Kaempferol Alleviates LD-mitochondrial Damage By Promoting Autophagy: Implications in Parkinson’s Disease Part 1

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Abstract: Emerging evidence indicates that unexpected lipid droplet (LD) deposition and peroxidation can accelerate organelle stress and plays a crucial role in the pathogenesis of neurodegenerative diseases (NDDs). In our previous study, we confirmed that kaempferol (Ka), a natural flavonoid small molecule, exhibited neuroprotective effects on mice with LPS-induced Parkinson's disease (PD). In addition, previous studies have shown that autophagy plays an important role in the regulation of cellular LD deposition. In the current study, we showed that Ka protected against neuronal loss and behavioral deficits in MPTP/p-induced PD mice, accompanied by reduced lipid oxidative stress in the substantia nigra pars compacta(SNpc). In cultured neuronal cells, Ka exhibited a relatively safe concentration range and significantly suppressed LD accumulation and cellular apoptosis induced by MPP+. Further study indicated that the protective effect of Ka was dependent on autophagy, specifically lipophagy. Critically, Ka promoted autophagy to mediate LD degradation in lysosomes, which then alleviated lipid deposition and peroxidation and the resulting mitochondrial damage, consequently reducing neuronal death. Furthermore, AAV-shAtg5-mediated Atg5 knockdown abolished the neuroprotective effects of Ka against lipid oxidation in PD mice. This work demonstrates that Ka prevents dopaminergic neuronal degeneration in PD via the inhibition of lipid peroxidation-mediated mitochondrial damage by promoting lip-ophagy and provides a potential novel therapeutic strategy for PD and related NDDs.

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1. Introduction

Parkinson's disease (PD)is a common neurodegenerative disease (NDD)that is pathologically characterized by the progressive loss of dopaminergic (DA)neurons in the substantia nigra pars compacta (SNpc). Although the exact etiology and natural course of this dis-ease have yet to be fully clarified, numerous system-level processes and dysfunctions, including mitochondrial functions, dopamine homeostasis, neuroinflammation, and autophagy, have been implicated in the pathogenesis of PD [1,2]. Recent reports revealed that lipid droplet (LD)-related lipotoxicity might participate in PD pathology [3,4]. LDs are highly dynamic organelles that emerge from the endoplasmic reticulum(ER)membrane and normally serve as intracellular sites of neutral lipid storage [5]. LDs were recently shown to play a much broader role than fatty acid (FA)storage and participate in many diseases. For example, myeloid cells, including macrophages, leukocytes, and eosinophils, form LDs in response to inflammation and stress, and LDs are sites of inflammatory cytokine production and storage and are further involved in antigen presentation and pathogen clearance [6]. Importantly, in atherosclerosis, LD-rich foam cells have been demonstrated to be deleterious in all stages of the disease [7].

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However, LDs have not been extensively studied in the central nervous system (CNS), with few papers reporting the histological presence of LDs in human brain tissue [8,9]. Even so, LDs may have important functions in the brain, as recent studies confirmed that aggregated LDs in glia accelerated neurodegeneration in a Drosophila model [10]. It has recently been reported that in mice, oil red O-positive lipid-laden cells, including neurons, glial fibrillary acidic protein (GFAP)+ astrocytes, and ionized calcium-binding adaptor molecule-1 (IBA-1)+ microglia, are present in many brain regions and have been shown to participate in age-associated processes [1]. Our previous work also demonstrated increased LD accumulation in the brain in a PD mouse model [12]. Taken together, these studies suggest that LDs may be involved in the pathogenesis of PD, as well as other NDDs.

