Indole Diterpenes from Mangrove Sediment-Derived Fungus Penicillium sp. UJNMF0740 Protect PC12 Cells against 6-OHDA-Induced Neurotoxicity via Regulating the PI3K/Akt Pathway

In our chemical investigation into Penicillium sp. UJNMF0740 derived from mangrove sediment, fourteen indole diterpene analogs, including four new ones, are purified by multiple chromatographic separation methods, with their structures being elucidated by the analyses of NMR, HR-ESIMS, and ECD data. The antibacterial and neuroprotective effects of these isolates were examined, and only compounds 6 and 9 exhibited weak antibacterial activity, while compounds 5, 8, and 10 showed protective effects against the injury of PC12 cells induced by 6-hydroxydopamine (6-OHDA). Additionally, compound 5 could suppress the apoptosis and production of reactive oxygen species (ROS) in 6-OHDA-stimulated PC12 cells as well as trigger the phosphorylation of PI3K and Akt. Taken together, our work enriches the structural diversity of indole diterpenes and hints that compounds of this skeleton can repress the 6-OHDA-induced apoptosis of PC12 cells via regulating the PI3K/Akt signaling pathway, which provides evidence for the future utilization of this fascinating class of molecules as potential neuroprotective agents.


Introduction
Indole diterpenes belong to an important class of alkaloids mainly isolated from species of the genera Penicillium, Aspergillus, Epichloe, Nodulisporium, and Tolypocladium, and they bear a cyclic diterpenoid core originating from geranylgeranyl diphosphate (GGPP) and an indole moiety deriving from tryptophan (Figure 1) [1][2][3].Indole diterpenes are generally divided into paxilline and non-paxilline types [2,3], and exhibit a range of biological activities involving an inhibition against the big potassium channel receptor [4,5], cholesterol acyl transferase (ACAT) [6], human kinesin [7], nitrogen monoxide (NO) production [8], bacteria [9], cancer cells [10,11], agricultural pests [12], and the H1N1 virus [13], which endows them promising application prospects in the fields of medicine or agriculture.However, the possible detriment to humans or animals has limited their widespread application, as indole diterpenes are often reported to be tremorgenic mycotoxins [1].Nevertheless, the work on the structural diversity, structure-activity relationship, or biosynthesis of indole diterpenes [1][2][3]14] still attracts considerable attention from the scientific community.During our investigation of Penicillium sp.UJNMF0740 (the nam Trichoderma citrinoviride UJNBHMF0740 [15]) obtained from mangrove paxilline-type indole diterpenes, including four new shearinine ana and structurally characterized (Figure 2), and their neuroprotective e against injury from 6-hydroxydopamine (6-OHDA), along with an against Staphylococcus aureus and Escherichia coli, were subsequently them, compounds 6 and 9 displayed mild growth inhibitory activity ag IC50 values of 48.8 and 47.6 µM, respectively, while compounds 5, 8, alleviated the 6-OHDA-induced injury of PC12 cells.Then, compo demonstrated to ameliorate cell damage through inhibiting the produ ygen species (ROS) and preventing the apoptosis mediated by the PI3 addition, the possible biosynthetic pathway of all the isolated metabo In summary, the current work not only presents new indole diterpene reports the neuroprotective effect of this class of fungal metabolites f panding the bioactivity spectrum and providing a new perspective fo tion of these interesting alkaloids.During our investigation of Penicillium sp.UJNMF0740 (the name was revised from Trichoderma