Chlorogenic Acid Alleviated Liver Dysfunction Through Activating PINK1/Parkin Dependent Mitophagy

Background: Chlorogenic acid (CGA) is a natural polyphenolic compound with anti-inammatory, antioxidant effects. It could improve mitochondrial dysfunction that was a key feature of acetaminophen (APAP) -induced liver injury. This study aimed to identify whether promoting mitophagy was associated with the hepatocyte protection for CGA. Methods: Acute hepatic injury model was induced by APAP in mice after CGA administration for 14 days. Survival rate was recoded within 24h of modeling. Serum aminotransferase, hepatic histopathology and TUNEL assays were simultaneously performed. The expression of apoptosis-related proteins (Bax and Bcl-2) and mitophagy-related genes and proteins (LC3(cid:0), P62, PINK1 and Parkin) were analyzed. The uorescence co-localization of LC3(cid:0) and Tom20 was analyzed with immunouorescence. Results: Compared with APAP group, CGA pretreatment signicantly increased survival rate of APAP-induced mice, inhibited the activity of ALT, AST and LDH in serum, and alleviated pathological features of liver such as inammatory cell inltration, necrosis of liver cells and vacuolation (p<0.05). Moreover, our data from the TUNEL and western blotting analysis showed that CGA signicantly decreased the number of apoptotic cells and reversed the elevated Bax level and decreased Bcl-2 level(p<0.05). Furthermore, we found that CGA promoted the uorescence co-localization of LC3(cid:0) and Tom20 and enhanced the protein expression of LC3(cid:0) (p<0.05). Finally, CGA signicantly promoted mitophagy by exhibiting the increased gene and protein expression of PINK1 and Parkin. Conclusions: Our results demonstrated that CGA promoted PINK1/Parkin dependent mitophagy and inhibited hepatic apoptosis to exert protection against liver damage in APAP-induced mice. NAPQI, N-acetyl-p-benzoquinone imine; ROS, reactive oxygen species; GSH, glutathione; APAP, acetaminophen; CGA, Chlorogenic acid; AG, Ammonium glycyrrhizinate; HE, hematoxylin and eosin; ALT, alanine transaminase; AST, aspartate transaminase; LDH, lactate dehydrogenase; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling; Cyt-c, cytochrome c.


Introduction
Acetaminophen (APAP) is the most commonly used antipyretic and analgesic. APAP could cause severe liver, kidney and heart injury or organ failure at a single overdose exceeding 15 to 25 g, although it is generally safe used at therapeutic dose (<4 g every 24 h) [1][2][3]. Acute liver injury is the most common side effect of acetaminophen overdose because liver is one of the most sensitive organ to acetaminophen. Recent studies have suggested multiple molecular mechanisms that contribute to APAP hepatotoxicity, including oxidative stress, sterile in ammation, mitochondrial dysfunction, especially impaired mitophagy [4] and apoptosis [5]. 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) in the liver and leads to mitochondrial dysfunction and subsequent oxidative stress [6]. Experiments have shown that autophagy can selectively remove NAPQI and reduce liver damage. On the contrary, autophagy inhibitor Chloroquine can aggravate APAP-induced liver damages [7,8].
Mitochondria are highly dynamic organelles that govern redox homeostasis, innate immunity and apoptotic cell death [9].In liver cells, mitochondria are the starting point for in ammatory signals and oxidative stress responses [10]. Mitochondrial reactive oxygen species (ROS) is one of the major sources of cellular ROS, which can damage lipids, proteins and DNA, leading to mitochondrial dysfunction [11,12]. It is now generally accepted that autophagy can clear the affected damaged mitochondria, reduce the production of ROS and eliminate in ammasomes (such as NLRP3 in ammasomes) [13]. At present, a large number of studies have shown that APAP can induce hepatocyte apoptosis [14][15][16][17]. When mitophagy is damaged, the impaired Mitochondria cannot be removed and accumulate in large quantities, which lead to a large number of hepatocytes apoptosis to aggravate the degree of liver injury.
Autophagy is a lysosomal degradation process responsible for degrading dysfunctional proteins and organelles [18]. Mitophagy, a selective autophagy of mitochondria, plays an important protective role in preventing DILI by timely removing damaged mitochondria [19]. Generally, mitophagy involves mitochondrial ssion, autophagosome formation, and fusion with lysosome in turn [20]. These processes are regulated by phosphatase and tensin homolog (PTEN)-induced kinase 1 (Pink1)/Parkin pathway.
Brie y, 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 [21]. Importantly, suppression of Pink1/Parkin augments acetaminophen hepatotoxicity via impairment of hepatic mitophagy, which indicates that Pink1/Parkin-mediated mitophagy plays a key role in alleviating acetaminophen hepatotoxicity [22,23]. Therefore, it is critically important to prevent acetaminophen hepatotoxicity through precise regulation of Pink1/Parkin pathway. Intriguingly, a recent study found that knockdown of Parkin augmented endplate chondrocyte apoptosis in H 2 O 2 -induced mitochondrial dysfunction cell model, which implied that Parkin-mediated mitophagy might involve in regulating apoptosis [24]. However, targeting mitophagy-mediated apoptosis for relieving DILI remains largely undetermined.
Chlorogenic acid (CGA, C 16 H 18 O 9 ) is a dietary polyphenol derived from coffee, apple, blueberry, tea and some natural medicines with multiple biological activities. Growing evidence has proved the protective effect of CGA on acetaminophen hepatotoxicity which involves multiple mechanisms including antioxidation, anti-in ammatory and anti-apoptosis [25][26][27][28]. More speci cally, they are concerned with attenuating liver mitochondrial injury and reducing mitochondrial HSP60 release, activating Nrf2 antioxidative signaling pathway, inhibiting MAPK signaling pathway, inhibiting CYP2E1 and CYP1A2 enzymatic properties and suppressing TLR3/4 and NF kappa B signaling pathway [25][26][27][28][29][30][31]. It's well known that mitophagy defect can perturb mitochondrial function and cause progressive accumulation of reactive oxide species (ROS) and the inability to supply energy, leading to oxidative stress, sterile in ammation and cell apoptosis, reinforcing a vicious cycle. However, whether promoting mitophagy was associated with the hepatocyte protection for CGA remains unclear. In this study, we determined the role of CGA on preventing acetaminophen 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/Parkin-depended mitophagy and inhibiting hepatic apoptosis.

