Protective effects of Chlorogenic Acid on acetaminophen-induced liver injury through regulating PINK1/Parkin pathway promoted mitophagy

doi:10.21203/rs.3.rs-1501143/v1

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Protective effects of Chlorogenic Acid on acetaminophen-induced liver injury through regulating PINK1/Parkin pathway promoted mitophagy

https://doi.org/10.21203/rs.3.rs-1501143/v1

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Background: Chlorogenic acid (CGA) is one of the most abundant dietary polyphenols, possessing well-known hepatoprotective capacity in acetaminophen (APAP)-induced hepatotoxicity mice, but whether its mechanisms are related to activating mitophagy remains unclear.

Methods and results: In this study, a mice model and HepG2 cells model induced by APAP were established, and then 24-hour survival rate, the serum levels of aminotransferases, liver histopathology in vivo and cell viability in vitro were firstly measured. Our results shown that CGA could significantly improve the 24-survival rate of mice and cell viability in vitro, and decrease the activities of serum enzymes including alanine transaminase (ALT), aspartate transaminase (AST) and lactate dehydrogenase (LDH) and reduced Pathological injury induced by excessive APAP in vivo. Moreover, TUNEL staining and apoptotic proteins (Bax, Bcl-2) showed that CGA inhibited liver cell apoptosis in vivo. Furthermore, our data showed that CGA promoted co-localization of Tom20 and LC3II in mitochondria, and simultaneously up-regulated the expression levels of mitophagy-related genes and proteins (PINK1, parkin, LC3Ⅱ/LC3Ⅰ) and down-regulated the expression level of p62 in vivo. Finally, the protective effect of CGA was largely abolished after PINK1 was knock-downed by siPINK1 in HepG2 cells and the expression of mitophagy-related proteins (PINK1, parkin, LC3Ⅱ/LC3Ⅰ and p62) was also reversed.

Conclusions: our study results suggested that CGA potentially ameliorated APAP-induced acute liver injury through enhancing PINK1/Parkin pathway promoted mitophagy.

Chlorogenic acid

Acute liver injury

Apoptosis

PINK1/Parkin pathway

Mitophagy

One of the primary reasons for acute liver disease is drug-induced hepatoxicity and composes around 5% of all hospital admissions in 50% of all acute liver failure cases[1]. Acetaminophen (APAP) is a common antipyretic and analgesic. Although it is generally safe used at therapeutic dose (< 4 g every 24 h), APAP could cause severe liver, kidney and heart injury or organ failure at a single overdose exceeding 15 to 25 g[2–4]. Since liver is one of the most sensitive organ to APAP, overdose APAP-induced acute liver injury is the most common adverse reaction. Recent studies have suggested multiple molecular mechanisms that contribute to APAP hepatotoxicity, including oxidative stress, sterile inflammation, mitochondrial dysfunction [5] and apoptosis [6].

It is well known that APAP induced liver toxicity starts from the production of toxic metabolite N-acetyl-p-benzoquinone imine (NAPQI). Excessive NAPQI accumulation leads to glutathione (GSH) depleted in the liver and leads to mitochondrial dysfunction and oxidative stress [7]. Mitochondria are highly dynamic organelles that govern cell redox homeostasis, innate immunity and apoptosis [8]. In liver cells, mitochondria are the starting point of inflammatory signals and oxidative stress responses [9]. Mitochondrial reactive oxygen species (ROS) can damage lipids, proteins and DNA, leading to impairment of mitochondrial structure and function [10, 11]. Emerging evidences indicated that mitophagy plays a key role in the liver's physiology and pathophysiology [12]. Mitophagy can selectively degrade damaged mitochondria, ensure its own function and metabolic stability.