Normally, intracellular LDs are degraded in lysosomes and deliver FAs to mitochondria for their consumption as an alternative energy source during periods of nutrient depletion [13]. However, neurons have a low capacity for mitochondrial FA consumption for energy production [14]. This characteristic makes neurons particularly sensitive to LD accumulation and peroxidation. Furthermore, the accumulation of LDs enhances the FA oxidation rate and imposes persistent pressure on the mitochondrial electron transport chain, which increases reactive oxygen species(ROS)production by electron transport chain complexes I and II [15] to amplify oxidative stress. Lipid overload also leads to ROS production by extramitochondrial sources such as nicotinamide adenine dinucleotide phosphate (NADPH)oxidases and other oxidative enzymes. Combined with the decreased expression of antioxidant enzymes and the low capacity for FA consumption in neurons, LD accumulation mediates oxidative stress and can further lead to mitochondrial damage, lysosomal dysfunction, defective autophagy, and the activation of inflammatory responses [6,17]. Unless the accumulated LDs can be inhibited or removed, highly active neurons undergo pathophysiology, giving rise to neurodegeneration [17]. Evidence indicates that the lysosome-mediated catabolic process called autophagy plays a pivotal role in maintaining cellular LD homeostasis in multiple tissues [17,18]. In detail, LDs can be selectively sequestered in autophagosomes and delivered to lysosomes for degradation by lysosomal acid lipases, a process specifically known as lipophagy [19]. Therefore, targeting LDs and their autophagic removal pathway could represent a novel approach to managing neurodegeneration.

The study of natural products and dietary agents as sources for potential NDD treatments and strategies has gained enormous interest in recent years [20]. Kaempferol (Ka) is a natural polyphenolic small molecule that can be found in a number of Chinese medicinal herbs and dietary sources [21]. Ka has been reported to exert antibacterial, anti-aging, and immunomodulatory effects, as well as antioxidant and neuroprotective activities [20,22,23]. Previously, Ka was reported to inhibit inflammation in BV2 microglial cells in vitro [24] and increase the resistance of DA neurons to neuroinflammation in vivo [25]. However, the effect of Kaon LDs in PD has not yet been investigated.

Given the importance of LDs and the intimate connection between LDs, mitochondria, and autophagy [16], which are all involved in PD, we hypothesize that Ka inhibits LD peroxidation and prevents DA neurodegeneration in PD. In the present study, we demonstrated that Ka significantly protected against neurodegeneration in a murine PD model by inhibiting LD accumulation. Further study revealed that Ka triggered autophagy and reduced the accumulation of oxidized LDs to alleviate mitochondrial dysfunction and mitochondrial ROS (mtROS)production, thereby preventing neuronal apoptotic death. The findings obtained in this study may help direct clinical decisions regarding the use of the natural molecule Ka in NDDs such as PD.

2. Materials and methods

2.1. Experimental animals

C57BL/6J mice (male,4-month old) were obtained from Nanjing Medical University Animal Core (Nanjing). Mice were maintained and bred in the Animal Resource Centre of the Faculty of Medicine, Nanjing Medical University, and in the animal facility at Nanjing Drum Tower Hospital, Mice are free to access food and water in a room with an ambient temperature of 22°C±2℃C and a light/dark cycle of12:12 h All animal procedures were carried out in strict accordance with the guideline of the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

2.2. Reagents

MPTP(M0896),3-MA (M9281),penicillin-streptomycin (V900929), sodium pentobarbital, and probenecid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Kaempferol (HPLC> 95%)for in vivo treatment was purchased from Nanjing Jingzhu Biotechnology Co., Ltd (Nanjing, China), kaempferol (HPLC>99.0%,96,353)for in vitro experiments was obtained from Sigma-Aldrich(St. Louis, MO, USA). BODIPY 493/503(D3922), BODIPY 581/591 C11 (D3861), DHE (D11347) were purchased from Thermo Fisher Scientific. For animal experiments, Ka was dissolved in a solution of 10% DMSO in 16% SBE-β-CD in sterile saline. For cell experiments, Ka was prepared in DMSO (Sigma-Aldrich) and PBS(final DMSO concentration is 0.01%), pH 7.4.