APAP-induced acute hepatotoxicity mice model and treatment
All mice were randomly divided into ve groups (n=20), the control (Ctrl) group, the APAP group (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). All the material under study is endotoxin free. 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 [32]. Then, all mice were sacri ced on day 16 and serum and liver tissues were collected.

Mortality in mice assay
After APAP (300mg/kg) treatment, the death period of mice was recorded within 24 h.

Biochemical analysis for blood
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).

Histopathology of liver tissue and TUNEL assay
Fresh liver tissues were immediately xed in 4% paraformaldehyde for 24 h, embedded in para n, and then cut into 4-μm-thick sections (n=5). The tissues were stained with hematoxylin and eosin (HE) for histological examination under a light microscopy. The histological scores (including in ammation, necrosis and apoptosis) indicating the degree of liver injury were determined according to Suzike's standard [33]. Simultaneously, these slides were subjected to terminal deoxynucleotidyl transferasemediated dUTP-biotin nick end labeling (TUNEL) for analyzing hepatocytes apoptosis according to the manufacturer's instructions of a TUNEL apoptotic detection kit. Images were captured by a uorescence microscopy (Leica Microsystems Wetzlar German), and calculated semi-quantitatively at magni cation ×200 to count the positive cells.

Immunohistochemistry analysis
The liver tissue was cut into 4-μm-thick sections then depara nization with xylene and gradient ethanol, antigen retrieval with trisodium citrate dihydrate for 30 minutes and blocked with 1% BSA at room temperature for 30 min. the sections were incubated with primary anti-Tom20 (1:100) and anti-LC3 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-uorescence quenching sealer and observed by confocal microscopy (Olympus, Japan).

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 signi cant.

Chlorogenic acid alleviated APAP-induced acute hepatotoxicity in mice
To con rm 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) signi cantly increased survival rates compared with APAP alone (p 0.05). Second, the effects of CGA on the degree of liver function injury was assessed after an APAP overdose of 300 mg/kg administration. Our data showed that the serum levels of ALT, AST and LDH in the APAP group were markedly increased, and were signi cantly 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. Moreover, the histopathologic analysis was performed on HE stained hepatic sections. As shown in Fig.1E, APAP administration signi cantly induced severe hepatocellular injury compared with the control mice, such as loss of hepatocyte architecture, vacuolization of hepatocytes, massive necrosis and mononuclear cell in ltration in the portal area. Conversely, treatment with CGA or AG signi cantly ameliorated the hepatocellular injury around in the portal area. Compared with the control group, necrosis scores and in ammation scores were increased signi cantly after APAP administration (p 0.05), while treatment of AG and CGA (40 mg/kg) signi cantly 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.

