The Neuroprotective Effect of Isotetrandrine on Parkinson's Disease via Anti-Inflammation and Antiapoptosis In Vitro and In Vivo

Parkinson's disease (PD) is one of the most influential diseases in the world, and the current medication only can relieve the clinical symptoms but not slow the progression of PD. Therefore, we intend to examine the neuroprotective activity of plant-derived compound isotetrandrine (ITD) in vitro and in vivo. In vitro, cells were cotreated with ITD and LPS to detect the inflammatory-related protein and mRNA. In vivo, zebrafish were pretreated with ITD and inhibitors prior to 6-OHDA treatment. Then, the behavior was monitored at 5 dpf. Our result showed ITD inhibited LPS-induced upregulation of iNOS, COX-2 protein expression, and iL-6, inos, cox-2, and cd11b mRNA expression in BV2 cells. The data in zebrafish also demonstrated a significant improvement of ITD on the 6-OHDA-induced locomotor deficiency. ITD also improved 6-OHDA-induced apoptosis in zebrafish PD. We also pharmacologically validated the mechanism with three inhibitors, including LY294002, PI3K inhibitor; LY32141996, ERK inhibitor, SnPP, and HO-1 inhibitors. All of these inhibitors could abolish the neuroprotective effect of ITD partially in locomotor activity. Besides, the molecular level also showed the same trend. Treatment of these inhibitors could significantly abolish ITD-induced antineuroinflammatory and antioxidative stress effects in zebrafish PD. Our study showed ITD possessed a neuroprotective activity in zebrafish PD. The mRNA level also supported our arguments. The neuroprotection of ITD might be through antineuroinflammation and antiapoptosis pathways via PI3K, ERK, and HO-1.


Introduction
Parkinson's disease (PD) is one of the most widespread neurodegenerative diseases in the world [1].Te patients typically exhibit some typical symptoms including tremors, stifness in the limbs and joints, slow movements, reduced coordination, and slow swallowing function [2].In clinical, the frst-line treatment for PD is still medication including levodopa, dopamine agonists, monoamine oxidase type B, or catechol-o-methyl-transferase inhibitor [3][4][5].However, some adverse efects occurred after the administration of these drugs such as vomiting, nausea, and low blood pressure [6,7].Besides, the administration of medication only can relive the symptoms but not slow the progression of PD [8].Terefore, there is an urgent demand on novel PD drugs.Among all the sources of novel compounds, the natural products derived from plants occupied a niche in recent years.
Nowadays, a rise of plant-derived therapies or nutritional supplements occurred under the coronavirus apex in a few years [9].Tere were more than 30 plants in extracts or compounds, such as Artemisia annua, Houttuynia cordata Tunb, and Sambucus formosana Nakai, which possessed antiviral activity via their own mechanism during the viral infection, respectively [10].In PD, more kinds of preclinical compounds were discovered in plant-based database.Tere were about 56 formulations or molecules which possessed the neuroprotective activity [11].Most of them protect dopaminergic cell against neurotoxin to reduce the risk of degeneration [12,13].Hence, our study also intended to verify a plant-based compound on its neuroprotective activity.
Isotetrandrine (ITD) is a botanic natural product that originally came from Berberis bealei and Berberis duclouxiana [14].It is considered as an analgesic, antimicrobial, immunosuppressive, and antimalarial agent [15].It is also known as a cell-permeable PLA2 inhibitor and has also shown to inhibit α1 adrenoceptor [16,17].However, the neuroprotective activity of ITD has not been investigated yet.Our study intended to realize the neuroprotective efect of ITD and clarifed its mechanism of action via various inhibitors.We hoped the data and subsequent research could provide a new choice for the sufering PD patients.

Cell
Maintain.BV2 murine microglia cell line was purchased from Elabscience Biotechnology Inc (Elabscience, USA, TX, No. CRL-2266).Te cells were maintained with BV2 cells complete medium (Elabscience, USA, TX, No. CM-0493) in a 5% CO 2 37 °C incubator.Te cells were cotreated with diferent concentrations (10, 100, and 200 μM) of ITD and LPS (1 μg/ml) for 24 hr and harvested the cells for the western blot and qPCR analysis.Te induction condition was referred to in a previous study [18].Te cell number used for western blot or qPCR was 1 × 10 6 per 6 cm dish.

