ZHANG Miao, GAO Xing-hong, HU Yuan

1. Department of Neurology, Zhongnan Hospital of Wuhan University, Wuhan 430071, China

2. School of basic medicine, Zunyi Medical University, Zunyi 5630062, China

3. Department of Neuropschology, Zhongnan Hospital of Wuhan University, Wuhan 430071, China

Keywords:

ABSTRACT Objective: To investigate the effect of honokiol on microglia polarization and the underlying mechanism.Methods: Inflammatory factors were detected using ELISA to determine the optimal concentration of cobalt chloride to induce, and that of honokiol to treat chronic hypoxia (48 h) in microglia cell line BV2 cells.BV2 cells were divided into four groups:control, chronic hypoxia, chronic hypoxia+honokiol, chronic hypoxia+honokiol+3-TYP (SIRT3 inhibitor).ELISA was used to measure the concentration of supernatant TNF and IL-1β proteins, qPCR was used to detect the expression of cellular M1 and M2 polarization markers,and biochemical assays were used to detect the level of reactive oxygen species in each group.Western Blot was used to detect protein levels of SIRT3 and upstream inflammatory molecules NLRP3 and caspase1.Results: Chronic cobalt chloride stimulation of BV2 cells at an optimal concentration of 100 μmol/L significantly increased the release of inflammatory fac- tors TNF and IL-1β after stimulation compared with the control group(P＜0.05); compared with the control group, cells in the chronic hypoxia group had down-regulation of SIRT3 protein expression, whereas the ROS levels, NLRP3 and caspase1 protein levels, the M1 polarization marker CD86, iNOS mRNA levels and CD16/32 ratio were upregulated.and honokiol (10 μmol/L) significantly up-regulated the SIRT3 protein and mRNA levels of M2 markers Arg-1 and CD206 in chronic hypoxic cells (P ＜ 0.05)and down-regulated levels of ROS, NLRP3/caspase1 protein, and mRNA levels of M1 markers(P ＜ 0.05), and this anti-oxidative and antiinflammatory effect was able to be reversed by SIRT3 inhibitor.Conclusion: Honokiol inhibits chronic hypoxia-induced microglia M1 polarization and inflammatory pathway activation, and its anti-inflammatory effects are SIRT3-de- pendent.

1.Introduction

Chronic cerebral hypoperfusion is a common pathophysiological basis for both Alzheimer’s disease (AD) and vascular cognitive impairment (VCI)[1].A longitudinal study involving brain imaging,blood, cerebrospinal fluid, and other biomarkers of 1,171 patients at different stages of AD revealed that cerebral hypoperfusion occurs earlier than the deposition of its key pathological marker,amyloid-beta protein, and the decrease in cerebral blood flow is associated with disease progression[2].On the other hand, carotid stenosis caused by atherosclerosis of the carotid arteries may lead to VCI through chronic cerebral hypoperfusion[3].In patients with carotid artery stenosis, stent implantation has been found to improve cerebral blood flow and delay the progression of vascular cognitive impairment[4].Our research group and others have discovered that chronic cerebral hypoperfusion can induce activation of central nervous system inflammatory cells, microglial cells, and astrocytes[5, 6].However, the mechanisms underlying neuroinflammation in chronic cerebral hypoperfusion are not fully understood at present,and animal and cell studies on this issue may contribute to the prevention and treatment of related dementia-related diseases.

Houpu is a traditional Chinese medicine derived from the leaves of the Magnoliaceae family.“Shennong Bencao Jing” describes houpu as having a bitter taste and being warm in nature.It is used to treat conditions such as stroke, febrile diseases, headaches,chills and fever, palpitations, and blood stasis.Over the years, it has been employed in Traditional Chinese Medicine for treating ailments like cerebral infarction and migraines.Honokiol (HNK),found in houpu, is one of its main active components.It is a smallmolecule phenolic acid compound that easily penetrates the bloodbrain barrier, exhibiting anti-inflammatory, antioxidant, and antitumor properties[7].Studies have shown that honokiol can activate endogenous stress-responsive and antioxidant enzyme SIRT3,improving cognitive and neurological damage associated with surgery[8].

In our previous research, we found that honokiol can ameliorate cognitive impairment and neural damage in rats with chronic cerebral hypoperfusion[9].In this experiment, we further established an in vitro model of chronic hypoxia to examine the effects of honokiol on the inflammatory pathways and polarization of chronic hypoxia-induced microglial cells.This research aims to provide new pharmaceutical options and treatment targets for dementia prevention and treatment.