Generally, mitophagy involves mitochondrial fission, autophagosome formation and fusion with lysosome in turn [13]. These processes are regulated by phosphatase and tensin homolog (PTEN)-induced kinase 1 (Pink1)/Parkin pathway. Briefly, Pink1 locates on the outer mitochondrial membrane of damaged mitochondria from the inner membrane, then removes the damaged mitochondria by recruiting Parkin in response to various stimuli [14]. Importantly, suppression of Pink1/Parkin augments APAP hepatotoxicity via impairment of hepatic mitophagy, which indicates that Pink1/Parkin-mediated mitophagy plays a key role in alleviating APAP hepatotoxicity [15, 16]. Therefore, it is critically important to prevent APAP hepatotoxicity through precise regulation of Pink1/Parkin pathway. Intriguingly, a recent study found that knockdown of PINK1 aggravated liver parenchymal cell damaged and significantly inhibited mitophagy in a cellular model of cadmium-induced mitochondrial dysfunction [17]. Another study proved that PINK1 siRNA augmented hypoxia/reoxygenation-induced apoptosis in human hepatocyte L02 cells, which implied that PINK1-mediated mitophagy might involve in regulating apoptosis [18]. However, targeting mitophagy-mediated apoptosis for relieving APAP-induced liver injury remains largely undetermined.

Growing evidences had proved the protective effect of Chlorogenic acid (CGA) on APAP hepatotoxicity which involves multiple mechanisms including anti-oxidation, anti-inflammatory and anti-apoptosis [19–23]. More specifically, they are concerned with attenuating liver mitochondrial injury and reducing mitochondrial HSP60 release, activating Nrf2 anti-oxidative signaling pathway, inhibiting MAPK signaling pathway, inhibiting CYP2E1 and CYP1A2 enzymatic properties and suppressing TLR3/4 and NF kappa B signaling pathway [20–26]. However, whether its mechanisms are related to activating mitophagy remains unclear. It’s well known that mitophagy defect can perturb mitochondrial function and cause progressive accumulation of ROS and the inability to supply energy, leading to oxidative stress, sterile inflammation and cell apoptosis, reinforcing a vicious cycle. In this study, we determined the role of CGA on preventing APAP hepatotoxicity based on targeting mitophagy-mediated apoptosis in mice. Our data showed pretreatment with CGA provided a protective role in APAP-induced acute hepatotoxicity mice via activating Pink1-depended mitophagy and inhibiting hepatic apoptosis.

2.1 Drugs and reagents

CGA (purify ≥ 98%) was purchased from Herbest (Baoji, China). APAP was obtained from Shanghai Johnson Pharmaceutical Co., Ltd. (Shanghai, China). AG (purify ≥ 98%) was provided by Abphyto (Chengdu, China). Commercial assay kits for alanine transaminase (ALT), aspartate transaminase (AST) and lactate dehydrogenase (LDH) were supplied by Medical System (Ningbo, China). Cell Total RNA Isolation Kit was acquired from Foregene. (Chengdu, China). Anti-Tom20 was provided by Santa Cruz Biotechnology, Inc. Anti-SQSTM1/p62 and anti-Parkin were offered by Cell Signaling Technology (Danvers, MA, USA). Anti-LC3, anti-Bcl2 and anti-Bax were furnished by Proteintech Group, Inc. (Wuhan, China). Anti-PINK1 was purchased from Abcam (Cambridge, UK). Anti-glyceraldehyde phosphate dehydrogenase (GAPDH) was produced by Servicebio (Wuhan, China). The BCA protein assay kit was acquired from Multi Sciences (Hangzhou China).

2.2 Animals and protocols

100 male 4–6 weeks Kunming mice (20 ± 2 g) were obtained from Byrness Weil biotech Co., Ltd. (Chengdu, China). All mice were acclimated for 3 days in a constant temperature and humidity room (24°C ± 1°C, 50% ±10% humidity) with standard diet, water and a 12-h light/dark cycle (lights on at 8:00 am and lights off at 8:00 pm). Animal care and experiments protocols were in accordance and approved by the Ethics Committee for Animal Experiments of the Institute of Material Medica Integration and Transformation for Brain Disorders in Chengdu University of Traditional Chinese Medicine and accorded with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Permit No: IBD2019008, Chengdu, China).

All mice were randomly divided into five groups (n = 20), the control group (Ctrl), APAP (300 mg/kg), the APAP (300 mg/kg) + AG group (200 mg/kg), and the APAP (300 mg/kg) + CGA group (20 mg/kg or 40 mg/kg). Mice in the intervention group were respectively pre-administered with CGA (20 mg/kg or 40 mg/kg) and AG (200 mg/kg) for 14 consecutive days. Simultaneously, the control and APAP groups were congruously administrated with same volume 0.9% saline. On day 15, mice were administered a single dose of APAP (300 mg/kg) to induce APAP acute hepatotoxicity except the control group was administered 0.9% saline[27]. Then, all mice were sacrificed on day 16 and serum and liver tissues were collected.