2.3. Induction and treatment of methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- induced PD mouse model

To evaluate the effects of Ka in the PD model of MPTP/p-toxicity, mice (male, aged 4-5 months old) were randomly divided into the saline-treated group, MPTP-treated group, MPTP+ Ka-treated group, and Ka alone treated group. In the MPTP-treated group, mice received chronic MPTP administration with a protocol similar to that described previously [26]: MPTP was dissolved in saline and injected subcutaneously at 25 mg/kg followed by 250 mg/kg probenecid(dissolved in dimethyl sulfoxide) intraperitoneally (i.p.)injection at intervals of 1 h every 3.5 days over a period of 5 weeks. Control mice received saline and probenecid injection. In the MPTP+ Ka-treated group, mice received Ka(50 mg/kg, a single injection/day during experiments, Jingzhu Biotechnology, Nanjing, China) intraperitoneally (i.p.) daily 3 d prior to treatment with MPTP and over the 5 MPTP injection weeks. One week after the last MPTP injection, mice were subjected to behavioral testing blinded to groups. After, all mice were anesthetized with 40 mg/kg sodium pentobarbital and sacrificed for brain proteins detection and immuno-histochemistry or qPCR analysis.

2.4. In vivo stereotaxic surgery and experimental treatments

Stereotaxic surgery under sodium pentobarbital anesthesia(40 mg/kg, i.p.)was performed as described [25]. Mouse Atg5 and control shRNA was transfected with packaging vectors (AAV-U6-CMV-shR-NA-EGFP)to generate AAV. For microinjection, anesthetized mice are placed in a stereotaxic apparatus. They were injected with the 1μL AAV by glass electrode aiming at DG(AP:-0.3 mm; ML:±0.13 mm; DV:-0.45 mm） at a rate of 0.25 μL/min. Then the needle was retained for an additional 2-min. 2 weeks after virus microinjection, mice were subjected to MPTP-induced PD model conduction and (or)Ka treatment.

Atg5 shRNA was obtained from Hanbio Biotechnology(Shanghai, China) Co., Ltd. For the Atg5 silence, the primers were shown in Supplementary Table 1.

2.5. Behavioral analysis

One week after the final injection of MPTP or saline, the rotarod test and pole test were performed as described before [27]. For the rotarod test, mice were acclimatized to the rotarod in two trials(6 min with an accelerated speed of 12-20 rpm) per day for 2 consecutive days before the start of the experiment. Then, the mice were tested at 20 rpm for 300 s for other 3 consecutive days. The latency to fall was recorded using Rotarod Analysis System (Jiliang, Shanghai, China).For the pole test, mice were placed head up on the top of a vertical wooden pole (rough-surfaced, height 50 cm, diameter 1 cm). All mice were accustomed to the apparatus 2 days before testing and then tested for three times in the third day. The total time (T-total, until the mouse reached the floor with its four paws) and the turn time(T-turn, for the mouse to turn completely head downward) were recorded. The experiment was blinded to mouse groups for each behavioral testing and the test was performed three times.

2.6. Brain sample collection

After the final behavioral tests, mice were anesthetized by sodium pentobarbital(40 mg/kg, i.p.). For qPCR, western blotting, and high-performance liquid chromatography (HPLC) analysis, the whole brains were rapidly extracted from animals then the midbrain and striatum tissues were quickly dissected, pre-frozen by liquid nitrogen. All samples were stored at -80°C until analysis.

For immunohistochemical analysis, mice were perfused trans-cardially with 4% paraformaldehyde (PFA). Brains were extracted,post-fixed, dehydrated, embedded in OCT (Tissue-Tek), and serial sections of the brains were cryosectioned （30 μm per slice） through each entire stratum and midbrain using a freezing microtome (Leica CM1950, Nussloch, Germany). All sections were collected in six separate series and brain slices were stored in 50% glycerin and frozen at -20°C until analysis.

For TEM, mice were perfused with 2.5% glutaraldehyde and 2%paraformaldehyde as described 【12】. A small portion （<1 mm²） of the mesencephalon was carefully sectioned and incubated in the same fixative for 2 h at 4°C. Specimens were postfixed in 1% osmium tetroxide, stained in aqueous uranyl acetate, and then dehydrated and embedded in epoxy resin. Ultrathin sections were stained using lead citrate and examined with a transmission electron microscope(JEM-1010, Tokyo, Japan).

