Part 4 Studies Have Shown That Autophagy Plays An Important Role in The Regulation Of Cellular LD Deposition

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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 2 μg/ml BOD-IPY 493/503 (InvitrogenTM, Cat D3922) or BODIPY 581/591 C11 (BD-C11）（2.0μM， InvitrogenTM， Cat D3861）in PBS for 15min at 37℃. 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 3342 (Sigma, Cat B2261) for 10 min before being covered on glass slides for imaging. how much cistanche to take Images were observed and photographs were captured by fluorescence microscopy (Olympus, Tokyo, Japan).

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2.16.Hoechst and PI staining

Cells were stained with Hoechst 33，342（1 μl diluted in 500μl PBS， Sigma， Cat B2261）and （or）PI （0.1 μl diluted in 500μl PBS， Solarbio，(cistanche sleep) 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 ul diluted in 500μl PBS）/PI （0.1 μl diluted in 500μl PBS) apoptosis kit (CA1020, Solarbio, Beijing, Chian) according to the manufacturer's protocol. After, apoptotic cells were analyzed using flow cytometry (guava easyCyterM 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 JC1 （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 analysis. Data were analyzed with the FCS Express software (Guava Easy CyterM8, 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 reach approximately 80% confluency at the time of the experiment.(cistanche tea) 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.

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2.19. Statistical analysis

Data were presented as mean ± SEM. The significance of the difference was determined by 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

Result1. 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). (cistanche tubulosa benefits) 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. 1c 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).(cistanche tubulosa extract) 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). superman herbs cistanche 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 (TH) 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.(cistanche tubulosa reddit) (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) was 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, bioflavonoids benefits 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 Kaon in 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]. buy cistanche 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 too, 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], distance that amplifies neurotoxicity and disease progression. 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,120 um. (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-tetrahydroloyridine.. (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 Nox1, 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 MPPt-induced neuronal apoptosis in vitro. As shown in Fig. 4a and b, MPPt(40 uM, 24h) induced the release of LDH from SH-SY5Y cells, and Ka(30 and 60 uM) 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 MPPt-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, MPP+ induced cell apoptosis, evidenced by the increased percentage of Annexin V-stained cells (AV /PI and AV+/PI+) 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 MPPt on cell survival.

Result 5. Ka suppresses MPPt-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)

Result 6. Ka promotes autophagy and decreases mtROS production in MPP+-treated SH-SY5Y cells

Normally, LDs are delivered to lysosomes via autophagy and can be broken down into FAs to avoid peroxidation. However, evidence indicates that autophagy is impaired, which plays a crucial role in PD [2]. When autophagy involves portioning LDs to be broken down in lysosomes (Fig. 6a), the process is specifically called lipophagy [2]. To investigate whether autophagy is involved in the neuroprotective effect of Ka, we examined the levels of autophagy-associated proteins. As shown in Fig. 6b-d, in cultured SH-SY5Y cells, the level of microtubule-associated protein light chain 3 (LC3)-II was markedly reduced, while the expression of p62 was markedly increased in MPP+-treated cells, as measured by immunoblotting, and this effect was significantly reversed by Ka. We further examined the changes in autophagy by immunostaining for LC3. To allow autophagosomes to accumulate within cells, bafilomycin was added to prevent autophagosome fusion with lysosomes. As shown, increased numbers of LC3b puncta were detected in MPP*-treated cells, indicating impaired autophagic degradation, and this effect was also reversed by Ka (Fig. 6e and f). These results indicate that LDs in SH-SY5Y cells are mobilized by autophagy for eventual removal and that Ka can promote autophagy to reduce oxidized lipid accumulation. Increased lipid peroxidation can exacerbate mitochondrial damage by increasing mitochondrial oxidative stress [4]. Additionally, Ka treatment significantly reduced the robust mtROS generation, as determined by MitoSOX staining (Fig. 6g and h), and decreased the number of dysfunctional mitochondria, as determined by mitochondria-specific labels to distinguish respiring (MitoTracker deep red) and total (MitoTracker green) mitochondria (Fig. 6i and j). In addition, to have a direct view of mitochondrial alterations, we further detected the oxygen consumption rate (OCR) by the Seahorse XF analyzer. As shown in Fig. 6k, the mitochondria respiration was reduced in MPPt-treated cells, while Ka treatment obviously reverses this damage caused by MPP+(Fig. 6k).

Result7. Autophagy inhibition reverses the suppressive effect of Kaon LD accumulation, mitochondrial dysfunction, and DA neuronal injury in vitro

To examine whether autophagy is one of the main mechanisms by which Ka mediates DA neuroprotection, the autophagy inhibitor 3-MA was used. We found that the inhibitory effects of Ka on MPP#-induced injury, including decreased LD accumulation (Fig. 7a and b), reduced mtROS production (Fig. 7c and d), and alleviated mitochondrial dysfunction (Fig.7e and f) in SH-SY5Y cells, were significantly blocked by 3-MA. Since LD oxidation and mitochondrial dysfunction are closely associated with DA neuronal death, we next examined primary cultured DA neurons. We treated cultured mesencephalon TH+ neurons with Ka and/or MPPf and quantified the number and length of TH+ neuronal processes by immunostaining. Morphological analysis revealed that the number and length of neuronal processes were markedly decreased by MPP+ and protected by Ka, and this protection was further reversed by 3-MA (Fig.7g-i). These data indicate that Ka alleviates DA neuronal damage by promoting autophagy.