2.4 Cell culture and cell viability assay

The human hepatocellular carcinoma HepG2 was purchased from the Cell Bank of the Chinese Academy of Science (Shanghai, China). HepG2 cells were cultured in DMEM with 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco) at 37°C with 5% CO₂. The cells were divided into Ctrl, APAP, APAP + CGA, APAP + CGA + si NC, APAP + CGA + si PINK1. The 1 × 10⁴ cells cultured in 96-well plates were pre-incubated with CGA at different concentrations for 15 min after adherent, and then incubated with APAP for an additional 24 h. The cell viability was measured by the conventional MTT assay according to the manufacturer's instruction.

2.5 Mortality in mice assay

After APAP (300mg/kg) treatment, the death period of mice was recorded within 24 h. During the period, the vital signs of the mice were observed every hour.

2.6 Biochemical analysis for serum

Serum was collected after centrifugation at 3500 rpm for 10 min at room temperature. Serum enzymatic activities of ALT, AST and LDH were measured with a HITACHI 7180 automatic biochemistry analyzer (Hitachi, Japan).

2.7 Histopathology of liver tissue and TUNEL assay

Fresh liver tissues were immediately fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, and then cut into 4-µm-thick sections. The tissues were stained with hematoxylin and eosin (HE) for histological examination under a light microscopy. The histological scores (including inflammation and necrosis) indicating the degree of liver injury were determined according to Suzike’s standard [28]. Simultaneously, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) was used to measure hepatic apoptosis according to the manufacturer's instructions of a TUNEL apoptotic detection kit. Images were captured by a fluorescence microscopy (Leica Microsystems, Wetzlar, German), and counted the positive cells.

2.8. PINK1 siRNA transfection

PINK1-siRNA (Hanheng Biotechnology, Shanghai, China) was used to inhibited the PINK1 gene and protein expression in HepG2 cells, according to the manufacturer's instructions. In addition, HepG2 cells transfected with non-silencing scrambled siRNA (Hanheng Biotechnology. Shanghai, China) were used as control. The respective siRNA senses and antisense sequences for PINK1 and control siRNA were as follows: siPINK1, 5′-CGCUGUUCCUCGUUAUGAATT-3′ and 5′-UUCAUAACGAG GAACAGCGTT-3′, and siNC, 5′-UUCUCCGAACGUGUCACGUdTdT-3′ and 5′-ACGUGACACGUUCGGAGAAdT dT-3′. After the cells were successfully transfected, the cells were treated according to the method shown in 2.2, and then subjected to Western blot analysis.

2.9 RT-qPCR analysis

Total RNA was extracted from liver samples using Trizol reagent. cDNA was synthesized with the SuperScript® IV First-Strand Synthesis System (Bio-Rad, Singapore). RT-qPCR was performed in the SYBR-Green PCR kit on Applied Biosystems StepOnePlus system. β-actin was used as the invariant control. The target gene expression levels by the 2^−ΔΔCt method. The sequences of primers used in this study are as follows: PINK1: Forward 5′-AGACTCCCAGTTCTCGCCT-3′, Reverse 3′- AGGGACAGCCATCTGAGTCC-5′; Parkin: Forward5′-AGCCAGAGGTCCAGCAGTT-3′, Reverse 3′-CTGGCACTCACCACTCATCC-5′; p62: Forward 5′-AGATAGCCTTGGAGTCGGTG-3′, Reverse 3′-CCGGGGATCAGCCTC TGTAG-5′; LC3-Ⅱ: Forward 5′-ACCCTAACCCCATA GGAGCC-3′, Reverse 3′-TGCAAGCG CCGTCTGATTA-5′; β-actin: Forward 5′-GCTCCGGCATGTGCAAAG-3′, Reverse 3′-TTCC CACCATCACACCCTGG-5′.