citrinoviride UJNBHMF0740 [15]) obtained from mangrove sediment, fourteen paxilline-type indole diterpenes, including four new shearinine analogs, were isolated and structurally characterized (Figure 2), and their neuroprotective effects on PC12 cells against injury from 6-hydroxydopamine (6-OHDA), along with antibacterial activities against Staphylococcus aureus and Escherichia coli, were subsequently evaluated.Among them, compounds 6 and 9 displayed mild growth inhibitory activity against S. aureus with IC 50 values of 48.8 and 47.6 µM, respectively, while compounds 5, 8, and 10 significantly alleviated the 6-OHDA-induced injury of PC12 cells.Then, compound 5 was further demonstrated to ameliorate cell damage through inhibiting the production of reactive oxygen species (ROS) and preventing the apoptosis mediated by the PI3K/Akt pathway.In addition, the possible biosynthetic pathway of all the isolated metabolites was proposed.In summary, the current work not only presents new indole diterpene derivatives but also reports the neuroprotective effect of this class of fungal metabolites for the first time, expanding the bioactivity spectrum and providing a new perspective for the future utilization of these interesting alkaloids.During our investigation of Penicillium sp.UJNMF0740 (the name was revised from Trichoderma citrinoviride UJNBHMF0740 [15]) obtained from mangrove sediment, fourteen paxilline-type indole diterpenes, including four new shearinine analogs, were isolated and structurally characterized (Figure 2), and their neuroprotective effects on PC12 cells against injury from 6-hydroxydopamine (6-OHDA), along with antibacterial activities against Staphylococcus aureus and Escherichia coli, were subsequently evaluated.Among them, compounds 6 and 9 displayed mild growth inhibitory activity against S. aureus with IC50 values of 48.8 and 47.6 µM, respectively, while compounds 5, 8, and 10 significantly alleviated the 6-OHDA-induced injury of PC12 cells.Then, compound 5 was further demonstrated to ameliorate cell damage through inhibiting the production of reactive oxygen species (ROS) and preventing the apoptosis mediated by the PI3K/Akt pathway.In addition, the possible biosynthetic pathway of all the isolated metabolites was proposed.In summary, the current work not only presents new indole diterpene derivatives but also reports the neuroprotective effect of this class of fungal metabolites for the first time, expanding the bioactivity spectrum and providing a new perspective for the future utilization of these interesting alkaloids.3), revealing the paxilline-type indole diterpene skeleton of 1 as 12.However, an additional methoxy group (δ H /δ C 3.64/53.3),the absence of the NH signal (δ H 7.20 in 12), and the remarkably upfield-shifted C-2 signal (∆δ C − 19.2) compared with 12 were observed, indicating that an imine fragment bearing a methoxy group in 1 replaced the amide moiety in 12 [5].The HMBC correlations from H 3 -32 and H 3 -41 to C-2 further confirmed the location of the methoxy group at C-2.The gross structure of shearinine R (1) was therefore constructed as shown in Figure 2. The relative configuration of 1 was deduced by the NOESY spectrum (Figure 4), in which the correlations of H-16/H-14β, H-16/H 3 -33, H 3 -32/H-17α, H 3 -32/H-5α, H 3 -32/H-15α, and H-14α/H-11 established the fusion of D/E and E/F rings as in 7 [5,16].The absolute configuration for 1 was determined as shown by comparing the calculated ECD spectrum for the (3S,4R,7S,9R,13S,16S) enantiomer to the experimental one for compound 1 (Figure 5), as well as the biosynthetic considerations [1,2].