2.6. Immunohistochemical analysis

Brain slices were rinsed in PBS followed by 3% H2O, for 10 min then incubated with 0.3% Triton X-100 in PBS supplemented 5% BSA for 1 h. After that, slides were incubated with the primary antibodies at 4°C overnight in PBS containing 5% BSA at 4°C overnight, then washed and incubated in secondary antibodies for 1 h at room temperature, followed by incubating with diaminobenzidine (DAB)or mounting in DAPI (Life Technologies, Cat P36931)as immunofluorescent staining for 5 min.For Nissl staining, the slides were merged in cresyl violet(CV)solution (0.1g cresyl violet, 99 ml H,O, and 1% acetic acid 1 ml)for 30 min at room temperature then dehydrated with alcohol and xylene. Images were observed and photographs were captured under an Olympus BX52 microscope(Olympus America Inc., Melville, NY, United States). The total number of TH-positive and Nissl's-positive neurons in the SNpc was obtained serologically by using the optical fractionator method with MicroBrightField Stereo-Investigator software (MicroBrightField, Williston, VT, USA).

2.7. High-performance liquid chromatography (HPLC) analysis

Mice striatal samples were homogenized with 0.1 M perchloric acid （1 mg tissues in 100 μL perchloric acid） and 0.1 mM EDTA， treated by ultrasonic and centrifuged at 20,000 rpm for 30 min as reported [27]. Then supernatant liquids were collected for measuring. The mobile phase is a mixed solution consisting of 90 mM sodium phosphate monobasic，1.7 mM 1-octane sulfonic acid， 50 mM citrate，50 μM EDTA，10% acetonitrile with the flow rate of 0.2 ml/min. In parallel, for quantitative standard curve calculation, stock standards solution for dopamine and dihydroxy-phenyl acetic acid (DOPAC)(Sigma-Aldrich, USA） were prepared in 0.1 M HClO4. An amount of 10 μl of prepared supernatant or standard solution was injected into the mobile phase and tested by ESA Coulochem ⅢI electrochemical detector(Coulochem III, Thermo Fisher Scientific). Samples were measured and peaks were quantified. The concentrations of monoamines were quantified by comparing with the standard using ClarityChrom software (Knauer, Germany) and then the contents in stratum were converted according to the dilution ratio. The standard curve of DA and DOPAC were shown in Supplementary Fig.1a and b.

2.8. Cells cultures and treatments

Mesencephalic primary neuron cultures were conducted as described previously [12].In brief, the mesencephalic tissues of C57BL/6 mice on embryonic day 14/15(E14/15) were carefully removed, mechanically dissociated to remove the membranes and large blood vessels, and then dissected. Next, they were digested with trypsin-EDTA(Amresco, Solon, OH， USA）and then filtered through a 100-μm filter to obtain a single-cell suspension. Subsequently, the cells were plated on poly-L-lysine-pre-coated 12-/24-well plates at 2.5×10° cells/ml containing Neurobasal medium(Cat 21103049,GibcoTM, Thermo Fisher Scientific, Rockford, USA) supplemented with B27(2% v:v, Cat 17504044,GibcoTM) and penicillin/streptomycin(0.5% v:v). The cultures were maintained in a humidified chamber (37°C, 5% CO2 incubator). The culture medium was changed every 3 days and cells can be used at 7-10 days.

Cell lines: SH-SY5Y cell lines were cultured in 10% FBS and 1%penicillin/streptomycin in a humidified incubator at 37°C and 5% CO2. In the experiments， SH-SY5Y cells were treated with MPP（200 μM） or different doses of Ka（0，3，30，60，100，300，600 μM） for 24 h. Ka （3，10，30，60μM，3 h）primed-SH-SY5Y cells were stimulated with MPp+（400 μM） for 24 h. Ka （30 μM，3 h） with （or without） 3-MA （3 mM，1 h）or α-tocopherol（50 μM，1h， Sigma-Aldrich，47783）primed-SH-SH5Y cells were stimulated with MPP+ （200 μM） for 24 h.