Western Blotting.
In accordance with the treatment plan, zebrafsh and cells were treated in 6 cm dishes.Cell pellets were collected, and an equivalent amount of lysis solution (containing 0.5 mM and 0.1% protease inhibitor cocktail) was used to lyse them.Using the Bradford protein assay, the lysed protein was then adjusted to the same concentration before being added to the sample bufer, which consists of 2% sodium dodecyl sulfate, 10% glycerol, 0.1% bromophenol blue, 2% 2-mercaptoethanol, and 50 mM Tris-HCl in pH � 7.2.SDS-PAGE electrophoretic separation was used to separate the prepared protein samples.Ten, target proteins were moved to a PVDF membrane that had been activated.PVDF membranes were treated with anti-iNOS (Cayman Chemical, USA, MI, No. 160862), anti-COX-2 (Cayman Chemical, USA, MI, No. 160106), and antiβ-actin for an overnight period at 4 °C after being blocked with 5% nonfat dried milk.Chemiluminescence was used to fnd a secondary antibody coupled to horseradish peroxidase (Millipore Corp).Te UVP BioChemi Imaging System was utilized to capture images, and LabWorks 4.0 software was used to calculate the relative densitometry.

Zebrafsh Maintenance.
In this work, we employed wildtype zebrafsh (AB strain) and maintained in the KMU Zebrafsh Core Facility at 28 were applied to zebrafsh embryos for 87 hours (9 hpf to 5 dpf ) in 24-well microplate.From 2 to 5 dpf, we cotreated with 6-OHDA (250 μM).At 5 dpf, the qPCR and protein samples were collected, respectively.Te zebrafsh behavioral tests alluded to in a prior work at 5 dpf and 6 dpf [24].We performed the behavior tests as previous study [25].In brief, zebrafsh larvae were placed in the cuvette, and their behavior was tracked using an automated video tracking system (Singa Technology Co.; Taiwan, No, TM-01).Te cuvette utilized in this investigation was 1-1-4.5 cm (L-W-H) in size and was held in front of the camera for 7.5 cm.Each zebrafsh in the test was given a 2-minute adaption period before the swimming pattern and the total distance of each fsh was recorded for 5 minutes.

Statistical Analysis.
Results were presented as mean-± SEM.For western blotting data, the intensity of each band was expressed as the relative OD divided by the average OD values from all internal controls.Data were analyzed using one-way analysis of variance followed by Dunnett's test.Te p values less than 0.05 were considered statistically signifcant.

Te Anti-Infammatory Efect of ITD on LPS-Induced
Infammation in BV2 Cell.We assessed the antineuroinfammation efect of ITD on LPS-induced BV2 cell in western blots.ITD (10, 100, or 200 μM) was cotreated with LPS (1 μg/ml) for 24 hr and harvested for the following western blots.Our data showed that 200 μM ITD

Te Anti-Infammatory Efect of ITD on 6-OHDA-Induced
Modulation in Zebrafsh PD.We then examined the mRNA level of infammation-related genes such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and tumor necrosis factor-alpha (TNF-α).Besides, we also examined tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine synthesis, to assess the number of dopamine neurons encoded by th1.Our data showed that 6-OHDA treatment could signifcantly reduce th1 expression, and pretreatment of ITD signifcantly reversed 6-OHDAinduced down-regulation of th1 expression (Figure 3

