Synergistic integration of dihydro-artemisinin with γ-aminobutyric acid results in a more potential anti-depressant

 

Yepu He a, Liyu Xu a, Yanbing Li a, Yinying Tang a, Shuwen Rao a, Rongtian Lin a, Zhijun Liu a, Heru Chen a,b,c

 

Abstract

 

Three hybrids of dihydro-artemisinin (DHA) with β-aminopropionic acid, γ-aminobutyric acid, and histamine have been designed and synthesized. The conjugate of DHA with GABA labelled as 5b was confirmed the most active candidate against both Cort- and SNP-induced PC12 cell impairments with EC50 value of 8.04 ± 0.35, and 9.38 ± 0.56 μM, respectively. 5b was clearly highlighted as a good modulator on protein expression of Akt, Bcl-2, and Bax, indicating its functions against programmed cell apoptosis. 5b significantly reversed the Cort-induced excessive calcium influx and release from internal organelles. It was demonstrated the ability to express increased levels of β-tubulin III and to up-regulate phosphorylation level of cAMP response element-binding protein (CREB), leading to cell differentiation. It can penetrate blood − brain barrier (BBB) with propriate stability. Altogether, these data strongly support that 5b is a potential anti-depressant.

 

Keywords:

Artemisinins

Anti-depressant

Depression

Cell differentiation

Drug design

 

1. Introduction

 

It is well known that, for many years, artemisinin and its derivatives (artemisinins) (Fig. 1) have been widely used as anti-malarial agents with few adverse side effects. Intriguingly, evidence has recently shown that artemisinins might have a therapeutic value for several other diseases beyond malaria, including cancers, inflammatory diseases, autoimmune disorders, and neurodegenerative disorders [1]. The reason why artemisinins might be used to treat neurodegenerative disorders is that they are able to decrease oxidation, inflammation, and amyloid beta protein (Aβ). It has been disclosed that artemisinin may have neuroprotective effects against oxidative stress through modulating various signaling pathways such as the extracellular signal-regulated kinases (ERK), cyclic-AMP response element binding protein (CREB), mitogen- activated protein kinase (MAPK), and Akt/mTOR pathways [2–6]. Also, artemisinins have been displayed to suppress inflammatory responses by the suppression of many pro-inflammatory cytokines including nuclear factor kappa B (NF-κB), and nuclear factor erythroid 2-related factor 2 (Nrf2) [7–9].

Therefore, artemisinin may probably be used as a structural moiety for developing drug to treat neurodegenerative diseases. In one of our previous works, dihydro-artemisinin (DHA) had been applied to hydride with memantine in compacted β conformation mode subsequently leading to enhanced effects against corticosterone (Cort)-induced insults in PC12 cells and might reverse memantine as an anti-depression NMDA antagonist [10].

As our concern, depression is the most common psychiatric illness affecting numerous people world-wide. Although there is a wide range of anti-depressants nowadays, the remission rates for monotherapy with an anti-depressant is only ~ 50% and 20–30% of the patients even do not respond to specific antidepressants [11]. Because of the complex mechanisms of underlying causes of depression and the limited knowledge about its pathogenetic risk factors, the progression of depression psychopharmacology has been restricted. Beyond all question, it is a challenging need to develop new drugs based on the understanding of novel molecular targets. Amongst them, glutamate/GABA systems are the most possible molecular targets.

It is a common sense that glutamate is the most abundant neurotransmitter in the human brain and is the major mediator of excitatory signals [12]. It involves in many physiological functions and participates in learning, memory and cognitive functions. Glutamate may bind to the cell surface receptors and activates the intracellular signal transduction system involved in downstream tropic pathways such as CREB and brain derived neurotrophic factor (BDNF) to preserve neuronal viability [13]; On the other hand, γ-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the brain. In conjunction with glutamate, GABA participates in balancing excitatory and inhibitory response, regulating activity in noradrenergic, dopaminergic, and serotonergic neurons. It is critical in proper brain functioning [14]. The receptors GABA binds to are of two types: ionotropic GABAa and GABAc receptors, and metabotropic GABAb receptors. Generally, GABAb receptors are the molecular targets focused on by a majority of preclinical antidepressant development studies [15]. It was found that pharmacological inhibition of GABAb receptors stimulated neural stem/progenitor cell proliferation, whereas knockout of GABAb1 receptor subunits increased neural cell proliferation and differentiation [16].

The idea to synergistically integrate DHA with GABA came naturally in our mind because of our previous sustained works and the acknowledgement of endogenous neurotransmitter GABA. Therefore, in the present study, DHA was applied as the building scaffold in a series of hybrids with endogenous neurotransmitters. The chemistry and biology of these new compounds will be described thereafter.