Structural Elucidation
Compound 2 was purified as an amorphous light-yellow powder and given a molecular formula of C 37 H 47 NO 8 based on the protonated molecular ion peak at m/z 634.3370 ([M + H] + , calcd 634.3374) in HR-ESIMS and the 1D NMR data (Table 1), accounting for 15 degrees of unsaturation.The similarity of the 1 H, 13 C NMR data for 2 to those for shearinine H (12) [5] disclosed that compound 2 was also an indole diterpenoid derivative, while the main differences between 2 and 12 were attributed to the 13 C chemical shifts of the C ring and C-2/C-3/C-17.A further analysis of the 2D NMR data for 2 (Figure 3) afforded identical structural units of A-C and E-H rings with 12.The planar structure of 2 was finally elucidated as a D ring opening analog derived from the amide bond hydrolysis of 12, based on the extra 18 mass units and the loss of one degree of unsaturation compared with 12.Although direct signals for the free amino and carboxylic acid groups in compound 2 were not observed in the 1 H NMR spectrum, the 13 C NMR data for related carbons in 2 were in good agreement with those of shearinine Q [17], an indole diterpenoid analog with the same ring-opening structural fragment, which further corroborated the structure of 2. Compound 2 shared similar NOESY data (Figure 4) as those of 1, and the experimental ECD spectrum matched well with the calculated spectrum of the (3S,4R,7S,9R,13S,16S) enantiomer.Therefore, the stereochemistry of compound 2 was deduced as depicted in Figure 2.      Compound 2 was purified as an amorphous light-yellow powder and given a mo ular formula of C37H47NO8 based on the protonated molecular ion peak at m/z 634.3 ([M + H] + , calcd 634.3374) in HR-ESIMS and the 1D NMR data (Table 1), accounting fo degrees of unsaturation.The similarity of the 1 H, 13 C NMR data for 2 to those shearinine H (12) [5] disclosed that compound 2 was also an indole diterpenoid derivat while the main differences between 2 and 12 were attributed to the 13 C chemical shift the C ring and C-2/C-3/C-17.A further analysis of the 2D NMR data for 2 (Figure 3) forded identical structural units of A-C and E-H rings with 12.The planar structure was finally elucidated as a D ring opening analog derived from the amide bond hydrol The molecular formula C 37 H 45 NO 8 of compound 3 with 16 degrees of unsaturation was evidenced by the HR-ESIMS ion at m/z 632.3220 ([M + H] + , calcd 632.3218) and the 1D NMR data (Table 1).The NMR signals of 3 highly resembled those reported for shearinine I [5], and the main difference between the two compounds was attributed to the fact that a methylene group (δ H 2.63/3.08;δ C 33.1) in shearinine I [5] was replaced by an oxygenated methine (δ H 4.91; δ C 76.3) in 3, allowing the location of an additional hydroxy group at C-22.The HMBC correlations from H-20 to C-22, and from H-22 to C-21, C-23, C-24, C-28, and C-29 (Figure 3), further confirmed the hydroxylated methine moiety of C-22.
Compound 4 was revealed to possess a molecular formula of C 37 H 43 NO 6 according to the HR-ESIMS ion peak at m/z 598.3166 ([M + H] + , calcd 598.3163) and the 1D NMR data (Table 1), corresponding to 17 degrees of unsaturation.The NMR data for 4 were very close to those of shearinine D (7) [5], indicating that their structures were highly related.However, a carbonyl signal (δ C 201.7) in 4 was observed instead of that for the oxymethine (δ H 4.86; δ C 76.7) in 7, indicating that the methine (C-22) in 7 was oxidized to carbonyl in 4, which was further verified by the HMBC correlations from H-20 and H-23 to C-22, along with the one-less-degree of unsaturation of 4 compared with that of 7.
A plausible biosynthetic pathway for 1-14 was postulated as shown in Scheme 1. Biosynthetically, compounds 13 and shearinine K could be produced through oxidation at C-13, as well as further prenylation and diprenylation on the aromatic ring of 14, respectively.Subsequently, oxidation and dehydration cyclization on the isopentenyls of shearinine K would afford 9, while the further oxidation at C-22 of 9 would proceed easily to generate 11 and 10.The double-bond isomerization from ∆ 23,28 of 9 to ∆ 27,28 would lead to 6, while the following oxidation and methylation occurring at C-22 of 6 would afford 7, 4, and 8.The oxidation of the respective intermediate (i) by the cleavage of the C-2-C-18 bond can produce an eight-membered ring fragment in 12, while the progress was reported to be automated [5].Then, the double-bond isomerization, like from 9 to 6, and the oxidation at C-22 in 12 would form 3, and 5 would be derived from the dehydration of 7. Finally, compound 12 could undergo the hydrolysis of the amide bond to yield 2, and keto-enol tautomerization and the methylation of 12 would produce 1.Scheme 1. Proposed biosynthetic pathway for compounds 1-14.