2.Materials and Methods

2.1 Main Reagents

Honokiol (MCE, HY-N0003), cobalt chloride (CoCl2, Sinopharm,10007216), 3-TYP (MCE, HY-108331), BV2 mouse microglial cell line purchased from Procell Life Science & Technology Co., Ltd.,ELISA kits obtained from elabscience, Trizol reagent (Invitrogen,CA, US No.15596026), reverse transcription kit (Azyme Biotech Co,Nanjing, China No.Q311-02).Primary antibodies against SIRT3,NLRP3, Caspase1, and Tubulin, along with corresponding secondary antibodies, were all purchased from Abcam.

2.2 Cell Culture, Treatment, and Examination

Composition of BV2 complete culture medium: DMEM high glucose + 10% fetal bovine serum + 1% penicillin/streptomycin,cultured at 37 ℃ under 5% CO2conditions.In hypoxic conditions and concentration screening experiments, cobalt chloride was added to the medium at different concentrations (0 μM, 50 μM, 75 μM, 100 μM 200 μM) to induce hypoxia, and honokiol was added at different concentrations (3 μM, 5 μM, 10 μM).Subsequently,BV2 cells were divided into four groups: the normal group, cobalt chloride group (model group, cobalt chloride concentration 100 μM), chronic hypoxia + honokiol group (concentration 10 μM), and chronic hypoxia + honokiol + 3-TYP group (following previous literature[10], concentration 5 μM).Both honokiol and 3-TYP were added simultaneously with cobalt chloride and treated for 48 h.

2.3 ELISA Detection of Inflammatory Factor Levels

Preheat each reagent for 20 min, dilute the standard solution in gradients, and sequentially add it to the enzyme-labeled strip precoated with antibodies along with the pre-diluted samples.Incubate for 1.5 h, then add biotinylated antibodies for another 1 h.After washing each well three times, add biotinylated working solution and incubate at 37 ℃ for 30 h.Wash five times in sequence, add TMB, incubate for 10 min, and immediately measure absorbance(OD value) at a wavelength of 450 nm using a preheated enzyme reader at 37 ℃ after adding the stop solution.

2.4 RT-PCR

After removing the cell culture supernatant, wash the cells three times with DPBS, then add Trizol.Add chloroform, centrifuge at 12,000 rpm for 15 min at 4 ℃, carefully aspirate the middle aqueous phase, mix with an equal volume of isopropanol, and let it stand for 10 min.Prepare 75% ethanol with DEPC water, centrifuge at 12,000 rpm for 10 min at 4 ℃, discard the supernatant, add 75% ethanol again, and centrifuge at 7,500 rpm for 15 min at 4 ℃.Discard the supernatant, carefully remove residual ethanol, add an appropriate amount of DEPC water, and incubate at 55 ℃ for 10 min for dissolution.Measure the concentration using nanodrop.Reverse transcription conditions: 42 ℃ for 2 min to remove genomic DNA,50 ℃ for 15 min, 85 ℃ for 5 seconds, followed by maintaining at 4 C to complete reverse transcription.Construct the qPCR system with SYBR Green fluorescent dye kit, setting up three replicates.qPCR conditions: initial denaturation at 95 ℃ for 30 seconds,cycling reaction at 95 ℃ for 5 seconds - 60 ℃ (adjusted according to the Tm value of different primers) for 30 seconds, repeating for 40 cycles, and melting curve at 95 ℃ for 30 seconds - 60 ℃ for 60 seconds - 95 ℃ for 15 seconds.See Table 1 for primer sequences.

2.5 Western Blot Detection

After centrifugation of cells in each group, RIPA lysis buffer was added, and after complete lysis, protein concentration was measured.After adding loading buffer and thorough mixing, the samples were boiled in a metal bath for denaturation.Protein samples were loaded onto SDS-PAGE gels based on protein concentration, with 20 μg of protein per well.The electrophoresis conditions were set at 75V for 30 min + 120 V for 60 min.Electrophoresis was stopped when bromophenol blue approached the bottom of the glass plate.Appropriately sized PVDF membranes were cut, and transfer to membranes was performed at a constant current of 200 mA.The transfer time was selected based on the molecular weight of the target protein.After membrane transfer, the primary antibody was incubated overnight at 4 ℃ in the refrigerator.The next day, after washing, the corresponding HRP secondary antibody was added,and ECL color development was performed after incubating at room temperature for 1 h.