Discussion
In our study, we demonstrated the antineuroinfammation activity of ITD in BV2 murine microglia with iNOS, COX-2, and iL-6 mRNA and protein levels.We further confrmed the neuroprotective activity of ITD in the zebrafsh PD model.Our data also showed a signifcant improvement of ITD on 6-OHDA-induced locomotor defciency in zebrafsh.Te qPCR analysis of apoptotic-related mRNA expression also demonstrated that ITD could improve 6-OHDA-induced apoptosis in the zebrafsh PD model.Besides, we also pharmacologically validated the mechanism  Parkinson's Disease with three inhibitors, included LY294002, PI3K inhibitor; LY32141996, ERK inhibitor; and SnPP, HO-1 inhibitor.All of these inhibitors could abolish the neuroprotective efect of ITD partially in locomotor activity.Te molecular level also showed the same trend.Treatment of these inhibitors could signifcantly abolish ITD-induced antiinfammatory and antioxidative stress efects in zebrafsh PD assay.Other antiinfammatory compounds were also discussed about their neuroprotection activity.Previous studies used BV2 murine microglia as an in vitro model to investigate the mechanism of neuroinfammation since 1990 [27].It was evaluated by microglia biomarkers and tested 90% positive for nonspecifc esterase activity.Besides, BV2 didn't show the signal of glial fbrillary acidic protein (GFAP) or galactocerebroside (GC) for astrocyte and oligodendrocytes, respectively, which is similar to primary microglia [28].Hyperactivated microglia serves as an essential function in regulating neuroinfammatory responses that induce neuronal injury.In recent years, there has been a lot of focus on fnding innovative ways to treat neuroinfammation [29,30].Velagapudi et al. demonstrated that tiliroside not only inhibited BV2 microglia activation by LPS/IFNc-induced neuroinfammation but also protected HT22 neuronal against damage via targeting Nrf2 antioxidative pathways.Te compound could inhibit NF-κB acetylation by activating SIRT1.In in vivo model, tiliroside also enhanced SIRT1 activity in mice hippocampus neurons [31].Besides, Park et al. demonstrated that Gyejibokryeonghwan (GBH), a traditional Korean medicine, could ameliorate LPS-stimulated BV2 activation in NO production, infammatory cytokines and reduced iNOS, COX-2, TNF-α, and IL-6 protein expression.In addition, infammatory transcription factor NF-) κB p65 was also decreased under the GBH treatment.GBH could also enhance nuclear factor-E2-related factor 2/cAMP response element-binding protein-dependent antioxidant pathway and the downstream HO-1 protein expression [21].Our data also revealed that 200 μM ITD could signifcantly inhibit LPS-induced upregulation of iNOS and COX-2 in mRNA and protein levels (Figure 1).Except for the in vitro model, some studies also used zebrafsh as a screening tool in antiPD drugs.
Bretaud et al. frst demonstrated that zebrafsh could be used as a PD animal model [32].Tey treated neurotoxins in zebrafsh larvae and intended to induce PD-like symptoms, including reducing swimming total distance or swimming velocity.Besides, Anichtchik et al. also showed that intracerebral injections of 6-OHDA and MPTP lead to a signifcant locomotor defcit in adult zebrafsh [33].Te studies mentioned above both tried to use zebrafsh to imitate clinical symptoms of PD, and they also confrmed the In addition, SvMEs revealed a signifcant antioxidant activity in vitro, which was also demonstrated in vivo on ktr4:NTR-hKikGR zebrafsh and they also discovered that SvMEs may reduce oxidative stress and α-synuclein aggregation which could alleviate PD-like symptoms.Antioxidant-related genes (sod1, gss, gpx4a, gclm, and cat) suggested that SvMEs possessed an anti-PD efect via an antioxidation mechanism [34].Our previous research in 2016 demonstrated how 11-dehydrosinulariolide (11-de) exerts its therapeutic efect via triggering cytosolic or mitochondrial DJ-1 expression, which subsequently activates the downstream Akt/PI3K, p-CREB, and Nrf2/HO-1 pathways.In addition, we discovered that 11-de might rescue the 6-OHDA-induced decrease in total swimming distance in a PD zebrafsh model.Utilizing a rat PD model, we demonstrated that an increase in the number of rotations and time spent on the beam caused by 6-OHDA could be reversed by 11-de treatment [35].Kozioł et al. demonstrated that the 7.5 μM xanthotoxin treatment restored locomotor activity defciency in zebrafsh larvae.Meanwhile, administration of xanthotoxin also reduced mobility defcits in a mouse model at a dose of 25 mg/kg.Te same action of the same chemical in two distinct animal models implies compatibility and strengthens the feasibility of zebrafshbased in vivo models.Teir fndings also suggested that xanthotoxin might be a promising lead compound [36].Our current fnding also depicted that ITD could signifcantly reverse 6-OHDA-induced locomotor defcit (Figure 2) and refected on the molecular level in apoptotic and infammation mRNA level (Figure 3).Indeed, ITD was mentioned as an antiinfammatory agent in previous studies.Liang et al. showed that the cytokine levels of TNF-α, IL-1β, and IL-6 in supernatant were reduced by ITD.Furthermore, their result showed that ITD signifcantly inhibited the activation of MAPK and NF-κB, which are induced by LPS in acute lung injury (ALI) model.Teir fndings showed that ITD reduced the severity of LPSinduced ALI via inactivating MAPK and NF-κB.ITD might inhibit oxidative stress and the pulmonary infammatory process [37].Sun et al. demonstrated that 100 μM ITD could ameliorated astrocyte cytotoxicity in neuromyelitis optica via inhibiting the binding of neuromyelitis optica- immunoglobulin G to aquaporin 4 [38].Moreover, ITD reduced tert-butyl hydrogen peroxide-induced oxidative damage via increasing HO-1 protein expression.Tis was accomplished by separating the nuclear translocation of factor-erythroid 2 p45-related factor 2 (Nrf2) from the Nrf2-Keap1 complex via activation of extracellular signalregulated protein kinase and c-Jun NH2-terminal kinase, as well as inactivation of Keap1 [26].In their study, they also used fve inhibitors, including SB203580 (p38 inhibitor), LY294002 (PI3K/Akt inhibitor), SP600125 (JNK inhibitor), U0126 (ERK inhibitor), or SnPP (HO-1 inhibitor) to further clarify the mechanism of ITD.To our surprise, the protective efect of ITD was abolished by the pretreatment of SnPP but not in LY294002.Our data showed that the neuroprotective efect of ITD was signifcantly inhibited by the treatment of SnPP, LY294002, and LY3214996 (Figures 4-6).We suggested that the integrality of in vivo model (zebrafsh) leads to diferences in results.
In conclusion, our results demonstrated that ITD possessed an antineuroinfammatory activity in LPS-induced BV2 murine microglia cell.Furthermore, it also showed a great neuroprotective activity in zebrafsh locomotor defcit.Te mRNA level also supported our arguments.We also verifed the mechanism of action via PI3K, ERK, and Parkinson's Disease 9 HO-1 inhibitors and proved ITD's efect on antineuroinfammation and antiapoptosis pathway.We believed ITD may be a promising candidate for PD therapy (Figure 8).