Antimicrobial Activities
The antibacterial activities of compounds 1-14 against Gram-positive Staphylococcus aureus ATCCC 25923 and Gram-negative Escherichia coli ATCC8739 were tested, and only 6 and 9 exhibited weak growth inhibitory effects against S. aureus with IC50 values of 48.8 and 47.6 µM, respectively, and ceftriaxone sodium was used as a positive control with an IC50 < 1.0 µM.

Neuroprotective Effects
The protective effects against 6-OHDA-induced neuronal injury on PC12 cells of the isolated 1-14 were evaluated by an MTT assay with N-acetylcysteine (NAC) as reference compound.In the initial test at 50 µM, compounds 5, 8, and 10 remarkably rescued the cell viability, with 5 showing the best activity (Figure 6A).In addition, 5 did not exhibit any obvious cytotoxicity toward the PC12 cells within the concentration range from 12.5 to 100 µM, and it even had a certain promoting effect on cell proliferation (Figure 6B).Additionally, compound 5 at 25 µM displayed the best ameliorating effect against the damage of PC12 cells within the testing range (Figure 6C).

Antimicrobial Activities
The antibacterial activities of compounds 1-14 against Gram-positive Staphylococcus aureus ATCCC 25923 and Gram-negative Escherichia coli ATCC8739 were tested, and only 6 and 9 exhibited weak growth inhibitory effects against S. aureus with IC 50 values of 48.8 and 47.6 µM, respectively, and ceftriaxone sodium was used as a positive control with an IC 50 < 1.0 µM.

Neuroprotective Effects
The protective effects against 6-OHDA-induced neuronal injury on PC12 cells of the isolated 1-14 were evaluated by an MTT assay with N-acetylcysteine (NAC) as reference compound.In the initial test at 50 µM, compounds 5, 8, and 10 remarkably rescued the cell viability, with 5 showing the best activity (Figure 6A).In addition, 5 did not exhibit any obvious cytotoxicity toward the PC12 cells within the concentration range from 12.5 to 100 µM, and it even had a certain promoting effect on cell proliferation (Figure 6B).Additionally, compound 5 at 25 µM displayed the best ameliorating effect against the damage of PC12 cells within the testing range (Figure 6C).

Antimicrobial Activities
The antibacterial activities of compounds 1-14 against Gram-positive Staphylococcus aureus ATCCC 25923 and Gram-negative Escherichia coli ATCC8739 were tested, and only 6 and 9 exhibited weak growth inhibitory effects against S. aureus with IC50 values of 48.8 and 47.6 µM, respectively, and ceftriaxone sodium was used as a positive control with an IC50 < 1.0 µM.