2.9. Cell viability CCK8 assay

The changed cell viability of Ka treatment was detected by Cell Counting Kit-8(CCK-8 Kit, Selleck, Houston, TX, USA).In brief, SHSY5Y cells were seeded in a 96-well plate and then treated with different concentrations of Ka（3，30，60，100，300，600 μM）for 24 h. Then 10μlof CCK-8 reagent was added to each well for 4 h. Finally, the absorbance was detected by the Multiskan Spectrum (Thermo Fisher Scientific) at 450 nm.

2.10.ATP assay

The cellular ATP levels were detected by an ATP Bioluminescence Assay Kit (Beyotime Biotechnology Co., China〕according to the manufacturer's instructions. After treatment, cells were lysed and centrifuged at 12,000 rpm for 5 min at 4°C and the supernatants were collected. Working solution （100μL）was added to 20μL of sample in the 96-well plate, and the luminescence was measured immediately on an auto-mated microplate reader. Measurements from all samples were normalized to protein concentration.

2.11. Na+-K -ATPase assay

For Na+-K+-ATPase measurement(A070-2-2, Jiancheng, Nanjing, China), cells were sonicated and centrifuged at 6000 rpm for 10 min to acquire supernatant. Reaction solutions of Na+-K+-ATPase detection were added and incubated according to the manufacturer's instructions. Then the absorbance at 660 nm was detected. Measurements of all samples were normalized to protein concentration.

2.12. Western blotting analysis

Cells or brain tissues were lysed in the RIPA buffer (50 mM Tris(pH 7.4),150 mM NaCl,1% NP-40(FNNO021,Thermo),0.5% sodium deoxycholate (D6750, Sigma), 0.1% SDS (74255, Sigma) supplemented with protease and phosphatase inhibitors(Roche, Shanghai, China). Protein concentrations were determined with the Micro BCA Kit （Beyotime， Shanghai， China）. A 30-μg protein of each sample were separated by SDS-PAGE using polyacrylamide TGX gels (Bio-Rad, Hercules, California, USA)and then transferred to polyvinylidene difluoride (PVDF)membranes (Millipore, Bedford, MA). After blocking, PVDF membranes were incubated with various specific primary antibodies in TBST at 4°C overnight then washed and incubated in corresponding horseradish peroxidase(HRP) conjugated secondary antibodies for 1 h at room temperature. Immunoreactive bands were visualized and detected by enhanced chemiluminescence(ECL)plus detection reagent (Pierce, Thermo Fisher Scientific, Rockford, IL) and analyzed using the ImageQuantM LAS 4000 imaging system(GE Healthcare, Pittsburgh, PA, USA).

2.13. Real-time quantitative(Q)-and reverse transcription (RT)-PCR

Total RNA was extracted from brain tissues and cultured cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription was carried out using the TAKARA PrimeScript RT reagent kit (TaKaRa, Japan). Realtime qPCR was carried out using SYBR Green Master Mix (Applied Biosystems) in a StepOnePlus instrument (Applied Bio-systems). The primers were purchased and validated by the General (Shanghai, China). The primers used for qPCR were shown in Supplementary Table 2.

2.14. Lactate dehydrogenase (LDH) assay

According to the manufacturer's instructions, cells were plated in a 96-well plate at 5000 cells/well, and a culture medium was collected to measure LDH levels with an assay kit (Nanjing Jiancheng Bioengineering Institute). Then the absorbance of samples was detected by the Multiskan Spectrum (Thermo Fisher Scientific) at 450 nm.

2.15.BODIPY493/503, BODIPY 581/591 C11, and MitoTracker deep red staining

Live cells were washed with PBS and incubated with 2ug/ml BOD-IPY 493/503(InvitrogenTM, Cat D3922)or BODIPY 581/591 C11 (BD-Cl1）（2.0 μM， InvitrogenTM， Cat D3861）in PBS for 15 min at 37°C. For MitoTracker Deep Red staining， live cells were incubated with 0.5 μg/ml MitoTracker Deep Red (InvitrogenTM, Cat M22426) in PBS for 30 min at 37°C. Then cells were washed twice in PBS and fixed in 3.5% PFA for 10 min followed by washing and counterstaining with Hoechst 33342 (Sigma, Cat B2261)for 10 min before being covered on glass slides for imaging. Images were observed and photographs were captured by fluorescence microscopy (Olympus, Tokyo, Japan).

