Huangqi Guizhi Wuwu decoction promotes M2 microglia polarization and synaptic plasticity via Sirt1/NF-κB/NLRP3 pathway in MCAO rats

Reagents

Crude herbs including Radix astragali, Ramulus cinnamomi, Radix paeoniae alba, Rhizoma zingiberis recens and Fructus jujubae, were all purchased from Puning Zequn Chinese Herbal Pieces Co., Ltd. Trizol and BCA assay reagent were both purchased from Beyotime Biotechnology (China). PrimeScript™RT reagent Kit and TB Green ® Premix Ex Taq™ II (TliRNaseH Plus) are purchased from TaKaRa. Antibodies against SIRT1, NLRP3, ASC, PSD-95, Synapsin-1, Iba1, CD16, CD206, p-NF-κB, GAPDH, anti-rabbit IgG (H+L) and Anti-mouse IgG (H+L) were bought from Cell Signaling Technology (Danvers, MA, USA). Cleaved caspase-1 was purchased from Abcam (Cambridge, UK). EX527, a SIRT1 inhibitor, was bought from Sigma-Aldrich (St. Louis, MO, USA).

HGWD preparation

Radix astragali 90 g, Ramulus cinnamomi 90 g, Radix paeoniae alba 90 g, Rhizoma zingiberis recens 180 g, and Fructus jujubae 90 g were weighed. The above herbs were soaked in water (8 times the total weight) and refluxed twice, 1 h each time. The liquid extracts were filtered, combined and concentrated to 100 mL by a rotary evaporator. The extract were the freeze-dried to powder (59.9 g), which were stored at −20° C.

Quality control analysis for HGWD extract

HGWD extract was analyzed by using high-performance liquid chromatography system (HPLC) in which paeoniflorin, calycosin-7-O-β-D-glucoside, ononin, calycosin, cinnamic acid, and formononetin were used as quantitative stands. HGWD power was dissolved in methanol, and then filtrated with 0.22 μm filter. HGWD solution (10 μL) was injected into HPLC, and separated at a flow rate of 1.0 mL/min with a Waters sunfire-C18 column (250 mm × 4.6mm, 5 μm). The mobile phase consisted of acetonitrile (A) and 0.1% formic acid (v/v) (B). The separation temperature was 30° C, with UV detection performed at 260 nm.

Animals

Sixty healthy male Sprague-Dawley rats (8 weeks old, weighing 290~310g) were purchased from SPF (Beijing) Biotechnology Co., Ltd. [license No. SCXK (Beijing) 2019-0010)]. This research scheme was approved by the Animal Ethics Committee of Nanjing University of Chinese Medicine. All rats were housed at 25° C with 60% relative humidity and 12-h light/dark cycle. Standard rodent chow diet and water were available. All research scheme was approved by the Animal Ethics Committee of Nanjing University of Chinese Medicine.

Middle cerebral artery occlusion (MCAO) surgery

The animals were randomly divided into 5 groups: the control group (the sham operation group), model group (the MCAO group), HGWD low-dose group (0.38 g /kg), HGWD high-dose group (0.76g/kg), and edaravone group (4 mg/kg). MCAO surgery was performed according to Longa’s method. Rats were anesthetized by 2-3% isoflurane. Then the right common carotid artery, external carotid artery and internal carotid artery were separated successively. The main external carotid artery was ligated and dissociated. A small incision was made in the external carotid artery, and the nylon cord (0.28mm) was gently inserted into the internal carotid artery to block the middle cerebral artery. After 90 mins, the nylon cord was gently pulled out to restore blood perfusion. Ligate the external carotid artery and suture the skin. The middle cerebral artery was not blocked in the sham operation group. Twenty-four hours after ischemia, rats in the HGWD groups were intragastrically administered HGWD extract, and the edaravone group was intraperitoneally injected for 7 days. The sham group and model group were given the same amount of normal saline by gavage.