Neuroprotective Effects
The protective effects against 6-OHDA-induced neuronal injury on PC12 cells of the isolated 1-14 were evaluated by an MTT assay with N-acetylcysteine (NAC) as reference compound.In the initial test at 50 µM, compounds 5, 8, and 10 remarkably rescued the cell viability, with 5 showing the best activity (Figure 6A).In addition, 5 did not exhibit any obvious cytotoxicity toward the PC12 cells within the concentration range from 12.5 to 100 µM, and it even had a certain promoting effect on cell proliferation (Figure 6B).Additionally, compound 5 at 25 µM displayed the best ameliorating effect against the damage of PC12 cells within the testing range (Figure 6C).This is the first manuscript describing the neuroprotective activity of diterpenoid indole alkaloids.As for the structure-activity relationship (SAR) for compounds 1-14, there was no regular pattern for this group of molecules, as reported for many classes of natural products.Nevertheless, it is interesting to note that several isolates, such as 1, 7, and 12, even displayed significant toxicity to PC12 cells at the testing concentration (50 µM).Particularly, the cases of 7 and 8 demonstrated the surprising effect of methylation in the conversion from toxicity to protection, which further revealed the complexity of the mechanism of natural products.
Apoptosis is the main mode of neuronal cell death in many nerve injuries.Hoechst 33258 staining, Annexin V-PI double staining, and Western blot experiments were then performed to assess the effect of 5 on PC12 cell apoptosis induced by 6-OHDA.According to Hoechst 33258 staining, chromatin condensation corresponding to apoptosis was clearly observed in PC12 cells exposed to 6-OHDA, which was notably improved in the groups treated with different concentrations of 5 for 12 h prior to administration with 6-OHDA (Figure 7A).Moreover, the quantitative analysis of cell apoptosis was conducted by double staining with Annexin V-PI on a flow cytometer.As shown in Figure 7B, the proportion of apoptotic cells significantly reduces in the pretreatment groups with compound 5 (13.79%, 23.96%, and 37.23% for 25, 50, and 100 µM, respectively) compared with the control group subjected to 6-OHDA-treatment alone (41.45%).This is the first manuscript describing the neuroprotective activity of diterpenoid indole alkaloids.As for the structure-activity relationship (SAR) for compounds 1-14, there was no regular pattern for this group of molecules, as reported for many classes of natural products.Nevertheless, it is interesting to note that several isolates, such as 1, 7, and 12, even displayed significant toxicity to PC12 cells at the testing concentration (50 µM).Particularly, the cases of 7 and 8 demonstrated the surprising effect of methylation in the conversion from toxicity to protection, which further revealed the complexity of the mechanism of natural products.
Apoptosis is the main mode of neuronal cell death in many nerve injuries.Hoechst 33258 staining, Annexin V-PI double staining, and Western blot experiments were then performed to assess the effect of 5 on PC12 cell apoptosis induced by 6-OHDA.According to Hoechst 33258 staining, chromatin condensation corresponding to apoptosis was clearly observed in PC12 cells exposed to 6-OHDA, which was notably improved in the groups treated with different concentrations of 5 for 12 h prior to administration with 6-OHDA (Figure 7A).Moreover, the quantitative analysis of cell apoptosis was conducted by double staining with Annexin V-PI on a flow cytometer.As shown in Figure 7B, the proportion of apoptotic cells significantly reduces in the pretreatment groups with compound 5 (13.79%, 23.96%, and 37.23% for 25, 50, and 100 µM, respectively) compared with the control group subjected to 6-OHDA-treatment alone (41.45%).Several members of the caspase family playing a vital role in apoptosis are often detected as marker proteins, while Bax and Bcl-2 are also important regulatory proteins for mitochondrial permeability in apoptosis [23].Therefore, the expressions of cleaved Several members of the caspase family playing a vital role in apoptosis are often detected as marker proteins, while Bax and Bcl-2 are also important regulatory proteins for mitochondrial permeability in apoptosis [23].Therefore, the expressions of cleaved Mar.Drugs 2023, 21, 593 9 of 14 caspase-3, cleaved caspase-9, Bax, and Bcl-2 were investigated by Western blotting.As indicated in Figure 7C-G, the exposure of PC12 cells to 6-OHDA leads to the upregulation of cleaved caspase-3, cleaved caspase-9, and Bax, as well as the downregulation of Bcl-2, while pre-treatment with 5 significantly protects PC12 cells against these influences, especially at 25 µM.Collectively, these data provide adequate evidence that compound 5 effectively reduces the apoptosis of PC12 cells stimulated by 6-OHDA.
The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway is well known to play a vital role in multiple cellular processes, such as cell metabolism, proliferation, differentiation, migration, and protein synthesis [24,25].Thus, we evaluated the effects of 6-OHDA and compound 5 on the expressions of PI3K, p-PI3K, Akt, and p-Akt in PC12 cells.The ratios of p-PI3K/PI3K and p-Akt/Akt were significantly downregulated in the 6-OHDA group compared with the control, while they were upregulated by pretreatment with 5 (25, 50, and 100 µM) compared with the 6-OHDA group (Figure 8A-C), suggesting that the PI3K/Akt pathway was involved in the suppression of 5 against the PC12 cell injury induced by 6-OHDA.
caspase-3, cleaved caspase-9, Bax, and Bcl-2 were investigated by Western blotting.As indicated in Figure 7C-G, the exposure of PC12 cells to 6-OHDA leads to the upregulation of cleaved caspase-3, cleaved caspase-9, and Bax, as well as the downregulation of Bcl-2, while pre-treatment with 5 significantly protects PC12 cells against these influences, especially at 25 µM.Collectively, these data provide adequate evidence that compound 5 effectively reduces the apoptosis of PC12 cells stimulated by 6-OHDA.
The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway is well known to play a vital role in multiple cellular processes, such as cell metabolism, proliferation, differentiation, migration, and protein synthesis [24,25].Thus, we evaluated the effects of 6-OHDA and compound 5 on the expressions of PI3K, p-PI3K, Akt, and p-Akt in PC12 cells.The ratios of p-PI3K/PI3K and p-Akt/Akt were significantly downregulated in the 6-OHDA group compared with the control, while they were upregulated by pre-treatment with 5 (25, 50, and 100 µM) compared with the 6-OHDA group (Figure 8A-C), suggesting that the PI3K/Akt pathway was involved in the suppression of 5 against the PC12 cell injury induced by 6-OHDA.
In neuronal damage, oxidative stress is thought to be an important factor [26].As 6-OHDA was reported to induce the production of reactive oxygen species (ROS) [27]