2.16. Hoechst and PI staining

Cells were stained with Hoechst 33，342（1 μl diluted in 500 μl PBS， Sigma， Cat B2261）and （or）PI （0.1 μl diluted in 500 μl PBS， Solarbio， CA1020)for 10 min and then fixed and observed by fluorescence microscopy(Olympus, Tokyo, Japan).

2.17. Flow cytometry

Cells were digested and rinsed with D-hank's then stained with Annexin V-FITC（5 μl diluted in 500μl PBS）/PI（0.1 μl diluted in 500 μl PBS) apoptosis kit(CA1020, Solarbio, Beijing, Chian) according to the manufacturer's protocol. After, apoptotic cells were analyzed using flow cytometry (guava easyCyteTM 8, Millipore, USA). Mitochondrial ROS and mitochondrial membrane potential measurements were performed as published 【12】. SH-SY5Y cells were stained with MitoSOX （2.5 μM， Invitrogen， USA）or with Jul（10 μg/ml， T-3168， Invitrogen， USA） at 37°C for 30 min. After washing with PBS twice, the cells were then resuspended in cold PBS containing 1% FBS for flow cytometric analyses. Data were analyzed with the FCS Express software(Guava Easy CyteTM8, Millipore, Hayward, CA, USA).

2.18. Seahorse respiration assays

Mitochondrial respiratory function in live SH-SY5Y cells was measured using the Seahorse extracellular flux (XFe96)analyzer (Agi-lent, Santa Clara, USA) via changes in the oxygen consumption rate (OCR).SH-SY5Y cells seeded at 3× 103 cells/well were allowed to adhere to the Seahorse cell culture plates and to reach approximately 80% confluency at the time of the experiment. The following day, cells were pretreated with Ka and then exposed to MPp+ for another 24 h. OCR was detected under basal conditions followed by sequential addition of 1 μM oligomycin， 1 μM FCCP， as well as 1 μM rotenone & antimycin A.

2.19. Statistical analysis

Data were presented as mean ± SEM. The significance of difference was determined by the Student's t-test, Two-way analysis of variance, or one-way analysis of variance(ANOVA)followed by Tukey's post hoc test. The difference was considered significant at P<0.05.

3. Results

Result 1. Ka restores motor dysfunction and increases dopamine levels in the striatum of MPTP/p PD model mice

To investigate the neuroprotective effect of Kaon PD pathogenesis, 4-month-old male C57BL/6 mice were injected with the neurotoxin MPTP to establish the MPTP/p PD model and were treated with Ka (Fig. la). After model induction, the rotarod test and the pole test were used to evaluate the motor and behavioral performance of mice in the different groups. As shown, the rotarod performance time was markedly reduced in MPTP-treated mice, and this effect was prevented by Ka (Fig. 1b). Ka also restored the behavioral deficits induced by MPTP, as indicated by the reductions in the turning time and total time in the pole test (Fig. lc and d), without affecting the body weights of the mice (Fig. le). Furthermore, HPLC analysis showed that the level of dopamine in the striatum was significantly decreased in MPTP/p-challenged mice, and this level was restored by Ka treatment (Fig. 1f. Ka treatment also alleviated the MPTP/p-induced reduction in dihydroxy-phenyl acetic acid (DOPAC, a metabolite of dopamine)levels in the striatum(Fig.1g). These results suggest that Ka restores motor dysfunction and increases dopamine levels in the striatum in MPTP-induced PD mice.

Result 2. Ka alleviates the loss of DA neurons in the SNpc of MPTP/p PD model mice

Next, to further evaluate the neuroprotective effect of Kaon MPTP/p-induced DA neuronal impairment, we examined tyrosine hydroxylase (THD+ neurons in the murine SNpc by immunostaining. As shown in Fig.2a and b, MPTP/p induced a significant reduction in the number of TH+ neurons, which was alleviated by the Ka treatment. Furthermore, stereological counts of total neurons in the SNpc, as defined by Nissl

Fig.1. Ka treatment alleviates motor dysfunction and increases dopamine levels in the striatum of MPTP/p PD model mice.