 

2. Results and discussion

 

2.1. Chemistry

 

The new hybrids consist of a DHA moiety, a spacer, and an endogenous neurotransmitter. The synthetic procedure was outlined in Scheme 1. Firstly, artemisinin was reduced by NaBH4 only at C-12, leading to DHA without destroying the peroxide bridge. The yield was 82.2%. DHA obtained via this process was a mixture of R- and S- iosmer at C-12. Without separation, DHA was then converted to 12-O-bromoethyl dihydro-artemisinin (1) in the presence of catalytic amount of dodecatungstophosphoric acid hydrate (H3O40PW12⋅×H2O) with a yield of 84.6%. Traditional catalysts such as BF3⋅Et2O or p-TsOH may play the similar role as dodecatungstophosphoric acid but with less efficiency. The workup in this step was quite trouble-free.

Compound 1 underwent nucleophilic substitution with NaN3 promoted by NaI, offering 12-O-triazoethyl dihydro-artemisinin (2) with a yield of 63.1%. By applying Staudinger reaction [17], the triazo group was turned into amino group resulted in 12-O-aminoethyl dihydro- artemisinin (3) with a yield of 85.4%. Formation of 3 into a water- soluble salt made the separation with triphenylphosphine oxide (Ph3PO) more easily. Compound 3 was then condensed with N-Fmoc- β-amino-propionic acid, and N-Fmoc-γ-aminobutyric acid, respectively, employed 2-(7-Azabenzotriazol-1-yl) -N,N,N’,N’-tetramethyluronium hexafluorophosphate (HATU) as coupling reagent. This step led to compounds 4a-b in a yield of 66.0–76.0%. Afterwards, removal of Fmoc group in 4a-b by piperidine offered compounds 5a-b in a very good yield (85.0–96.0%). Finally, a traditional nucleophilic substitution between 1 and histamine conveniently resulted in compound 6 in 76.4% yield.

All the desired DHA hybrids were isolated after silica gel column chromatography with a high purity degree (>96.3%). Their structures were confirmed by 1H NMR, 13C NMR, and HRESIMS.

 

2.2. Neuroprotection against depression cell models and the structure–activity relationship (SAR)

 

Compounds 5a-b, as well as 6 were evaluated for their in vitro neuroprotection against depression cell models. Two models were set up here, corticosterone (Cort)- and sodium nitroprusside (SNP)-induced rat pheochromocytoma (PC12) cell models. Firstly, cytotoxicity of three hybrids against PC12 cells were investigated. As shown in Fig. S1, compounds 3 and 5a were non-toxic up to 100 μM; whilst compounds 5b and 6 were toxic only at or above a dose of 100 μM. This may probably ascribe to the aggregation of 5b and 6 at high dose. However, further investigation is needed to disclose the truth. By the way, it was confirmed in Fig. S2 that the propriate concentration of Cort, and SNP for depression cell model was 400, and 500 μM, respectively.

Cort is a stress hormone. It has been applied to develop a rat model of depression by exogenous administration [18]. Since the PC12 cell line possesses typical neuron features and expresses a high level of glucocorticoid receptors, the Cort-impaired PC12 cell model may be used to screen antidepressant candidate and to study the mechanisms involved. As indicated in Fig. 2, only 5b was displayed dose-dependently neuroprotective activity against Cort-induced insults in PC12 cells with statistically significant (Fig. 2C). Although only one methylene difference between 5a and 5b, 5a was not demonstrated neuroprotective activity (Fig. 2B). Quite interestingly, artemisinin showed neuroprotective activity only at the dose of 25 μM (Fig. S3A), whilst 12-O-aminoethyl dihydro-artemisinin (3) lost the activity (Fig. 2A). The EC50 value of 5b against Cort-induced insults in PC12 was 8.04 ± 0.35 μM.

As we know, pathological high levels of nitric oxide (NO) have been considered as one of the primary mediators of brain ischemic damage, glutamate neurotoxicity and neurodegeneration [19,20]. Sodium nitroprusside (SNP), an inorganic compound with the formula Na2[Fe (CN)5NO], will release NO upon the reduction of the [Fe(CN)5NO]2- anion. Overexposure of different type of cell lines to SNP causes cytotoxicity and activates apoptotic cascade in a variety of neuronal injury models in vitro providing an opportunity to investigate mechanisms of NO-induced oxidation and cell death [21,22]. Therefore, SNP-induced neurotoxicity in PC12 cells is suitable to evaluate neuroprotective candidates. In Fig. 3, it was found that only 5b was demonstrated dose- dependently neuroprotective activity against SNP-induced insults in PC12 cells with statistically significant (Fig. 3C). The EC50 value was 9.38 ± 0.56 μM. Compound 3 was shown with activity only at the dose of 50 μM. However, compounds 5a and 6 had no activity. Similar to reported [2], we found that artemisinin showed neuroprotective activity against SNP-induced impairments with EC50 value of 14.62 ± 0.46 μM.

