neuroinflammation by inhibiting microglial activation and the levels of pro-inflammatory mediators. In addition, GZFL could inverse the tau hyperphosphorylation and the lessened expression of synaptic proteins in hippocampus of PS cDKO mice. Furthermore, GZFL prevented LPS-induced neuroinflammatory responses in primary microglia by decreasing the levels of pro-inflammatory mediators. It is noteworthy that therapeutic effects of GZFL on memory impairment are depended on the inhibition of neuroinflammatory responses by the blockage of JAK2/STAT3 signaling pathway. Taken together, GZFL may be an effective compound Chinese medicine for the improvement and postponement of neurodegenerative progression in AD.

Keywords: Alzheimer’s disease; Guizhi Fuling capsule; JAK2/STAT3 pathway; memory deficits; microglial activation; neuroinflammation.

Figure 1

Guizhi Fuling capsule (GZFL) improves recognition memory impairment in presenilin1/2 conditional double knockout (PS cDKO) mice without locomotor alteration. (A) The experimental timeline of GZFL treatment and behavioral tests. (B, C) An open field test was used to detect the moving distances (B) and percentage of time spent in the margin zone (C). (D) During novel object recognition task, preference indexes were measured. (E) Preference indexes were in comparison in each session within the different groups. Data are the mean ± SEM (n = 16), *P < 0.05; **P < 0.01.

Figure 2

Guizhi Fuling capsule (GZFL) relieves spatial and associative memory impairment in presenilin1/2 conditional double knockout (PS cDKO) mice. (A, B) Duration (A) and frequency (B) entered into the novel arm of Y maze. (C) The escape latency to find the hidden platform during the first 5-d training period of Morris water maze (MWM). (D, E) During the probe phase of MWM, the percentage of time (D) and distance (E) were tested in the target quadrant. (F)The escape latency to seek out the visible platform in MWM. (G) The swimming speed during the test period of MWM. (H) The freezing response in the fear conditioning task. Data are the mean ± SEM (n = 16), *P < 0.05; **P < 0.01.

Figure 3

Guizhi Fuling capsule (GZFL) represses microglial activation in the hippocampus (HPC) CA1 of presenilin1/2 conditional double knockout (PS cDKO) mice. (A) Representative images of Iba1⁺ microglia in HPC CA1. The location of HPC CA1 was displayed in overview images by the box. Magnification of overview image is 5×, scale bar, 200 μm. Magnification of other four images is 40×, Scale bar, 50 μm. (B) The percentage of amoeboid cells in total Iba1⁺ cells were calculated to conduct a quantitative analysis of microglial activation in HPC CA1. (C–E) The branches (C), the average branch length (D) and longest branch length (E) in HPC CA1. Data are the mean ± SEM (n = 6), *P < 0.05; **P < 0.01.

Figure 4

Guizhi Fuling capsule (GZFL) decreases the increased levels of pro-inflammatory mediators in the hippocampus (HPC) of presenilin1/2 conditional double knockout (PS cDKO) mice. (A) Representative Western blot for COX-2 and iNOS in HPC. (B, C) Quantification of Western blot for COX-2 and iNOS in HPC. (D–F) The levels of TNF-α (D), IL-1β (E) and IL-6 (F) were measured by ELISA in HPC. (G–K) The mRNA levels of COX-2 (G), iNOS (H), TNF-α (I), IL-1β (J) and IL-6 (K) in HPC were analyzed by qRT-PCR. Uncropped immunoblots are shown in Supplementary Figure 1 . These values were showed as relative changes to the respective wild-type (WT) mice, which was set to 1. Data are the mean ± SEM (n = 6), *P < 0.05; **P < 0.01.

Figure 5

Guizhi Fuling capsule (GZFL) raises the expressions of synaptic proteins and decreases the phosphorylation of tau protein in the hippocampus (HPC) of presenilin1/2 conditional double knockout (PS cDKO) mice. The brain tissues of mice in each group were lysed, quantified and then analyzed by Western blot to detect the expressions of synaptic proteins (NR2A, NR2B, PSD95, MAP2 and SYP) and phosphorylated tau protein (ser396/404). (A) Representative Western blot for synaptic proteins in HPC. (B–F) Quantification of Western blot for synaptic proteins in HPC. (G) Representative Western blot for phosphorylated tau proteins in HPC. (H, I) Quantification of Western blot for phosphorylated tau proteins in HPC. The protein expressions of synaptic proteins and phosphorylated tau proteins were standardized based on the respective expressions of β-actin or total tau. Uncropped immunoblots are shown in Supplementary Figure 2 . These values were showed as relative changes to the respective wild-type (WT) mice, which was set to 1. Data are the mean ± SEM (n = 6), *P < 0.05; **P < 0.01.