(a) Schematic diagram of the experimental design. (b) Time on the rod was measured for the rotarod test on three consecutive days.(c-d) The time is taken to turn around(time of turning)and descend a pole(time of climbing)were recorded for the pole test. (e)Mouse body weights were measured at the end of the study. (f-g)Dopamine (DA) and dihydroxy-phenyl acetic acid (DOPAC) in the striatum were analyzed by high-performance liquid chromatography. The data are expressed as the mean ± SEM.n =9-10 for each group in (b,c,d); n =6-8 for each group in (f, g).ns, not significant, *P<0.05,**P<0.01,***P<0.001,by one-way ANOVA followed by Tukey's post hoc test. MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Ka, kaempferol.

staining, verified that the loss of TH cells reflected cellular death but not the downregulation of TH expression and showed that Ka treatment restored the decrease in the number of Nissl-positive neurons in the SNpc of MPTP-treated mice from 43.3% to 25.4%(Fig.2c and d).In addition, Ka significantly ameliorated the MPTP-induced reduction in TH and DAT protein expression, as measured by Western blotting (Fig. 2e-g). This evidence confirms the neuroprotective effect of Ka on the PD mouse model.

Result 3. Ka reduces the accumulation of LD vacuoles and oxidative stress in the SNpc of MPTP/p-treated mice

Increasing evidence has shown the involvement of LDs in NDDs[10], consistently, our previous work identified increased LDs in the SNpc of MPTP-induced PD mice[12]. LDs can be easily identified and show round, low-density structures with homogenous amorphous contents [28]. Normally, LDs can be degraded via autophagy to avoid accumulation [29]. To investigate whether Ka could regulate LDs in PD, the accumulation of LDs was analyzed by transmission electron microscopy (TEM) in the SNpc of control, MPTP/p, and MPTP/p+ Ka mice. As shown in our previous work [12], the number and size of LDs were increased in MPTP/p mice compared to saline-treated controls(Fig. 3a and b) and were significantly reduced in Ka-treated mice (Fig.3c-e). Moreover, in Ka-treated mice, LDs were more frequently observed nearby to, enclosed in, or degraded by autolysosomes, which were defined as electron-dense lipofuscin granules. Further histological staining of TH* neurons with BODIPY, a dye that specifically labels neutral lipids and is commonly used to detect LDs [30], showed that both the number and size of BODIPY LDs in the SNpc were higher in PD mice than in controls, and were significantly decreased by Ka treatment (Fig.3f-h).

If LDs cannot be degraded in a timely manner, the accumulated LDs may undergo peroxidation and contribute to mitochondrial ROS-mediated stress [4], which amplifies neurotoxicity and disease pro-gression. Hence, we further examined the expression profiles of genes associated with protection against free FA toxicity (Gpx8), neutralizing oxidative species, superoxide radicals (Sod3)and hydrogen peroxide (Cat), and FA metabolism (Acsbg1 and Dbi) as reported [31], all of which were strikingly decreased in the SNpc of MPTP-treated mice compared with controls, and these effects were ameliorated in Ka-treated PD mice (Fig.3i). Similarly, MPTP/p also increased the mRNA expression levels

Fig.2. Ka ameliorates the loss of DA neurons in the SNpc of MPTP/p PD model mice.

(a-b) Microphotographs of tyrosine hydroxylase(TH)-positive neurons (a) and stereological counts of TH-positive neurons in the substantia nigra pars compacta (SNpc)(b).Scale bars are as indicated.(c-d) Microphotographs of cresyl violet-positive cells(c) and stereological counts of cresyl violet-positive cells in the SNpc(d). Scale bar，120μm.（e-g） Western blot analysis of TH and DAT protein expression in the SNpc（e） and quantitative analysis（f，g）. The quantified data are normalized to the saline control group. The data are expressed as the mean ± SEM.*P<0.05, **P<0.01,by one-way ANOVA followed by Tukey's post hoc test.n=4-5 for each group. Ve, vehicle, Ka, kaempferol; MPTP,1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine..(For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) of NADPH oxidase subunits such as Noxl, Nox2, and Nox4 in the SNpc of MPTP/p mice, which were further blunted by Ka treatment (Fig. 3j). Taken together, these data suggest that Ka alleviates MPTP/p-induced LD accumulation, peroxidation, and ROS-mediated stress.