Generally speaking, histamine is a biogenic amine involved in many physiological and pathological processes. It is most prominently known as an inflammatory mediator. Herein, we could see that incorporation of histamine with DHA did not enhance the anti-depression effects of DHA.

Apparently, results from Cort- and SNP-impaired PC12 cell models indicated that the integration of DHA with GABA brought about synergistic effect. This was further confirmed in Fig. S4. Clearly, compound 5b was identified as the most active candidate against Cort-induced injuries in PC12 cells. It was more active than DHA, GABA, and their combination DHA/GABA (in 1:1 mol ratio). Therefore, we decided to explore the potential of 5b as an antidepressant and the possible mechanisms involved. The Cort-induced PC12 cell model was chosen.

However, as an essential prerequisite of drug candidate, propriate stability of the compound must be guaranteed. Herein, the stability of 5b in cell culture medium and mouse serum was studied. It was found that the half life time (t1/2) of 5b in cell culture medium and mouse serum was 18.0 h, and 5.4 h, respectively (Fig. 4). Obviously, 5b possesses an acceptable stability although there is an unstable peroxide bridge inside the molecule.

 

2.3. 5b attenuated Cort-induced apoptosis in PC12 cells

 

Considering that high concentrations or a long-term sustained rise in plasma glucocorticoid levels are important pathological processes of mental disorder [23]. Depression may be associated with glucocorticoid- induced hippocampal injury. Several studies have demonstrated that corticosterone exacerbates excitotoxic and metabolic neuronal injury [24,25]. We evaluated the attenuation of 5b against Cort-induced cell apoptosis.

As demonstrated in Fig. 5, we found that overexposure of PC12 cells to 400 μM Cort did increase the cell apoptosis rate from 3.3% in Control group to 46.4% in Model group, and 45.3% of cell death belonged to early apoptosis. Interestingly, pre-treatment of PC12 cells with 25 μM of DHA, GABA, DHA/GABA (in 1:1 mol ratio), and 5b, respectively for 4 h decreased the cell apoptosis rate to 40.8%, 30.5%, 35.3%, and 20.8%. Among them, 5b showed the most potent to make cells survive from the stress-induced apoptosis. Unexpectedly, the DHA/GABA combination showed only equivalent activity to DHA.

 

2.4. 5b lowered the Cort-elevated Ca2+ influx

 

Sustained Ca2+ influx through glutamate receptor channels is thought to represent a common pathway of neuronal cell death. Investigations showed that Cort can markedly facilitate Ca2+ influx into the hippocampal neuron, leading to neurotoxicity [26]. As shown in Fig. 6, excess levels of Cort in PC12 cells did elevate intramolecular Ca2+ influx (Model group). More detail, 400 μM Cort exposure induced an increase in the fluorescence intensity from 100% in control group to 283% in model group. When the cells were pre-treated with 25 μM of DHA, GABA, DHA/GABA, and 5b, the fluorescence intensity was reduced from 283% to 235%, 202%, 234%, and 171%, respectively. The activity of 5b was the best amongst all the tested groups with statistically significant.

 

2.5. 5b elevated the phosphorylation levels of Akt

 

Since the phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) pathway is a main survival signaling pathway which mediates the effect of many growth factors and other stimuli [27], we would like to make clear whether the neuroprotective effect of 5b is related to this signal pathway.

As indicated in Fig. 7, exposure of PC12 cells to 400 μM Cort down- regulated p-Akt/Akt protein ratio, implying the increase of cell apoptosis. However, pre-treatment of 25 μM 5b significantly elevated the p-Akt/Akt protein ratio (p < 0.05), indicating the ability of 5b to reduce cell apoptosis via Akt pathway. Unexpectedly, no other group (DHA, GABA, and DHA/GABA) was demonstrated the activity to up- regulate the phosphorylation levels of Akt statistically.

It is well-known that Akt is a serine/threonine kinase. It blocks cell death by both impinging on the cytoplasmic cell death machinery and by regulating the expression of genes involved in cell death and survival [28]. Among molecules, members of the Bcl-2 family of proteins are central to the regulation of cell death in eukaryotes. The mammalian proto-oncogene homolog Bcl-2 have been shown by genetic means to promote cell survival; while other members (including Bcl-XS, Bad, Bax, and Bak) promote cell death. When pro-apoptotic Bcl-2 family members such as Bax, Bim, Bad or Bid are in excess over anti-apoptotic members, they promote the release of at least two proteins: cytochrome c and Diablo/Smac from the mitochondria. Cytochrome c binds to APAF1 which leads to the activation of caspase 9 and, subsequently, to the activation of caspase 3. This is consistent to the results indicated in Fig. S5 and Fig. S6. Overexposure of PC12 cells to Cort down-regulated Bcl-2/Bax protein ration, and raised the phosphorylation level of caspase 3, implying the increase of cell apoptosis; while 5b significantly reversed this situation, which led to the reduction of cell apoptosis.