Neurological evaluation

The neurological deficit of rats was evaluated according to the modified neurological severity score (mNSS) criteria [24]: 0, no deficit; 1-6, mild deficit; 7-12, moderate deficit; 13-18, severe deficit. Neurological severity score (mNSS) of rats was performed at at 24 h, 3 d, and 7 d after the MCAO surgery, by researcher who was blind to the drug treatment.

2, 3, 5-triphenyltetrazolium chloride (TTC) staining

At the end of the experiment, rats were thoroughly perfused with saline under deep anesthesia. Then the brains were quickly removed and frozen at -20° C. The brain was sectioned coronally into 2mm slices, which were incubated with 1%TTC for 10 min in the dark. After staining, the un-infarcted part in the brain visualized to be red, while the infarcted tissue was white. After taking pictures, the Image J software was used to analyze the area of cerebral infarction.

Hematoxylin-eosin (HE) staining

The collected brain tissue was fixed with 4% paraformaldehyde, embedded in paraffin, and sliced into sections with a thickness of 5 μm. After routine dewaxing to water, HE staining was performed, and histopathological changes of brain tissues of each group were observed under an optical microscope (Olympus Optical Ltd., Tokyo, Japan).

Oxidative stress and inflammatory cytokines detection

MDA, SOD and GSH levels in the brain tissues were measured with their commercial assay kits (Nanjing Jiancheng, Nanjing, China). The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1β in brain tissue were determined by enzyme-linked immunosorbent assay.

RT-PCR to detect CD16, iNOS, CD206 and Arg-1 mRNA expression in hippocampus

Total RNA was extracted from the brain tissues by TRIzol reagent and reversely transcribed into cDNA according to the instructions of reverse transcription kit. The mRNA level of CD16, iNOS, CD206 and Arg-1 were determined by qRT-PCR. The reaction conditions were set as follows: pre-denaturation at 95° C for 5 min, denaturation 95° C for 10 s, and annealing under 60° C for 30 s, 45 cycles. The level of CD16, iNOS, CD206 and Arg-1 was quantitatively analyzed by 2-ΔΔCT. The primers used were as follows: CD16: forward, 5’-TCCGTGGCAGTCTATGAGGA-3’, and reverse, 5’-CAGATGGTGAGGTCGCAAGT-3’; iNOS: forward, 5’-AGTCAACTA CAAGCCCCACG-3’, and reverse, 5’GCAGCTTGTCCAGGGATTCT-3’; CD206: forward, 5’-GGTTCCGGTTTGTGGAGCAG-3’, and reverse, 5’-TCCGTTT GCATTGCCCAGTA-3’; Arg-1: forward, 5’-TCCTTAGAGATTATCGGAGCG-3’, and reverse, 5’-GTCTT TGGCAGATATGCAGG-3’; GAPDH: forward, 5’-GCCAAGGCTGTGGGCAAGGT-3’, and reverse, 5’-TCTCCAGGCGGC ACGTCAGA-3’.

Immunofluorescence staining to examine the expression of Iba1, CD16 and CD206 in hippocampus

After cardiac perfusion, the brain tissue was rapidly removed with cold PBS, and then embedded in Tissue Tek OCT (Sakura Finetek, Torrance, CA, USA), sectioned into slices (7 μm thickness). Immunofluorescence staining was performed to detect microglia polarization. In brief, the sections were blocked with 1%BSA and 0.3%Triton X-100 at room temperature for 1 h, and incubated with primary antibodies including Iba1 (1:500), iNOS (1:500), Arg1 (1:500), CD206 (1:500) overnight at 4° C. Then, slides were washed with PBS, followed by incubation with Goat Anti-mouse labeled Alex Flour ®488 or Goat Anti-Rabbit IgG labeled Alex Flour ®594 (1:000). After sealing with DAPI reagent, the slides were observed and photographed using fluorescence microscopy (BX63, Olympus Optical, Ltd., Tokyo, Japan).