General Experimental Procedure
The ESIMS data were measured on an Agilent 6460 Triple Quad LC-MS instrument (Agilent Technologies Inc., Waldbronn, Germany), while the HR-ESIMS spectra were obtained from an Agilent 6545 Q-TOF mass spectrometer (Agilent Technologies Inc., Waldbronn, Germany).The NMR experiments were performed on a Bruker Avance DRX600 spectrometer (Bruker BioSpin AG, Fallanden, Switzerland) with the residual solvent peaks at δH 7.26 and δC 77.2 (CDCl3).ECD and UV spectra were acquired on a Chirascan circular dichroism spectrometer with a 1 mm pathway cell (Applied Photophysics Ltd., Surrey, UK).Optical rotations were recorded on a Rudolph VI polarimeter (Rudolph Research Analytical, Hackettstown, USA) with a 10 cm-length cell.Semi-preparative HPLC was performed on a Shimadzu LC-20A instrument (Shimadzu, Tokyo, Japan) with YMC-Pack ODS-A columns (5 µm, 10 × 250 mm, YMC Co. Ltd., Tokyo, Japan).D-101 macroporous resin (Sinopharm Chemical Reagent Co. Ltd., Shanghai, China), silica gel (Qingdao In neuronal damage, oxidative stress is thought to be an important factor [26].As 6-OHDA was reported to induce the production of reactive oxygen species (ROS) [27] resulting in cell damage and apoptosis, intracellular ROS was then detected by fluorescence dye DCFH-DA and flow cytometry to examine the effect of 5 on 6-OHDA-induced oxidative damage.The results in Figure 8D indicate that supplementing 5 in 6-OHDA-induced PC12 cells can significantly reduce the intracellular generation of ROS compared with the 6-OHDA group, and the pre-treatment of the 25 µM compound 5 presents the best effect.