 

2.6. 5b maintained the activity to induced neurite outgrowth

 

Artemisinin and DHA have been reported with activity to induce neurite outgrowth in PC12 cells [29]. Compounds that can stimulate neurite formation would be meaningful for developing new therapeutics against both neurodegenerative disorders and trauma-induced neuronal injuries. As an early neuronal commitment marker [30], the protein level of β-tubulin isotype III (β-tubulin III) is changed during neurite growth. Moreover, cAMP response element-binding protein (CREB) is involved in cell differentiation. It was demonstrated that the phosphorylation of Ser-133 stimulates the ability of CREB to activate transcription of CRE-dependent genes that is required for differentiation of nuclear growth factor (NGF)-treated cells [31].

In the current experiments, we found that, as well as DHA, 5b maintained the activity to induce neurite outgrowth (Fig. 8A) and increase the expression levels of β-tubulin III in PC12 cells with statistically significant (Fig. 8B). Interestingly, GABA was shown the activity to induce neurite growth but only had the tendency to express increased levels of β-tubulin III. Considering that Cort was reported the effects to disturb neurite growth [32], we did not use the Cort-induced PC12 cell model in this study. No doubt, CREB did involve in cell differentiation as indicated in for 5 min. Then the supernatant was submitted to HPLC for content analysis. Each experiment was repeated 4 times.

 

2.7. Blood − Brain barrier (BBB) permeability assay

 

It is a common sense that penetration of the blood − brain barrier (BBB) is a critical factor for successful CNS drugs. In order to determine the BBB penetration of the candidate, the parallel artificial membrane permeability (PAMPA) assay was employed, which was initially established by Di et al. [33]. Here, 7 commercial drugs through a lipid extract of porcine brain were determined using a mixture of PBS and ethanol in the ratio of 70:30 (Table S1, Supporting Information). A plot of experimental data versus reported values produced the linear correlation Pe (exp) = 1.1719Pe(bibil.)-0.0221 (R2 = 0.9404) (Fig. S8). According to this equation and taking into account the described limits by Di et al. for BBB permeation, we determined that compounds with permeability above 4.67 × 10− 6 cm/s may penetrate into the CNS by passive diffusion. Based on this assay, the permeability of 5b was determined with a value of (14.87 ± 3.52) × 10− 6 cm/s, indicating excellent potent to cross BBB.

 

3. Conclusion

 

As a conclusion, three conjugates of DHA with β-aminopropionic acid, γ-aminobutyric acid, and histamine have been designed and synthesized. Their neuroprotection against both Cort- and SNP-induced PC12 cell impairments were evaluated. We confirmed compound 5b as the most active candidate with EC50 value of 8.04 ± 0.35, and 9.38 ± 0.56 μM, respectively.

By using Cort-induced PC12 cell model to explore the anti-depression mechanism involved, it was found that exposure of PC12 cells to 400 μM Cort elevated cell apoptosis rate, and cleaved caspase/caspase protein ratio; Whilst in the meantime, down-regulated the ratios of p-Akt/Akt, and Bcl-2/Bax. It also induced excessive calcium influx and release from internal organelles. Interestingly, pretreatment of 25 μM 5b significantly reversed all these Cort-induced dysfunctions. Candidate 5b was confirmed the most potent if compared to DHA, GABA, and DHA/GABA combination. Furthermore, 5b was demonstrated the ability to express increased levels of β-tubulin III and up-regulate the phosphorylation level of cAMP response element-binding protein (CREB), leading to cell differentiation. It was indicated the BBB permeability by passive diffusion, and with propriate stability. Altogether, these data support that 5b is a reliable and potent drug candidate for treating depression.

 

4. Experimental section

 

4.1. General methods for chemistry

 

All reagents and solvents were used as purchased from commercial sources or indicated otherwise. Flash chromatography was performed using silica gel (300 mesh). All reactions were monitored by TLC, using silica gel plates with the fluorescence F254 displayed by UV light visualization. 1H NMR and 13C NMR spectra were recorded on a Bruker AV- 400 spectrometer or Bruker AV-300. Coupling constants (J) are expressed in Hertz (Hz). Chemical shifts (δ) of NMR are reported in parts per million (ppm) units relative to an internal control (TMS). Low resolution ESI-MS data were recorded on a Finnigan LCQ Advantage MAX mass spectrometer and high resolution ESI-MS data on an Applied Biosystems Q-STAR Elite ESI-LC-MS/MS mass spectrometer. The purity of the compounds was determined by reverse phase high performance liquid chromatography (HPLC) analysis to be >95%. HPLC was performed on either LC-100 liquid chromatograph equipped with a tunable LC-100 UV detector (Shanghai Wufeng Inc., China) or Agilent 1200 series liquid chromatograph equipped with an Agilent 1200 Series UV detector (Agilent Technologies, USA). The columns used were Cosmosil 5C18 (Nacalai Tesque Inc., Japan) for general purification. A flow rate of 1.0 mL/min was used with mobile phase of MeOH in H2O with 0.1% modifier (ammonia or trifluoroacetate, v/v).