Conclusions
6-OHDA treatment signifcantly increases intracellular ROS, which activates the resting microglia.Te infammatory cytokine such as TNF-α and iL-6 leads to dopaminergic cell death in zebrafsh.Te reduction of dopamine neurons is also refected in locomotor defciency.Te pretreatment of ITD could protect cells from 6-OHDA damage in two ways.First, ITD inhibited LPS or 6-OHDA-induced upregulation of iNOS, COX-2, iL-6, cd11b, and TNF-α expression.In addition, ITD also protects neurons in the antiapoptosis pathway, including PI3K and p-ERK pathways.We also verifed that ITD could inhibit intracellular ROS through the upregulation of HO-1 expression.Trough the antiapoptotic and antineuroinfammation efect of ITD shown in BV2 cells and zebrafsh, ITD could protect cells from damage and rescue zebrafsh from the locomotor defciency induced by exposure to 6-OHDA treatment.Parkinson's Disease

Figure 1 :
Figure1: Efect of isotetrandrine (ITD) on LPS-induced iL-6, iNOS, and COX-2 mRNA and protein expression in BV2 cell.BV2 microglia cells were cotreated with LPS and diferent concentrations of isotetrandrine (ITD) (200, 100, and 10 μM) for 24 hr and harvested for western blots and qPCR.(a) Western blots for iNOS, COX-2, and β-actin protein expression in each group.(b) Quantifcation results of relative density of iNOS and COX-2 expression in each group.Te relative density of the LPS-induced group was taken to be 100%.β-actin was used as an internal control.(c-f ) Quantitative PCR of interleukin-6 (Il-6), inducible nitric oxide synthase (Inos), and cyclooxygenase-2 (Cox-2) and cd11b mRNA expression in each group.Gapdh was used as an internal control.Data are presented as mean ± SEM (n � 3).* Signifcantly diferent from the control group; p < 0.05; # signifcantly diferent from the LPS group.

Figure 8 :
Figure 8: Schematic diagram of mechanism of ITD in 6-OHDA-induced cell death.