2.10 Protein preparation and western blotting analysis

The total proteins of liver were extracted by RIPA lysis buffer containing protease and phosphatase inhibitor (100:1:1). Protein concentrations were determined using the BCA protein assay kit. Equal amounts of total protein (30 µg) were separated by 8%-15%SDS-PAGE gels before transferred to the PVDF membranes. Then, the membranes were blocked with 5% fat-free milk at room temperature for 90 min and incubated with primary antibody at 4°C overnight, including anti-GAPDH (1:5000), anti-Parkin (1:1000), anti-PINK1 (1:1000), anti-p62 (1:1000), anti-LC3 (1:1000), anti-Bcl2 (1:1000) and anti-Bax (1:1000). After washing with TBST, the membranes were incubated with secondary goat anti-rabbit IgG (1:5000) or anti-mouse IgG (1:5000) at room temperature for 90 min, and were conjugated with horseradish peroxidase. Then, proteins were detected by chemiluminescence reagent. The relative protein levels were normalized to GAPDH level.

2.11 Immunohistochemistry analysis

The liver tissue was cut into 4-µm-thick sections then deparaffinization with xylene and gradient ethanol, antigen retrieval with trisodium citrate dihydrate for 30 min and blocked with 1% BSA at room temperature for 30 min. the sections were incubated with primary anti-Tom20 (1:100) and anti‐LC3Ⅱ(1:300)at 4℃ overnight and then with secondary antibodies (1:500) in the dark for 1 h. Hoechst 33258 was used to counterstain the nucleus. At last, the sections were mounted with anti-fluorescence quenching sealer and observed by confocal microscopy (Olympus, Japan).

2.12 Statistics

Statistical analysis was performed by one-way analysis of variance (ANOVA) and all values were expressed as mean ± standard error of the mean (SEM), with the exception of survival rate. p༜0.05 was considered to be statistically significant.

3.1 Chlorogenic acid alleviated APAP-induced acute hepatotoxicity in mice

To confirm the potential hepatic protective effects of CGA, mice were pretreated with CGA (20 or 40 mg/kg) and AG (200 mg/kg) for 14 days prior to APAP administration (300 mg/kg). Firstly, we evaluated protective effect of CGA on the survival rate. As shown in Fig. 1A, APAP administration resulted in an obvious decline in survival rates to 65% within 6 h, which remained at this level up to 24 h. Pretreatment of CGA (40 mg/kg) significantly increased survival rates compared with APAP alone (p༜0.05). Moreover, the effects of CGA on the degree of liver function injury were assessed after an APAP overdose of 300 mg/kg administrations. Our data showed that the serum levels of ALT, AST and LDH in the APAP group were markedly increased, and were significantly decreased by AG and CGA (40 mg/kg) treatment (p༜0.05, Fig. 1B, C and D, which suggested that CGA could alleviate liver damage by reducing the activity of ALT, AST and LDH. Furthermore, the histopathologic analysis was performed on HE stained hepatic sections. As shown in Fig. 1E, APAP administration significantly induced severe hepatocellular injury compared with the control mice, such as loss of hepatocyte architecture, vacuolization of hepatocytes, massive necrosis and mononuclear cell infiltration in the portal area. Conversely, treatment with CGA or AG significantly ameliorated the hepatocellular injury around in the portal area. Compared with the control group, necrosis scores and inflammation scores were increased significantly after APAP administration (p༜0.05), while treatment of AG and CGA (40 mg/kg) significantly decreased all the scores (p༜0.05, Fig. 1F and G), these results suggested that pretreatment with CGA provided a protective role on APAP-induced acute hepatotoxicity in mice.

3.2 Chlorogenic acid suppressed APAP-induced apoptosis of hepatocyte in mice

To further elucidated the role of CGA on APAP-induced apoptosis of hepatocyte in mice, TUNEL staining and western blotting were used. As shown in Fig. 2A and B, there were little apoptotic cells in the normal liver tissue. However, significantly increased apoptotic cells could be observed in the APAP group, which was significantly reduced by treatment of AG and CGA (40 mg/kg, p༜0.05). To further demonstrate the effect of CGA on APAP-induced apoptosis, the expressions of apoptosis-associated proteins (Bax and Bcl-2) were measured by western blotting. We found that APAP mice presented a significantly elevated expression of Bax and decreased expression of Bcl-2 (p༜0.05, Fig. 2C, D and E) and reduced Bcl-2/Bax rate (p༜0.05, Fig. 2F) compared to the control mice, which was significantly reversed after treatment with 40 mg/kg of CGA (p༜0.05, Fig. 2C-F). Taken together, these data indicated that CGA attenuated APAP-induced apoptosis of hepatocyte through regulating Bcl-2 family proteins levels in mice.