Result 4. Ka protects SH-SY5Y cells against MPP+-induced apoptosis

MPP+, the active metabolite of MPTP, inhibits mitochondrial com-plex enzymes and causes the cell death that is directly associated with PD[32]. We next investigated whether Ka could prevent MPP+-induced neuronal apoptosis in vitro. As shown in Fig. 4a and b， MPPt（400 μM，24 h) induced the release of LDH from SH-SY5Y cells, and Ka(30 and 60 μM） significantly attenuated LDH release without affecting cell survival (Fig.4a). Since Ka induced a substantial protective effect at a concentration of 30 μM（Fig.4b）， we used this concentration in subsequent experiments. The Hoechst/PI staining results also showed that Ka significantly reduced the percentage of cells with MPP+-induced chromatin condensation(Fig.4c and d)and cellular apoptosis, as evidenced by the increased Hoechst fluorescence intensity(Fig. 4c) and the percentage of Hoechst/PI-stained cells (Hoechst+/PI+)(Fig.4e). Also, as detected by flow cytometry, MPPt induced cell apoptosis, evidenced by the increased percentage of Annexin V-stained cells(AV+/PI- and AV+/Prt) was protected by Ka(Fig. 4f and g). Additionally, Ka notably reversed MPP+-induced changes in apoptosis-related protein expression, as evidenced by the upregulation of Bcl-2, downregulation of Bax (Fig.4hi-j), and reduced cleavage of caspase-3(Fig.4h, k). These data suggest that Ka can abolish the detrimental effects of MPp+ on cell Survival.

Result 5. Ka suppresses MPp+-induced LD accumulation and lipid peroxidation, which mediate mitochondrial damage in SH-SY5Y cells

Recent studies have shown that unexpected LD accumulation can result in increased lipid peroxidation-mediated stress and accelerate mitochondrial dysfunction, which promotes neurodegeneration [10]. Our previous work also showed that the increased LDs in the SNpc were associated with PD pathology in mice [12]. To investigate whether the protective effect of Ka was related to LD accumulation in vitro, we first performed LD-specific staining in SH-SY5Y cells using BODIPY 493/503 and observed increased LD deposition (enhanced levels of neutral lipids)

Fig. 3. Ka reduced the accumulation of electron-dense LD vacuoles and lipid oxidation in the SNpc of MPTP/p-treated mice.

(a-c)Representative EM images showing electron-bright vacuoles (lipid droplets), electron-dense autophagic vacuoles containing autolysosomes(ALS), or lipofuscin-containing lysosomes (Lys)in the SNpc of mice treated with saline (a), MPTP/p (b), and MPTP/p+ Ka (c).Scale bars,500 nm. With(a,b) as indicated in our previous work (PMID:29967574). (d-e)Quantification of electron-bright LD vacuoles per neuron in the SNpc in the indicated mice. n =3 mice,10-12 neurons per group. (f-h)Midbrain sections from saline-, MPTP/p-, and MPTP/p + Ka-treated mice were stained for BODIPY (LDs)and TH (DA neurons). The rightmost panels show magnifications of BODIPY + TH+ neurons. Arrows indicate LDs.(g-h), Quantification of BODIPY+ LD number and size. (i-i)mRNA expression of FFA toxicity-related genes(Gpx8, Sod3, Gat, Acsbg1, and Dbi)(i)and oxidative stress genes(Nox1, Nox2, and Nox4)(j)in the SNpc of the indicated mice was analyzed by real-time qPCR.n = 4-5 per group, three independent experiments. The data are expressed as the mean ± SEM; ns, not significant, *P<0.05,**P<0.01,***P<0.001, by one-way ANOVA followed by Tukey's post hoc test. Ka, kaempferol; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

This article is extracted from https://doi.org/10.1016/j.redox.2021.101911 Received 23 November 2020