 

4.3.1. Cell culture

 

PC12 cells (Shanghai Cell Bank, Chinese Academy of Sciences, Shanghai, China) were cultured in DMEM medium containing 10% (v/v) fetal bovine serum (FBS), 5% horse serum, 100 μg/ml of streptomycin and 100 U/ml of penicillin in a water-jacketed incubator at 37 ◦C in a humidified atmosphere of 5% CO2. Cells were fed every 3 days and sub- cultured once they reached 80–90% confluence.

 

4.3.2. MTT assay

 

The viability of PC12 cells mediated by drugs was measured using 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 570 nm) assay. The process was described below: Cells in suspension were plated in 96-well plates at a density of 5 × 103 cells/well and were treated with either vehicle (0.1% DMSO) or drugs for 4 h after 24 h culture. The final DMSO concentration in all experiments was less than 0.1% in medium. The medium was discharged and the cells were washed with PBS. Then, PC12 cells were treated with 400 μM Cort for 24 h in Cort-impaired cell model; whilst in SNP-impaired cell model, PC12 cells were treated with 500 μM SNP for 24 h. Afterwards, the medium was discharged and the cells were washed with PBS. Then 10 μL of 5 mg/mL MTT solutions were added to each well, and the plate was incubated for an additional 4 h. The absorbance was measured at 570 nm using a microplate reader (Bio-Rad; Hercules, CA, USA) after 150 μL of DMSO was added. All of the tests were repeated at least 3 times.

 

4.3.3. Detection of cell apoptosis by Annexin-V/PI double dyed assay

 

PC12 cells (2 × 105 cells/ml) were plated in 6-well plates and then treated with either vehicle, or pretreated with 25 μM DHA, GABA, DHA/ GABA, and 5b, respectively for 4 h, after 24 h culture, following the treatment of 400 μM Cort. The cells were incubated at 37 ◦C, 5% CO2 for 24 h. Then the cells were collected by centrifugation at r. t. and washed twice with ice-cold PBS. Afterwards, the cells were suspended in 100 µL annexin V binding buffer and 5 µL each of annexin V and PI were added to these samples. Next, these samples were incubated for 30 min at room temperature and then assayed by flow cytometric analysis (FACScan, Bection Dickinson, San Jose, CA). All of the tests were repeated at least 3 times.

 

4.3.3.1. Measurement of intracellular Ca2+ ([Ca2+]i).

 

[Ca2+]i was monitored using the fluorescent Ca2+-sensitive dye, Fura 3-acetoxymethy ester (Fura 3-AM). PC12 cells in the exponential phase were plated on a 96-well cell culture plate with 2 × 105 cells/well treated with either vehicle (0.1% DMSO), or pretreated with 25 μM DHA, GABA, DHA/GABA, and 5b, respectively for 4 h, after 24 h culture. Cells were treated with 400 μM Cort for another 24 h. Briefly, cells were trypsinized, pelleted, resuspended in medium and incubated with 5 μM Fura- 3/AM in PBS containing 1.3 mM CaCl2 for 40 min at 37 ◦C and then washed twice to remove any extracellular dye. All the dyed cells were submitted to a flow cytometry (BD FACS Calibur, Franklin Lakes, CA, USA) for fluorescence detection. Excitation wavelength was 340/380 nm; emission wavelength was 510 nm. The fluorescence ratio (F340/ F380) was calculated as an indicator of [Ca2+]i.

 

4.3.3.2. Western blotting analysis.

 

PC12 cells were collected and washed with PBS after the treatment followed the procedure as described above. Then, the cells were lysed with RIPA buffer for 45 min on ice and then centrifuged at 12,000g at 4 ◦C for 15 min. Afterwards, the protein content of the supernatant was determined with a Pierce BCA protein assay kit (Thermo Fisher Scientific, USA) to ensure equal sample loading. Protein lysates were separated in 12% SDS-PAGE and blotted onto nitrocellulose membranes (Amersham Biosciences, USA). Proteins were detected using the primary monoclonal antibody of β-Actin (1:3000), Caspase 3 (1:1000), Cleaved Caspase 3 (1:1000), Akt (1:2000), Bcl-2 (1:1000), Bax (1:1000), CREB (1:1000), p-CREB (1:1000),-tubulin III (1:3000) with the corresponding diluted ratio given in parentheses, respectively for at least 16 h at 4 ◦C, and then washed and incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. Protein bands were visualized using enhanced chemiluminescence detection reagents (Bio-Rad, USA). The resulting images were scanned using a scanner (Epson V330 Photo, Japan).