Figure 6

Influences of Guizhi Fuling capsule (GZFL) on cell viability, and the levels of pro-inflammatory mediators in primary microglia stimulated by LPS. (A) Primary microglia were treated with GZFL (12.5-400 μg/ml) for 24 h followed by MTT assay to measure cell viability. (B) Primary microglia were stimulated with LPS (100 ng/ml) with or without GZFL at the indicated concentration (6.25-400 μg/ml) for 24 h followed by Griess assay to detect the level of nitrite in medium. (C) Representative Western blot for iNOS and COX-2 in primary microglia pretreated with GZFL (12.5-400 μg/ml) followed by the stimulation of LPS (100 ng/ml). (D, E) Quantification of Western blot for iNOS and COX-2 in primary microglia. (F–H) Cell media were gathered to analyze the levels of TNF-α, IL-1β and IL-6 by ELISA. The protein expressions of iNOS and COX-2 were standardized based on the respective expressions of β-actin. Uncropped immunoblots are shown in Supplementary Figure 3 . These values were showed as relative changes to the respective Control, which was set to 1. Data are the mean ± SEM from three independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001 vs untreated Controls; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs LPS alone.

Figure 7

Guizhi Fuling capsule (GZFL) inhibits the mRNA expressions of pro-inflammatory mediators in LPS-stimulated primary microglia with morphological changes. Primary microglia were stimulated by LPS (100 ng/mL) in the absence or presence of GZFL (50 μg/ml) for varying times. (A) Morphological changes of primary microglia were observed under a phase-contrast light microscope. Magnification is 40×, Scale bar, 25 μm. Control (Ctrl): untreated primary microglia. Ctrl+GZFL: GZFL (50 μg/ml) treatment alone. LPS: LPS (100 ng/ml) stimulation alone. LPS+GZFL: combined treatment of LPS (100 ng/ml) and GZFL (50 μg/ml). (C–E) Total RNA was extracted to quantitate the mRNA expressions of COX-2 (B), iNOS (C), TNF-a (D) and IL-1β (E) by qRT-PCR. These values are expressed as fold change over the respective Controls. Data are the mean ± SEM from three independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001 vs untreated Controls; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs LPS alone.

Figure 8

Guizhi Fuling capsule (GZFL) blocks the JAK2/STAT3 signaling pathway in LPS-stimulated primary microglia. Primary microglia were pretreated with GZFL (50 μg/ml) for 1 h followed by the stimulation of LPS (100 ng/ml) for varying times. Microglia were lysed, and the total protein expression of JAK2, STAT3, and their respective phosphorylated forms were assayed by Western blot. (A) Representative Western blot for the expressions of JAK2, STAT3, and their respective phosphorylated levels at 2 h, 4 h, 6 h, 12 h and 24 h of LPS stimulation. (B, C) Quantification of Western blot for phosphorylated JAK2 (B) and STAT3 (C). The expressions of phosphorylated JAK2 and STAT3 were standardized based on the respective total JAK2 and STAT3. Uncropped immunoblots are shown in Supplementary Figure 4 . These values were showed as relative changes to the respective Control, which was set to 1. Data are the mean ± SEM from three independent experiments, **P < 0.01, ***P < 0.001 vs untreated Controls vs untreated Controls; ^#P < 0.05, ^###P < 0.001 vs LPS alone.

Figure 9

Guizhi Fuling capsule (GZFL) inhibits neuroinflammatory responses in LPS-stimulated primary microglia by intercepting the JAK2/STAT3 signaling pathway. Control (Ctrl): untreated primary microglia. LPS: LPS (100 ng/ml) stimulation alone. LPS+GZFL: combined treatment of LPS (100 ng/ml) and GZFL (50 μg/ml). LPS+Ruxolitinib: combined treatment of LPS (100 ng/ml) and Ruxolitinib (5 μM). LPS+C188-9: combined treatment of LPS (100 ng/ml) and C188-9 (10 μM). LPS+GZFL+Ruxolitinib: combined treatment of LPS (100 ng/ml), GZFL (50 μg/ml) and Ruxolitinib (5 μM). LPS+GZFL+C188-9: combined treatment of LPS (100 ng/ml), GZFL (50 μg/ml) and C188-9 (10 μM). (A) Representative Western blot for the total of JAK2, STAT3, and their respective phosphorylated levels. (B, C) Quantification of Western blot for phosphorylated JAK2 (B) and STAT3 (C). (D) Representative Western blot for the total of STAT3 and phosphorylated STAT3. (E) Quantification of Western blot for phosphorylated STAT3. (F) Representative Western blot for the expressions of iNOS and COX2. (G, H) Quantification of Western blot for iNOS (G) and COX2 (H). The expressions of phosphorylated JAK2 and STAT3 were standardized based on the respective total of JAK2 and STAT3. The protein expressions of iNOS and COX2 were standardized based on the respective expressions of β-actin. Uncropped immunoblots are shown in Supplementary Figure 5 . These values were showed as relative changes to the respective Control, which was set to 1. Data are the mean ± SEM from three independent experiments, ***P < 0.001 vs untreated Controls; ^###P < 0.001 vs LPS alone.

Figure 10

Summary diagram representing the effects of Guizhi Fuling capsule (GZFL) on improving memory deficits in presenilin1/2 conditional double knockout (PS cDKO) mice by inhibiting microglial neuroinflammation through blockage of JAK2/STAT3 signaling pathway.