Western blotting

Brain tissue protein was extracted using RIPA lysis buffer. The protein concentration was detected by BCA kit. Protein samples were separated by SDS-PAGE electrophoresis, transferred to 0.22 μm PVDF membrane, and blocked with 5% non-fat milk. Then the membrane was incubated with primary antibodies overnight at 4° C. The membrane was washed with TBST, incubated with the secondary antibody IgG (1:10000) at room temperature for 1.5 h. After washing, the brands were visualized with the ECL chromogenic agent. The gray value of each protein was analyzed by Image J software.

Statistical analysis

All statistical analyses were performed using Graphpad Prism 8 software. Experimental data are expressed as mean±SD. One-way analysis of variance was used for comparison between groups. P < 0.05 suggest the difference was statistically significant.

Availability of data and material

The authors hereby declare that the data and materials in this study will be presented upon request from the corresponding author.

Quantitative analysis of HGWD extract

Firstly, six major ingredients in HGWD extract were identified for the quality control using HPLC-UV method, including paeoniflorin, calycosin-7-O-β-D-glucoside, ononin, calycosin, cinnamic acid, and formononetin. The chromatographic condition was optimized, and representative chromatograms of standard compounds and HGWD extract was presented in Figure 1A, 1B. With the validated HPLC-UV method, the concentrations of paeoniflorin, calycosin-7-O-β-D-glucoside, ononin, calycosin, cinnamic acid, and formononetin were determined to be 1.9728, 0.1134, 0.0741, 0.0129, 0.0325, and 0.0056 μg/mg, respectively, in HGWD extract.

Figure 1. Representative chromatograms obtained by HPLC-UV. (A) standard compounds. (B) HGWD extract. 1, paeoniflorin; 2, calycosin-7-O-β-D-glucoside; 3, ononin; 4, calycosin; 5, cinnamic acid; 6, formononetin.

HGWD protects against brain damage after MCAO in rats

Next, we determined the neuroprotective effects of HGWD in vivo. MCAO model rats were established, and modified Neurological Severity Score (mNSS) was applied to evaluate the neurological behavior of rats at day 1, 3, and 7 after MCAO. As presented in Figure 2A, 2B, rat in the MCAO group showed the highest mNSS scores and low survival rate, while HGWD and edaravone treatment significantly attenuated the neurological deficits in MCAO rat. Meanwhile, MCAO/R performance caused notably increased infarct volume according to the TTC staining (Figure 2C). A significant reduction of infarct volume was observed in rats administrated with HGWD at doses of 0.38 and 0.76 g/kg, from 42.65 ± 2.48% to 33.08 ± 4.96%, and from 42.65 ± 2.48% to 30.95 ± 1.95%, respectively, when compared with the MCAO model group (Figure 2C). Besides, we examined the histopathological changes in the ischemic brain section by HE staining. The results showed that HGWD exhibited a protective effect against tissue injury, characterized by less degeneration and necrosis of nerve cells (Figure 2D). These above data suggest that HGWD can relieve cerebral injury induced by MCAO.

Figure 2. HGWD improved neurological deficits in MCAO rats. (A) mNSS score. (B) Survival rate. (C) Infarct size observed by TTC staining. (D) Histopathological changes in the ischemic portion of the rat brain observed by HE staining (scale bar= 100 μm). ^##P < 0.01 vs. control group; *P< 0.05, **P< 0.01 vs. model group.

HGWD decreased inflammatory and oxidative stress responses in MCAO rats

To understand the effects of HGWD on inflammatory response, we measured the levels of inflammatory cytokines such as IL-6, TNF-α, and IL-1β using ELISA kits. The results displayed that, compared with the sham group, MCAO can distinctly enhanced the release of TNF-α, IL-1β, IL-6 in the brain. HGWD treatment can obliviously inhibit inflammation in MCAO rat (Figure 3A–3C). Additionally, we investigated the effect of HGWD on the oxidative stress mediators, including SOD, MDA and GSH in the brain tissue. As Figure 3D–3F displayed, MCAO rats presented reduced SOD and GSH levels while promoted MDA level. However, HGWD at doses of 0.38 and 0.76 g/kg decreased the elevation of MDA and enhanced SOD and GSH levels. These data indicated that HGWD could weaken inflammatory and oxidative stress responses in rats with MCAO.