General Experimental Procedure
The ESIMS data were measured on an Agilent 6460 Triple Quad LC-MS instrument (Agilent Technologies Inc., Waldbronn, Germany), while the HR-ESIMS spectra were obtained from an Agilent 6545 Q-TOF mass spectrometer (Agilent Technologies Inc., Waldbronn, Germany).The NMR experiments were performed on a Bruker Avance DRX600 spectrometer (Bruker BioSpin AG, Fallanden, Switzerland) with the residual solvent peaks at δ H 7.26 and δ C 77.2 (CDCl 3 ).ECD and UV spectra were acquired on a Chirascan circular dichroism spectrometer with a 1 mm pathway cell (Applied Photophysics Ltd., Surrey, UK).Optical rotations were recorded on a Rudolph VI polarimeter (Rudolph Research Analytical, Hackettstown, USA) with a 10 cm-length cell.Semi-preparative HPLC was performed on a Shimadzu LC-20A instrument (Shimadzu, Tokyo, Japan) with YMC-Pack

Assay of Antimicrobial Activity
Liquid growth inhibition in the 96-well microplate assay [28] was applied to evaluate the initially antimicrobial activity against Staphylococcus aureus ATCCC 25923 and Escherichia coli ATCC8739 strains for the obtained fourteen metabolites at 50 µM, while a 2-fold serial dilution method in 96-well microplates [28] was used to determine the IC 50 values for the compounds with inhibition rates higher than 50% in the primary test.

Cell Line and Culture
A rat PC12 cell line was obtained from the Procell Life Science & Technology Co., Ltd.(Wuhan, China).The cells were maintained in RPMI-1640 media supplemented with 10% FBS and 1% penicillin/streptomycin (Genview, China) in a humidified incubator with a 5% CO 2 atmosphere at 37 • C.

Cell Viability Assay
The MTT assay was performed to measure the viability of the PC12 cells with different treatments.PC12 cells were seeded in a 96-well plate at a density of roughly 1.5 × 10 5 cells per well and cultured for 24 h.Then, the cells were pre-treated with DMSO or the tested compounds for 12 h, and then exposed to 75 µM of 6-OHDA for 12 h.After incubation, the MTT solution was added to the cells with a final concentration of 0.5 mg/mL and further incubated at 37 • C for 4 h.The supernatants were carefully removed and 100 µL of DMSO was added to each well to dissolve formazan crystals.Finally, the absorbance was detected at 490 nm with a Microplate Reader (Tecan, Switzerland).The cell viability was presented as the percentage of the absorbance measured in the vehicle-treated cells.

Hoechst 33258-Staining Assay
Hoechst 33258 staining was used to assess the chromatin condensation, a typical feature of cellular apoptosis.PC12 cells were plated in 6-well plates and treated with different concentrations of compound 5 (25,50, and 100 µM) for 12 h, and exposed to 75 µM of 6-OHDA for a further 12 h.Then, a 4% paraformaldehyde solution was applied to fix the cells, which were stained with 10 µg/mL of Hoechst 33258 (Sigma-Aldrich, St Louis, MO, USA) for 15 min at 37 • C, followed by washing twice with 1× PBS.The stained cells were observed under a fluorescence microscope (Leica DMI8, Wetzlar, Germany).

Flow Cytometric Analysis
The effect on the apoptosis of PC12 incubated with 6-OHDA from compound 5 was evaluated by Annexin V-FITC/PI double staining associated with flow cytometry.The PC12 cells were first pretreated with different concentrations of 5 and 75 µM of 6-OHDA, as previously indicated.The harvested cells were washed with PBS and resuspended in the binding buffer to be about 1 × 10 6 cells/mL.Then, the cells were incubated with Annexin V-FITC and PI in the dark for 15 min and detected with a flow cytometer (ACEA Biosciences, San Diego, CA, USA).