 

4.3.4. Measurement of neurite outgrowth

 

PC12 cells from stock cultures, trypsinized and suspended in the medium described in the previous section, were seeded in 96-well plates coated with porcine tendon Type I collagen (Nitta Gelatin, Osaka, Japan) at a density of 4 × 103 cells/well. After 24 h, the medium was replaced with a fresh medium or the fresh medium containing 25 μM DHA, GABA, DHA/GABA, and 5b, respectively. Cells were then cultured for 48 h, fixed with 1% glutaraldehyde, and stained with Giemsa. Three hundred to 400 cells were microscopically examined in randomly chosen fields and scored as having a neurite if they had at least one neurite that was longer than the cell body width. The data were expressed as percentage of neurite-bearing cells. In some experiments, images of the cells were captured, and the total length of the neurite extension per positive cell and average length of neurites in positive cells were determined in randomly chosen fields using Motic Images Plus software (version 2.0S; Motic Instruments Inc., Richmond, Canada).

 

4.3.5. BBB permeation assay

 

The BBB penetration of compounds was evaluated using the parallel artificial membrane permeation assay (PAMPA) described by Di et al. [33]. Commercial drugs were purchased from Sigma and Alfa Aesar. Porcine brain lipid (PBL) was obtained from Avanti Polar Lipids. The donor microplate (PVDF membrane, pore size 0.45 mm) and acceptor microplate were both from Millipore. The 96-well UV plate (COSTAR) was from Corning Incorporated. The acceptor 96-well microplate was filled with 300 μL PBS/EtOH (7:3), and the filter membrane was impregnated with 4 μL PBL in dodecane (20 mg/mL). Compounds were dissolved in DMSO at 5 mg/mL and diluted 50-fold in PBS/EtOH (7:3) to a final concentration of 100 μg/mL. Then, 200 μL of the solution was added to the donor wells. The acceptor filter plate was carefully placed on the donor plate to form a sandwich, which was left undisturbed for 10 h at 25 ◦C. After incubation, the donor plate was carefully removed, and the concentration of compounds in the acceptor wells was determined using the UV plate reader (Flexstation 3). Every sample was analyzed at five wavelengths in four wells and in at least three independent runs. Pe was calculated by the following expression: Pe = − Vd × Va/[(Vd + Va)A × t] × ln(1 − drugacceptor/drugequilibrium), where Vd is the volume of donor well; Va, volume in acceptor well; A, filter area; t, permeation time; drugacceptor, the absorbance obtained in the acceptor well; and drugequilibrium, the theoretical equilibrium absorbance. The results are given as the mean ± standard deviation. In the experiment, 7 quality control standards (Table S1) of known BBB permeability were included to validate the analysis set. A plot of the experimental data versus literature values gave a strong linear correlation, Pe (exp.) = 1.1719Pe (lit.) − 0.0221 (R2 = 0.9404) (Figure S6). From this equation and the limit established by Di et al. (Pe (lit.) = 4.0 × 10− 6 cm/s) for BBB permeation, we concluded that compounds with a permeability > 4.67 × 10− 6 cm/s could cross the BBB (Table S2).

 

4.3.6. Stability of 5b

 

5b was dissolve in PBS to be a stock solution with a concentration of 20 μM. Then 0.2 mL 5b solution and 1.8 mL serum (Kunming mice from the animal research center at medical laboratory animal center of Guangdong Province in China) were suspended together for 1 min, and the suspension was put into the incubator at 37 ◦C. At the set time point, which was 0.5, 1, 2, 4, 8, and 12 h, 10 μL sample was taken out, respectively, and pretreated with methanol and perchloric acid 20% to eliminate the proteins. The mixture was centrifuged at 10,000 r/min for 5 min. Then the supernate was submitted for RP-HPLC analysis. The instrument used was Agilent 1200 (Agilent Co., Germany). Column: COSMOSIL 5C18-MS-II, 4.6 × 250 mm; Flow rate: 1.0 mL/min; Wavelength: 254 nm; Eluant: methanol : H2O = 55 : 45 (isocratic).

In another case, 0.2 mL 5b solution and 1.8 mL 10% FBS DMEM were suspended for 1 min, and the suspension was put into the incubator at 37 ◦C. At the set time point, which was 0.5, 1, 2, 4, 8, 12, 28, and 36 h, 10 μL sample was taken out, respectively, and pretreated with methanol and perchloric acid 20% to eliminate the proteins. The mixture was centrifuged at 10,000 r/min for 5 min. Then the supernate was submitted for RP-HPLC analysis.

The content percentage of 5b was set 100% at 0 h time point. The content percentage at any time point was calculated as following: At/A0 × 100%, where At and A0 are the peak area at t time point and 0 h time point, respectively. Each experiment was repeated 4 times.