Figure 3. Effect of HGWD treatment on inflammatory cytokines and oxidative stress responses in brain tissue of rats. (A–C) The levels of IL-1β, IL-6, and TNF-α. (D–F) oxidative stress factors SOD, MDA, and GSH levels in the brain tissue. ^##P < 0.01 vs. control group; *P< 0.05, **P< 0.01 vs. model group.

HGWD shifts microglia polarization towards M2 in MCAO rats

Many evidences have highlighted the important role of microglia M1/M2 polarization in the progression of inflammation in cerebral injury. Therefore, we investigated whether the neuroprotective effects of HGWD were associated with altered microglia polarization. qRT-PCR experiments revealed the elevated expression of M1-like markers (CD16 and iNOS) and M2-like markers (CD206 and Arg-1) in the ischemic brain of MCAO rats (Figure 4A). However, HGWD could shifted microglia polarization towards M2 phenotype, decreasing CD16 and iNOS expression but increasing CD206 and Arg-1 expression, compared with MCAO model group. Double immunostaining further confirmed that the number of CD16+ M1 microglia and CD206+ M2 microglia were both significantly increased in the brain of rat after MCAO (Figure 4B, 4C). Consistent with qRT-PCR results, HGWD could decreased number of CD16+ M1 microglia while increased CD206+ M2 microglia, suggesting that HGWD promoted M2 polarization in MCAO rats.

Figure 4. HGWD promotes the M2 phenotype in microglia after ischemic stroke. (A) M1 marker (CD16 and iNOS) mRNA and M2 marker (CD206 and Arg-1) mRNA levels. (B) Representative double-immunofluorescence staining for CD16 (red) and Iba-1 (green) markers in hippocampus. (C) Quantification of the fluorescence intensity of CD16⁺/Iba-1⁺ cells. (D) Representative double-immunofluorescence staining for CD206 (red) and Iba-1 (green) markers in hippocampus. (E) Quantification of fluorescence intensity of CD206⁺/Iba-1⁺ cells. Scale bar: 50 μm. ^#P< 0.05, ^##P < 0.01 vs. control group; *P< 0.05, **P< 0.01 vs. model group.

HGWD increased dendritic spine density and enhanced synaptic plasticity in MCAO rats

Previous studies have reported that microglia could modify synaptic plasticity involving “find-me” and “eat-me” pathways [25]. Considering the promotion of HGWD on M2 polarization, we further tested whether HGWD produce beneficial effect on synaptic plasticity. As displayed in Figure 5A, 5B, the density of dendritic spines decreased in the MCAO group compared to the Sham group (P < 0.001). HGWD could dose-dependently increased the dendritic spine density when compared to the MCAO group. The expression levels of synapse marker proteins (PSD95, Synapsin I) were also determined to confirm the effect of HGWD on synaptic plasticity. The results showed that MCAO significantly decreased the protein levels of PSD95 and Synapsin 1 (P< 0.01, both) compared to the Sham group. The expression of PSD95 and Synapsin I were obviously upregulated in HGWD-treated rat compared to the MCAO rat.

Figure 5. HGWD promoted dendritic spine density and synaptic plasticity. (A) Examples of dendritic spines. Scale bar=10 μm. (B) Density of dendritic spines. (C) Representative western blot bands of PSD95 and synapsin I in each group. (D, E) The quantitative analysis of PSD95 and synapsin I expression in hippocampus. ^##P < 0.01 vs. control group; *P< 0.05, **P< 0.01 vs. model group.

HGWD modified Sirt1/NF-κB/NLRP3 pathway in the hippocampus of MCAO rats

Infammasome NLRP3 plays pivotal roles in regulating microglia polarization and neuroinflammation. To study the underlying mechanism of neuroprotective action of HGWD, NLRP3 inflammasome-associated proteins in the hippocampi of rat were probed. Western blotting revealed that, in comparison with Sham group, the expression of the main components of NLRP3 inflammasome, NLRP3, ASC, and cleaved caspase-1 were enhanced by cerebral ischemia-reperfusion injury. Notably, administration of HGWD significantly mitigated the activation of NLRP3 inflammasome in MCAO rats (Figure 6A–6D).