Result 8. Silencing Atg5 abolished the Ka-mediated prevention of neuronal loss in vivo

To further confirm that Ka protects against MPTP/p-induced TH+neuronal loss in an autophagy-dependent manner, we silenced Atg5 by microinjecting an adeno-associated virus (AAV)-expressing a previously characterized shRNA against Atg5 into the bilateral mesencephalon (Fig.8a) as previously described [25].In preliminary experiments, we observed that Atg5 shRNA transfection for 2 weeks achieved sufficient GFP expression in TH+ neurons (Fig. 8b) and inhibited ATG5 expression in the SNpc of the injected mice (Fig. 8c). We found that Atg5-knockdown reversed the neuroprotective effect of Ka, as evidenced by the upregulated protein expression of TH (Fig. 8d and e) and increased number of TH+ neurons (Fig. 8f and g). These data further support the contribution of autophagy to the protective effect of Ka in PD mice. In conclusion, we confirmed that Ka protects against MPTP/p-induced injury by reducing LD oxidation and mitochondrial dysfunction by promoting autophagy.

4. Discussion

The present study showed that the natural small molecule Ka was a key factor by which autophagic signaling could inhibit lipid oxidative toxicity. Our results demonstrated that the autophagy pathway is a key regulatory loop through which Ka alleviates LD peroxidation and subsequent mitochondrial damage and focused on the role of autophagy-regulated LD toxicity in alleviating mitochondrial damage while pre-venting neurodegeneration due to excessive oxidative stress (Fig. 9).

LDs are the intracellular sites of neutral lipid storage [5]and are critical for lipid metabolism and energy homeostasis, while LD dysfunction has been linked to many diseases [16]. Accumulating evidence has suggested that the roles LDs play in biology and pathology are significantly broader than previously thought. In the CNS, evidence has shown that increased LDs in neurons and glia are implicated in neurodegenerative pathology [10,36].In detail, LDs have been detected both in the axons of Aplysia neurons cultured in vitro [37] and in brain sections from Huntington's disease models [38].In addition, it was shown that neurons are particularly sensitive to lipid toxicity, and the accumulation of LDs that cannot be removed in a timely manner leads to accelerated LD peroxidation and can give rise to neurodegeneration [17].

Lipid accumulation in hyperactive neurons is toxic not only because of the susceptibility of these lipids to peroxidation. Moreover, the contents of the excess LDs may enter nonoxidative metabolic pathways, triggering excessive production of ceramide, which is toxic to cells [39]. Recent studies have also shown that in LD-defective cells, FAs can be converted into acylcarnitines, which then cause mitochondrial dysfunction and ROS production [40]. These processes are likely to have specific and profound consequences in hyperactive neurons because dysfunctional mitochondria compromise the capacity for FA consumption, and mtROS production further causes membrane lipid peroxidation.

Thus, LD homeostasis should be well controlled in neurons, which is critical for avoiding toxicity and ultimately maintaining the health of the brain. In the present study, we have found that the small natural molecule Ka inhibits neuronal LD toxicity that manifested as increased LD accumulation, elevated lipid peroxidation, and upregulated lipid toxicity-related gene expression in MPP#-insulted SH-SY5Y cells. More importantly, we have also shown that Ka treatment reduces the 45].In the present study, we observed defective autophagy/lipophagy in MPPt-injured neuronal cells, including reduced LC3-II levels and elevated p62 expression, and an increased number of LC3B puncta deposited in cells. Defective lipophagy in injured SH-SY5Y cells was reversed by treatment with Ka. As a consequence, Ka decreased the accumulation of oxidized LDs, damaged mitochondria, and mtROS generation in MPP#-treated SH-SY5Y cells. Importantly, the inhibition of autophagy by 3-MA abolished the Ka-mediated elimination of oxidized LDs and damaged mitochondria and the alleviation of DA neuron injury in vitro. Moreover, the inhibition of autophagy by Atg5 knockdown in vivo reversed the protective effects of Kaon DA neuronal degeneration in the MPTP-induced PD mouse model. Collectively, these findings indicate that Ka inhibits LD toxicity-related mitochondrial damage by promoting autophagy in an experimental PD model.

Accumulating evidence indicates that increased mtROS generation is a primary characteristic of high-activity neurons and has been observed in many NDDs, including PD [46]. Reports about whether ROS is a cause or consequence of LD formation are contradictory [47,48]. Interestingly, our in vitro results demonstrated that pharmacological inhibition of LD peroxidation with o-tocopherol prevented mtROS generation and mitochondrial membrane potential abnormalities and alleviated mitochondrial reductions, as evidenced by Tomm20 staining in SH-SY5Y cells, which supports the idea that LD peroxidation plays a causal role in MPP-induced mitochondrial dysfunction and the generation of mtROS. However, it is possible that elevated ROS initially accelerates LD formation, and subsequently, LDs induce further ROS generation and exacerbate intracellular and mitochondrial ROS levels.

In the present study, we showed that mice with MPTP-induced PD had high levels of LD accumulation, which was accompanied by the increased loss of DA neurons, accelerated ROS-mediated stress, and behavioral deficits. Ka treatment prevented LD toxicity in vitro and ameliorated DA neuronal loss and behavioral deficits in the PD mouse model, and these effects were autophagy-dependent. Therefore, it is reasonable to conclude that Ka promotes autophagy, and this enhanced autophagy decreases oxidized LD accumulation, reduces mtROS pro-duction, and alleviates mitochondrial damage in DA neurons, which contributes to the inhibition of LD toxicity, thereby attenuating PD pathology (Fig. 9).

Funding

This research was supported by grants from the National Natural Science Foundation of China (No. 81903587), the Project funded by the China Postdoctoral Science Foundation (No.2019M661807), the Natural Science Foundation of Jiangsu Province(No. BK20190120), and the Open Project of Chinese Materia Medica First-Class Discipline of Nanjing University of Chinese Medicine (No. 2020YLXK006), and the Drug Innovation Major Project (No.2018ZX09711001-003-007).

This article is extracted from Redox Biology 41 (2021) 101911