 

4.3.7. Statistical analysis

 

Experimental values were given as means ± SD. Statistical analysis of the data was performed using the SPSS 18.0 statistical software. One- way analysis of variance (ANOVA) was applied to analyze for difference in data of biochemical parameters among the different groups, followed by Dunnett’s significant post-hoc test for pairwise multiple comparisons. Differences were considered as statistically significant at *P < 0.05, **P < 0.01.

 

References

 

[1]          B.-W. Lu, L. Baum, K.-F. So, K. Chiu, L.-K. Xie, More than anti-malarial agents: therapeutic potential of artemisinins in neurodegeneration, Neural Regen Res 14 (9) (2019) 1494–1498.

[2]          W. Zheng, C.M. Chong, H. Wang, X. Zhou, L. Zhang, R. Wang, Q. Meng, P. Lazarovici, J. Fang, Artemisinin conferred ERK mediated neuroprotection to PC12 cells and cortical neurons exposed to sodium nitroprusside-induced oxidative insult, Free Radic Biol Med 97 (2016) 158–167.

[3]          U.P. Okorji, R. Velagapudi, A. El-Bakoush, B.L. Fiebich, O.A. Olajide, Antimalarial drug artemether inhibits neuroinflammation in BV2 microglia through Nrf2- dependent mechanisms, Mol Neurobiol 53 (2016) 6426–6443.

[4]          F. Yan, H. Wang, Y. Gao, J. Xu, W. Zheng, Artemisinin protects retinal neuronal cells against oxidative stress and restores rat retinal physiological function from light exposed damage, ACS Chem Neurosci 8 (2017) 1713–1723.

[5]          Z. Zeng, J. Xu, W. Zheng, Artemisinin protects PC12 cells against beta-amyloid- induced apoptosis through activation of the ERK1/2 signaling pathway, Redox Biol 12 (2017) 625–633.

[6]          S.P. Lin, W. Li, A. Winters, R. Liu, S.H. Yang, Artemisinin prevents glutamate- induced neuronal cell death via Akt pathway activation, Front Cell Neurosci 12 (2018) 108, https://doi.org/10.3389/fncel.2018.00108.

[7]          P. He, S. Yan, J. Zheng, Y. Gao, S. Zhang, Z. Liu, X. Liu, C. Xiao, Eriodictyol attenuates LPS-induced neuroinflammation, amyloidogenesis, and cognitive impairments via the inhibition of NF-kappa B in male C57BL/6J mice and BV2 microglial cells, J Agric Food Chem 66 (2018) 10205–10214.

[8]          H. Lu, B. Wang, N. Cui, Y. Zhang, Artesunate suppresses oxidative and inflammatory processes by activating Nrf2 and ROS dependent p38 MAPK and protects against cerebral ischemia reperfusion injury, Mol Med Rep 17 (2018) 6639–6646.

[9]          M. Abdalkader, R. Lampinen, K.M. Kanninen, T.M. Malm, J.R. Liddell, Targeting Nrf2 to suppress ferroptosis and mitochondrial dysfunction in neurodegeneration, Front Neurosci 12 (2018) 466, https://doi.org/10.3389/fnins.2018.00466.

[10]        L. Zhang, F. Zhou, L.-T. Zhang, L. Peng, C. Guo, C. Luo, H. Chen, Incorporation of Dihydroartemisinin into Memantine Through a Propriate Spacer to Make Hybrid with Enhanced Effects to Protect PC12 Cells from Corticosterone-caused Impairments, Chem. Res. Chinese Universities 33 (4) (2017) 611–622.

[11]        D.A. Mrazek, J.C. Hornberger, C.A. Altar, I. Degtiar, A review of the clinical, economic, and societal burden of treatment-resistant depression: 1996–2013, Psychiatr Serv 65 (8) (2014) 977–987.

[12]        B.S. Meldrum, Glutamate as a neurotransmitter in the brain: review of physiology and pathology, J Nutr 130 (4S Suppl) (2000) 1007S–1015S.

[13]        J.L. Martin, C. Finsterwald, Cooperation between BDNF and glutamate in the regulation of synaptic transmission and neuronal development, Commun Integr Biol 4 (1) (2011) 14–16.

[14]        O.A.C. Petroff, Book Review: GABA and Glutamate in the Human Brain, Neuroscientist 8 (6) (2002) 562–573.

[15]        C. Mombereau, K. Kaupmann, W. Froestl, G. Sansig, H. van der Putten, J.F. Cryan, Genetic and pharmacological evidence of a role for GABA(B) receptors in the modulation of anxiety- and antidepressant-like behavior, Neuropsychopharmacology 29 (6) (2004) 1050–1062.