3.3 CGA promoted PINK1-dependent mitophagy in APAP treated mice

To verified the effects of CGA on autophagy, immunofluorescence co-localization analysis was firstly used to examine the colocalization of Tom20 (a specific mitochondria marker) and LC3Ⅱ (autophagy marker). Our data showed that pretreatment of CGA significantly promoted the colocalization of Tom20 and LC3Ⅱ to exert enhancing autophagy effect compared with APAP mice (Fig. 3A). The expression decrease of Tom20 is often considered to the reduction of mitochondria damage and the activation of autophagy. As shown in Fig. 3B, We found that APAP mice presented a significantly elevated expression of Tom20 compared to the control mice, which was significantly reversed after treatment with CGA(p༜0.05). Moreover, we tested the expression of autophagy-related genes and proteins (LC3Ⅱ and p62). As expected, after pretreatment with CGA, the expressing of LC3II mRNA and protein were dramatically increased while p62 was the exact opposite compared with APAP mice. Simultaneously, the ratio levels of LC3II/I were significantly decreased in APAP mice while they were remarkably increased in CGA mice (p༜0.05, Fig. 3C-E). These results indicated that CGA promoted autophagy flux in APAP-induced liver injury mice. As a type of mitochondria-specialized autophagy, PINK1-dependent mitophagy was recently recognized as a novel target for treating alcoholic liver disease and APAP hepatotoxicity, due to its maintaining role in mitochondrial homeostasis [14]. Therefore, we further determine whether CGA promoted PINK1-dependent mitophagy. RT-qPCR and Western blot assay were used to measure the genes and proteins expression levels of the mitophagy marker PINK1 and Parkin, and an obvious inhibition of PINK1 and Parkin expressions were found in APAP mice (p༜0.05, Fig. 3C-E). Interestingly, pretreatment with CGA at the dose of 40 mg/kg significantly increased the levels of PINK1 and Parkin (p༜0.05, Fig. 3C-E), suggesting that CGA alleviated APAP-induced hepatotoxicity via activating PINK1/Parkin-dependent mitophagy.

3.4 PINK1 deficiency reversed the protective effect of CGA

To further to confirm the protective effect of CGA on APAP-induced mice through promoting PINK1-dependent mitophagy, APAP-induced HepG2 cells were utilized. Firstly, we investigated the effect of different concentrations of chlorogenic acid on the viability of HepG2 cells, and the results showed that 0-100 µM of chlorogenic acid showed no toxicity in HepG2 cells (Fig. 4A). MTT results showed that CGA significantly inhibited APAP-induced cytotoxicity (p༜0.05, Fig. 4B). The previous animal experiments indicated that the protective effect of CGA was dependent on PINK1/Parkin-mediated mitophagy. Moreover, to investigates whether altered PINK1 expression plays an important role in the cytoprotective effect conferred by CGA, PINK1 was knocked down by specific siRNA. As shown in Fig. 4C and D, transfection with PINK1 siRNA resulted in significantly reduced gene and protein level of PINK1 compared with the si NC groups. PINK1 siRNA attenuated the protective effect of CGA in APAP-induced HepG2 cell death (p༜0.05, Fig. 4B). In addition, Western blot results showed that knockdown of PINK1 expression abolished the effect of CGA on up-regulation of LC3B II/I ratio, PINK1 and Parkin expression (p༜0.05, Fig. 4E and F). These results indicated that CGA alleviated APAP-induced hepatotoxicity was activated mitophagy in PINK1/Parkin-dependent way.

APAP could induce acute liver injury at excessive doses [29]. Our present study demonstrates that treatment with CGA (ⅰ) alleviates hepatocellular injury induced APAP in vivo and in vitro. (ⅱ) inhibits hepatocyte apoptosis through regulating Bcl-2 family proteins under APAP overdose conditions, and (ⅲ) promotes PINK1-dependent mitophagy releasing attenuate acute liver injury induced by APAP. Collectively, our results firstly revealed that CGA inhibited hepatocyte apoptosis in response to APAP excess by activating PINK1- mediated mitophagy.