2.6 ROS Measurement

The kit was purchased from Beyotime Company.Cells were seeded and cultured in a 96-well plate.After drug intervention in each group of cells, the cell culture medium was discarded, and an appropriate amount of DCFH-DA was added to each well to achieve a final concentration of 10 μM.Incubation was carried out at 37 ℃ in a cell culture incubator for 20 min, shaking every 3-5 min to ensure thorough mixing of the reagent with the cells.After incubation, cells were washed three times with serum-free cell culture medium for 5 min each time.After washing, fluorescence intensity was detected using a fluorescence microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

2.7 Statistical Analysis

The experimental results were analyzed using GraphPad Prism 9.5 statistical software.For Elisa and PCR data, the blank group was set as 1, and all other groups were normalized accordingly.Data are presented as mean ± standard deviation.One-way analysis of variance (ANOVA) was employed for multiple comparisons, and Bonferroni correction was applied for inter-group comparisons.Statistical significance was set atP＜ 0.05.

Tab 1 Primer Sequences for PCR

3.Results

3.1 Effects of Chronic Cobalt Chloride Treatment on BV2 Microglial Cell Line

Compared to the control group, BV2 cell viability showed no significant reduction after 48 h of treatment with 50 μM, 75 μM, and 100 μM cobalt chloride (CoCl2).However, in the group treated with 200 μM cobalt chloride, cell viability was significantly decreased compared to the control group (Figure 1A, P ＜ 0.05), indicating that excessive hypoxia led to cell death.After treatment with 50 μM,75 μM, and 100 μM cobalt chloride, the levels of the inflammatory factors TNF-α and IL-1β in the cell culture supernatant were significantly elevated compared to the control (Figure 1B,P＜ 0.05).The concentration of 100 μM cobalt chloride, which showed the highest inflammatory factor levels, was selected for subsequent experiments.

3.2 Effects of Honokiol on Inflammatory Factor Release in Chronic Hypoxia BV2 Cells

ELISA was used to assess the impact of different concentrations of honokiol (HNK) on the levels of inflammatory factors in the supernatant of BV2 cells treated with 100 μM cobalt chloride.It was found that concentrations of 5 μM and 10 μM honokiol significantly inhibited cobalt chloride-induced inflammatory factor release in BV2 cells (Figure 2,P＜ 0.05).The concentration of 10 μM honokiol was selected for subsequent treatments.

Fig 1 Impact of chronic hypoxia on BV2 cell viability and inflammatory factors.

Fig 2 Effects of different concentrations of honokiol on inflammatory factor secretion in chronic hypoxia BV2 cells.

3.3 Honokiol Suppresses M1 Polarization of BV2 Cells under Chronic Hypoxia, and This Effect is Reversed by 3-TYP

Compared to the cobalt chloride group, the chronic hypoxia+ honokiol group showed a downregulation of M1 polarization markers CD86, iNOS mRNA levels, and CD16/32 ratio, along with an upregulation of M2 markers Arg-1 and CD206 mRNA levels (Figure 3A,P＜ 0.05).This suggests that honokiol can inhibit chronic hypoxia-induced M1 polarization of microglial cells.However, when the SIRT3 inhibitor 3-TYP was added, the inhibitory effects of honokiol on inflammatory factor release and M1 polarization in chronic hypoxia BV2 cells disappeared (Figure 3A,3B).

3.4 Honokiol inhibits chronic hypoxia-induced ROS-NLRP3-Caspase1 pathway activation in BV2 cells via SIRT3

In the control group, BV2 cell morphology was normal and appeared round-shaped.In the cobalt chloride group, BV2 cell morphology changed and became spindle-shaped.Compared to the cobalt chloride group, the chronic hypoxia + honokiol group showed a tendency towards normal cell morphology (Figure 4A).SIRT3 is a mitochondrial deacetylase that plays a role in anti-oxidant free radical (ROS) formation, which is closely related to microglial cell activation and inflammatory pathways.Molecular analysis of SIRT3,ROS, and inflammatory pathway in cells from each group revealed that, compared to the control group, the cobalt chloride group had significantly downregulated SIRT3 protein expression, while ROS levels, NLRP3, and Caspase1 protein expression were significantly increased (P＜ 0.05).In comparison to the cobalt chloride group,the chronic hypoxia + honokiol group showed upregulated SIRT3 protein expression and significantly decreased ROS levels, NLRP3,and Caspase1 protein expression.The SIRT3 inhibitor 3-TYP could reverse the antioxidant stress and anti-inflammatory effects of honokiol on BV2 cells under chronic hypoxia.

Fig 3 Reversal of the Effects of Honokiol on Inflammatory Factor Secretion and Polarization Markers in Chronic Hypoxia BV2 Cells by 3-TYP.