Figure 7 .
Figure 7. Effect of compound 5 on 6-OHDA-induced cell apoptosis in PC12 cells.Cells were pretreated with different concentrations of 5 and then exposed to 6-OHDA for 12 h.(A) Morphology of nuclear chromatin was observed by Hoechst 33258 staining using a fluorescence microscope, while the red arrows mean representative chromatin condensations in nuclei.(B) The apoptosis rates were detected by an Annexin V-FITC/PI apoptosis detection kit on flow cytometry.(C) The apoptosisrelated protein expressions of cleaved caspase-3, cleaved caspase-9, Bax, and Bcl-2 by Western blot.(D-G) Quantitative analyses of the levels of cleaved caspase-3, cleaved caspase-9, Bax, and Bcl-2 by GraphPad Prism 8.0 software.The values are means ± SDs, ### p < 0.001 compared with the control group; * p < 0.05, ** p < 0.01, *** p < 0.001; and ns (no significance) compared with the 6-OHDAtreatment group.

Figure 7 .
Figure 7. Effect of compound 5 on 6-OHDA-induced cell apoptosis in PC12 cells.Cells were pretreated with different concentrations of 5 and then exposed to 6-OHDA for 12 h.(A) Morphology of nuclear chromatin was observed by Hoechst 33258 staining using a fluorescence microscope, while the red arrows mean representative chromatin condensations in nuclei.(B) The apoptosis rates were detected by an Annexin V-FITC/PI apoptosis detection kit on flow cytometry.(C) The apoptosis-related protein expressions of cleaved caspase-3, cleaved caspase-9, Bax, and Bcl-2 by Western blot.(D-G) Quantitative analyses of the levels of cleaved caspase-3, cleaved caspase-9, Bax, and Bcl-2 by GraphPad Prism 8.0 software.The values are means ± SDs, ### p < 0.001 compared with the control group; * p < 0.05, ** p < 0.01, *** p < 0.001; and ns (no significance) compared with the 6-OHDA-treatment group.
resulting in cell damage and apoptosis, intracellular ROS was then detected by fluorescence dye DCFH-DA and flow cytometry to examine the effect of 5 on 6-OHDA-induced oxidative damage.The results in Figure 8D indicate that supplementing 5 in 6-OHDA-induced PC12 cells can significantly reduce the intracellular generation of ROS compared with the 6-OHDA group, and the pre-treatment of the 25 µM compound 5 presents the best effect.

Figure 8 .
Figure 8. Effects of 5 on the PI3K/Akt pathway and ROS level in PC12 cells.PC12 cells were incubated with different concentrations of 5 for 12 h, and then 6-OHDA (75 µM) was added for an additional 12 h.Protein expressions were detected by Western blot, while the quantification analysis was conducted by GraphPad Prism 8.0 software.(A) The protein expressions of Akt, p-Akt, PI3K, and p-PI3K.(B,C) Quantitative analysis of the ratios of p-PI3K/PI3K and p-Akt/Akt.(D) Intracellular ROS was detected by the DCFH-DA reagent and flow cytometry and stated as the fluorescence intensity.All experiments were performed in triplicate.Results are described as means ± SDs, ## p < 0.01, ### p < 0.001 vs. the control group; *** p < 0.001 vs. 6-OHDA-treatment group.

Figure 8 .
Figure 8. Effects of 5 on the PI3K/Akt pathway and ROS level in PC12 cells.PC12 cells were incubated with different concentrations of 5 for 12 h, and then 6-OHDA (75 µM) was added for an additional 12 h.Protein expressions were detected by Western blot, while the quantification analysis was conducted by GraphPad Prism 8.0 software.(A) The protein expressions of Akt, p-Akt, PI3K, and p-PI3K.(B,C) Quantitative analysis of the ratios of p-PI3K/PI3K and p-Akt/Akt.(D) Intracellular ROS was detected by the DCFH-DA reagent and flow cytometry and stated as the fluorescence intensity.All experiments were performed in triplicate.Results are described as means ± SDs, ## p < 0.01, ### p < 0.001 vs. the control group; *** p < 0.001 vs. 6-OHDA-treatment group.