Natural products in the protection and prevention of liver injury are particularly well-documented, such as CGA. It has been shown to protect against liver damage caused by different drugs, including carbon tetrachloride, methotrexate, and APAP [21, 23, 26]. our data indicated that CGA pretreatment significantly improved survival rate and liver function demonstrated with inhibiting APAP overdose caused elevation of serum ALT, AST and LDH. At the same time, Chlorogenic acid enhanced cell viability of APAP-induced HepG2 cells. It has been well-documented that hepatocyte apoptosis coexisted with necrosis was critically important to APAP overdose induced hepatotoxicity, due to perpetuating liver damage [30, 31]. Conventionally, apoptosis is initiated by mitochondrial dysfunction and regulated by Bax (a proapoptotic protein) and Bcl-2 (an anti-apoptotic protein) [32]. In present study, results from the TUNEL and western blotting analysis showed that CGA significantly decreased the amount of apoptotic cells and reversed the elevated Bax level and decreased Bcl-2 level, clearly indicating that CGA exhibited anti-apoptosis effects on APAP hepatotoxicity.

Recent evidence suggests that hepatocyte apoptosis overlaps with autophagy in liver injury [33]. Mitochondrial dysfunction leads to insufficient ATP production, increases mitochondrial ROS, excess cytochrome c (Cyt-c) release, upregulation of Bax, caspase-3 and caspase-9, and further initiates the apoptosis pathway [34]. Moreover, hepatocyte apoptosis is regulated by autophagic activity. As expected, we found APAP overdose dramatically inhibited shifts of LC3 protein from cytoplasmic form (LC3Ⅰ) to autophagosome form (LC3Ⅱ), and markedly increased p62 and Tom20 level, while CGA treatment reversed the detrimental effects induced by APAP overdose, and the expression of LC3Ⅱ on mitochondria increased significantly. Interestingly, the current findings showed that mitophagy played a critical role in removing damaged mitochondria in APAP hepatotoxicity [35], especially PINK1-mediated mitophagy [36]. CGA significantly activated Mitophagy in APAP-induced mice, manifested by elevated levels of PINK1 and Parkin. In vitro, CGA increased the expression of PINK1, Parkin and LC3B in HepG2 cells, whereas PINK1-specific knockdown attenuated the protective effect of CGA. In conclusion, this study suggests that CGA may attenuate APAP-induced liver injury by promoting mitophagy through activating PINK1/Parkin pathway.

Author contributions Bang-yan Hu and Jin Li performed the research; Bang-yan Hu wrote the original manuscript; Dao-yin Gong completed histopathology researches; Yan Dai analyzed the data; Shi-jun Xu conceived, designed the study and critically reviewed and revised the paper; Li-hong Wan designed the study and critically revised the paper.

Funding This study was supported by Applied basic Research project of Sichuan Province (No.2019YFS0173, No.2021YJ0009), Chengdu Technological Innovation R & D Project (No.2021-YF05-01985-SN) and Xinglin Scholar Research Promotion Project of Chengdu University of TCM (No. CXTD2018013).

Conflict of interest All authors have no conflict of interest to disclose.

Ethical approval The study was approved by the Ethics Committee for Animal Experiments of the Institute of Material Medica Integration and Transformation for Brain Disorders in Chengdu University of Traditional Chinese Medicine.

Consent to participate Not applicable.

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Protective effects of Chlorogenic Acid on acetaminophen-induced liver injury through regulating PINK1/Parkin pathway promoted mitophagy

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Drugs and reagents

2.2 Animals and protocols

2.4 Cell culture and cell viability assay

2.5 Mortality in mice assay

2.6 Biochemical analysis for serum

2.7 Histopathology of liver tissue and TUNEL assay

2.8. PINK1 siRNA transfection

2.9 RT-qPCR analysis

2.10 Protein preparation and western blotting analysis

3. Results

3.1 Chlorogenic acid alleviated APAP-induced acute hepatotoxicity in mice

3.2 Chlorogenic acid suppressed APAP-induced apoptosis of hepatocyte in mice

3.3 CGA promoted PINK1-dependent mitophagy in APAP treated mice

3.4 PINK1 deficiency reversed the protective effect of CGA

4. Discussion

Declarations

References

Status:

Version 1