Figure 6. HGWD modified Sirt1/NF-κB/NLRP3 pathway in the hippocampus of MCAO rats. (A) Representative western blot bands of NLRP3, ASC and Cleaved Caspase-1 in each group. (B–D) The quantitative analysis of NLRP3, ASC and Cleaved Caspase-1 protein expression. (E) Representative western blot bands of Sirt1 and p-NF-κB in each group. (F, G) The quantitative analysis of Sirt1 and p-NF-κB protein expression. ^##P < 0.01 vs. control group; *P< 0.05, **P< 0.01 vs. model group.

Recently, it has been reported that NLRP3 inflammasome could be inhibited through Sirt1-dependent pathway [26]. Selective Sirt1 antagonist was reported to suppress NF-κB activity, and subsequently blunted NLRP3 inflammasome activation [27]. In order to further detect whether HGWD affected Sirt1/NF-κB pathway, the relative protein levels of Sirt1 and p-NF-κB in in the hippocampi of rat were measured (Figure 6E–6G). Consistently, HGWD-treated groups significantly enhanced Sirt1 protein but down-regulated p-NF-κB levels.

Inhibition of Sirt1 weakened the neuroprotective effect of HGWD in vivo

Finally, we further explored the role of Sirt1 in the therapeutic effect of HGWD. Sirt1 specific inhibitor EX527 (10mg/kg) was injected into the subarachnoid space prior to MCAO operation. As shown in Figure 7A–7C, the infarct volume and mNSS score in the MCAO+H-HGWD (0.76mg/kg) group was significantly decreased compared with MCAO group (P < 0.01). The effects of HGWD on reduction of infarct size and improvement of neurological functions were markedly abolished by EX527. Besides, the number of CD16+ M1 microglia decreased and CD206+ M2 microglia increased in the MCAO+H-HGWD (0.76mg/kg) group compared with that of the MCAO group (P < 0.01, both; Figure 7D–7G). However, there was no significant difference between the microglia M1/M2 polarization in the MCAO+EX-527+H-HGWD (0.76mg/kg) group and the MCAO group. Next, the expressions of PSD95, Sirt1 and NLRP3 were detected. Western blot revealed that, compared with the MCAO+H-HGWD group, the expression levels of PSD95, Sirt1 (P <0.01) were pronouncedly decreased and NLRP3 was higher (P<0.01) in the MCAO+EX-527+H-HGWD group (Figure 8A–8C). The above data indicated that inhibiting SIRT1 could notably weakened the therapeutic effect of HGWD against MCAO.

Figure 7. EX527 dramatically blocked the efficacy of HGWD on neurological deficits and microglia polarization in MCAO rats. (A) Representative TTC staining section. (B) Quantitative analysis of infarct regions. (C) The neurological scores (mNSS). (D) Representative double-immunofluorescence staining for CD16 (red) and Iba-1 (green) markers in hippocampus. (E) Quantification of the fluorescence intensity of CD16⁺/Iba-1⁺ cells. (F) Representative double-immunofluorescence staining for CD206 (red) and Iba-1 (green) markers in the hippocampus. (G) Quantification of fluorescence intensity of CD206⁺/Iba-1⁺ cells. Scale bar: 50 μm. ^##P < 0.01 vs. control group; **P< 0.01 vs. model group.

Figure 8. EX527 notably weakened the modulation of HGWD on PSD95, Sirt1 and NLRP3 expression. (A) PSD95 expression in each group. Left, representative western blot. Right, Quantitative analysis of PSD95. (B) Sirt1 expression in each group. Left, representative western blot. Right, Quantitative analysis of Sirt1. (C) NLRP3 expression in each group. Left, representative western blot. Right, Quantitative analysis of NLRP3. (D) Schematic representation of mechanisms by which HGWD protect against MCAO injury. ^##P < 0.01 vs. control group; **P< 0.01 vs. model group. ^##P < 0.01 vs. control group; **P< 0.01 vs. model group.