[16]        C. Giachino, M. Barz, J.S. Tchorz, M. Tome, M. Gassmann, J. Bischofberger, B. Bettler, V. Taylor, GABA suppresses neurogenesis in the adult hippocampus through GABAb receptors, Development 141 (1) (2014) 83–90.

[17]        A. Koziara, A. Zwierzak, Optimized Procedures for One-pot Conversion of Alkyl Bromides into Amines via the Staudinger Reaction, Synthesis-Stuttgart 11 (1985) 1063–1065.

[18]        Y. Zhao, R. Ma, J. Shen, H. Su, D. Xing, L. Du, A mouse model of depression induced by repeated corticosterone injections, Eur J Pharm 581 (2008) 113–120.

[19]        V.L. Dawson, V.M. Kizushi, P.L. Huang, S.H. Snyder, T.M. Dawson, Resistance to neurotoxicity in cortical cultures from neuronal nitric oxide synthase-deficient mice, J. Neurosci. 16 (1996) 2479–2487.

[20]        G.A. Benavides, Q. Liang, M. Dodson, V. Darley-Usmar, J. Zhang, Inhibition of autophagy and glycolysis by nitric oxide during hypoxia-reoxygenation impairs cellular bioenergetics and promotes cell death in primary neurons, Free Radic. Biol. Med. 65 (2013) 1215–1228.

[21]        M. Sani, H. Sebai, N.A. Boughattas, M. Ben-Attia, Time-of-day dependence of neurological deficits induced by sodium nitroprusside in young mice, J. Circadian Rhythms 9 (2011) 5, https://doi.org/10.1186/1740-3391-9-5.

[22]        J.P. Silva, F. Proenca, O.P. Coutinho, Protective role of new nitrogen compounds on ROS/RNS-mediated damage to PC12 cells, Free Radic. Res. 42 (2008) 57–69.

[23]        S.J. Lupien, B.S. McEwen, M.R. Gunnar, C. Heim, Effects of stress throughout the lifespan on the brain, behavior and cognition, Nat. Rev. Neurosci. 10 (2009) 434–445.

[24]        Y. Goodman, A.J. Bruce, B. Cheng, M.P. Mattson, Estrogens Attenuate and Corticosterone Exacerbates Excitotoxicity, Oxidative Injury, and Amyloid β-Peptide Toxicity in Hippocampal Neurons, J. Neurochem. 66 (1996) 1836–1844.

[25]        F. Mo, Y. Tang, P. Du, Z. Shen, J. Yang, M. Cai, Y. Zhang, H. Li, H. Shen, GPR39 protects against corticosterone-induced neuronal injury in hippocampal cells through the CREB-BDNF signaling pathway, Journal of Affective Disorders 272 (2020) 474–484.

[26]        J.Z. Zhou, J.Q. Zheng, Y.X. Zhang, J.H. Zhou, Corticosterone impairs cultured hippocampal neurons and facilitates Ca2+ influx through voltage-dependent Ca2+ channel, Acta Pharmacol Sin 21 (2000) 156–160.

[27]        H. Wang, X. Zhou, J. Huang, N. Mu, Z. Guo, Q. Wen, R. Wang, S. Chen, Z. Feng, W. Zheng, The role of Akt/FoxO3a in the protective effect of venlafaxine against corticosterone-induced cell death in PC12 cells, Psychopharmacology 228 (2013) 129–141.

[28]        A. Brunet, S.R. Datta, M.E. Greenberg, Transcription-dependent and -independent control of neuronal survival by the PI3K–Akt signaling pathway, Current Opinion in Neurobiology 11 (2001) 297–305.

[29]        Y. Sarina, O. Yagi, T. Nakano, K. Hashimoto, Y. Kimura, M. Asakawa, S. Zhong, E. Narimatsu, Gohda, Induction of neurite outgrowth in PC12 cells by artemisinin through activation of ERK and p38 MAPK signaling pathways, Brain Res 1490 (2013) 61–71.

[30]        H.C. Joshi, D.W. Cleveland, Differential utilization of β-tubulin isotypes in differentiating neurites, J Cell Biol 109 (1989) 663–673.

[31]        D.D. Ginty, A. Bonni, M.E. Greenberg, Nerve growth factor activates a Ras- dependent protein kinase that stimulates c-fos transcription via phosphorylation of CREB, Cell 77 (1994) 713–725.

[32]        M. Ohmoto, Y. Shibuya, S. Taniguchi, T. Nakade, M. Nomura, Y. Ikeda-Matsuo, T. Daikoku, Protective effects of butein on corticosterone-induced cytotoxicity in Neuro2A cells, IBRO Reports 8 (2020) 82–90.

[33]        L. Di, E.H. Kerns, K. Fan, O.J. McConnell, G.T. Carter, High throughput artificial membrane permeability assay for blood-brain barrier, Eur J Med Chem 38 (2003) 223–232.