Fig 4 Honokiol inhibits chronic hypoxia-induced ROS-NLRP3-Caspase1 pathway activation in BV2 cells via SIRT3

4.Discussion

Chronic cerebral hypoperfusion serves as a common pathological basis for Alzheimer’s disease (AD) and vascular dementia(VaD), promoting the progression of diseases such as AD or VaD.Investigating this phenomenon is of significant importance.Honokiol, derived from the leaves of Magnolia officinalis, is a monomer extract.In our previous study, we found that honokiol could improve cognitive function in rats with chronic cerebral hypoperfusion and alleviate hippocampal neuroinflammatory reactions[9].This study further explores the anti-inflammatory mechanisms of honokiol.

Cobalt can induce mild cellular hypoxia and is suitable for constructing long-term treatment cell models.In this experiment,we observed that prolonged treatment (48 h) with 0-100 μM cobalt chloride did not cause death of microglial cells and, in a dosedependent manner, increased the secretion of neuroinflammatory factors.Specifically, 100 μM cobalt chloride induced the morphological activation of microglial cells into branched structures and increased the transcription of M1 polarization markers.These characteristics are similar to the biological behavior of microglial cells in chronic cerebral hypoperfusion animal models reported in other studies[11].Therefore, chronic treatment with cobalt chloride can simulate the activation of microglial cells induced by chronic hypoxia in vivo.

Microglial cells, as central nervous system macrophages, play an immune defense role.Activated microglial cells exhibit two phenotypes, where M1-type releases a large number of inflammatory factors and can promote the activation of astrocytes, leading to the release of toxic substances that directly cause neuronal damage and subsequent brain function impairment.M2-type microglial cells have neuroprotective and reparative effects[12].In a mouse model of chronic cerebral hypoperfusion, the corpus callosum exhibits M1-type polarization of microglial cells.Using the immunosuppressant fingolimod promotes the transformation of microglial cells from M1 to M2 type, thereby facilitating white matter injury repair[11].Another study found that knockout of the voltage-gated proton channel specific to mouse microglial cells reduces intracellular ROS production induced by chronic cerebral ischemia, promotes the phenotypic transition from M1 to M2, and improves white matter damage[13].Therefore, M1-type polarization of microglial cells plays a central role in chronic cerebral hypoperfusion-related cognitive impairment, and interventions targeting this process represent an important direction for future research.

SIRT3 is a mitochondrial antioxidant enzyme primarily expressed in mitochondria.It regulates key enzymes in the tricarboxylic acid cycle and the electron transport chain through deacetylation,thereby exerting antioxidant stress effects[14].Previous studies have indicated that honokiol can activate SIRT3, increase its expression,and has anti-neuroinflammatory effects in various central nervous system disease models, including depression, Alzheimer’s disease,and postoperative cognitive dysfunction[8, 15, 16].Our research has also found that honokiol can inhibit hippocampal neuroinjury and cognitive impairment in rats with chronic cerebral hypoperfusion,although the specific mechanism remains unclear[9].In this study,we discovered that honokiol upregulates the protein expression of SIRT3 in chronically hypoxic microglial cells, reduces ROS, and decreases the transcription levels of M1 markers CD86 and iNOS.Simultaneously, it upregulates the transcription of M2 markers such as Arg-1 and CD206.The use of a SIRT3 inhibitor negates these effects, suggesting that under chronic hypoxia, honokiol promotes the phenotypic transition of microglial cells through SIRT3.In our previous study, we found that while SIRT3 activity decreased in the hippocampus of chronically cerebral ischemic rats, overall protein expression showed no significant change[9].However, in this in vitro experiment, we observed a downregulation of SIRT3 expression in microglial cells under chronic hypoxia, indicating that the regulation of SIRT3 expression under chronic hypoxia may be cell-specific.

NLRP3 inflammasome is a multiprotein complex composed of NLRP3 protein, adaptor protein ASC, and caspase-1 precursor protein.Hypoxia can affect the electron transport chain in microglial cells, leading to the generation of excessive ROS in mitochondria,thereby promoting the oligomerization of NLRP3 protein and its activation.Activated NLRP3 interacts with the adaptzor protein,leading to the conversion of caspase-1 precursor into caspase-1 effector protein.The latter promotes the release of various inflammatory factors and can induce the activation and secretion of neurotoxic components by astrocytes[17].We found that honokiol,while reducing intracellular ROS, can inhibit the expression of the NLRP3 inflammasome and downstream caspase-1 effector protein,as well as M1 polarization markers.This effect is SIRT3-dependent,suggesting that the ROS-NLRP3-caspase1 pathway may be a key pathway for M1 polarization of microglial cells under chronic hypoxia conditions.