Chlorogenic acid suppressed APAP-induced apoptosis of hepatocyte in mice
To further elucidate 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, signi cantly increased apoptotic cells could be observed in the APAP group, which was signi cantly 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 signi cantly 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 signi cantly 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.

Chlorogenic acid increased autophagy in APAP treated mice
Previous studies have demonstrated that autophagy and apoptosis exist in overlap with many pathophysiology conditions [34]. Enhancing autophagy can prevent the progression of acute liver injury [35]. To veri ed the effects of CGA on autophagy, immuno uorescence co-localization analysis was rstly used to examine the colocalization of Tom20 (a speci c mitochondria marker) and LC3 (autophagy marker). Our data showed that pretreatment of CGA signi cantly promoted the colocalization of Tom20 and LC3 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 signi cantly elevated expression of Tom20 compared to the control mice, which was signi cantly reversed after treatment with CGA(p 0.05). Moreover, we tested the expression of autophagy-related genes and proteins (LC3 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 was signi cantly 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 ux in APAP-induced liver injury mice.

Chlorogenic acid promoted mitophagy via PINK1/Parkin pathway
As a type of mitochondria-specialized autophagy, PINK1-dependent mitophagy was recently recognized as a novel target for treating alcoholic liver disease and acetaminophen hepatotoxicity, due to its maintaining role in mitochondrial homeostasis [21]. Therefore, we further determine whether CGA promoted PINK1-dependent mitophagy. qRT-PCR 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 or p 0.01, Fig.4A, B and C). Interestingly, pretreatment with CGA at the dose of 40 mg/kg signi cantly increased the levels of PINK1 and Parkin (p 0.05, Fig.4A, B and C), suggesting that CGA alleviated APAP-induced hepatotoxicity via activating PINK1/Parkin-dependent mitophagy.

Discussion
APAP is a commonly used antipyretic and analgesic, which induces acute liver injury at excessive doses, APAP (0.15-3.0 g/kg, po) can cause dose-dependent death in mice [36]. In this study, mice were given 300mg/kg APAP treatment to induce liver injury. Our present study demonstrates that pretreatment with CGA ( ) promotes PINK1-dependent mitophagy, ( ) inhibits hepatocyte apoptosis through regulating Bcl-2 family proteins under APAP overdose conditions, and ( ) reduces mortality in mice and alleviates APAP-induced acute hepatotoxicity. Collectively, our results were the rst to reveal that CGA preserved hepatocyte survival in response to APAP overdose via activating PINK/Parkin mitophagy inhibited hepatocyte apoptosis, indicating CGA might be a promising therapeutic natural polyphenol for the detoxi cation of APAP-induced liver injury. Graphical summary of the results is schematized in Fig. 5.
It has been well-documented that hepatocyte apoptosis coexisted with necrosis was critically important to acetaminophen overdose induced hepatotoxicity, due to perpetuating liver damage [15,37]. Conventionally, apoptosis is initiated by mitochondrial dysfunction and regulated by Bax (a proapoptotic protein) and Bcl-2 (an anti-apoptotic protein) [38]. In present study, results from the TUNEL and western blotting analysis showed that CGA signi cantly 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.
Natural product 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 [25,26,31]. Existing studies have shown that CGA has anti-in ammatory and anti-oxidative stress effects in liver injury. Mitochondrial dysfunction, in ammation and oxidative stress are interdependent processes. Mitophagy helps regulate mitochondrial homeostasis by removing dysfunctional mitochondria. Inhibiting mitochondrial autophagy can lead to the accumulation of damaged mitochondria and sometimes the activation of in ammasomes.
Recent evidence suggests that hepatocyte apoptosis overlaps with autophagy in liver injury [39]. Mitochondrial dysfunction leads to insu cient 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 [40]. Moreover, hepatocyte apoptosis is regulated by autophagic activity. As expectated, we found APAP overdose dramatically inhibited shifts of LC3 protein from cytoplasmic form (LC3 ) to autophagosome form (LC3 ), and markedly increased p62 and Tom20 level, while CGA treatment reversed the detrimental effects induced by APAP overdose, and the expression of LC3 on mitochondria increased signi cantly. Interestingly, the current ndings showed that mitophagy played a critical role in removing damaged mitochondria in APAP hepatotoxicity [19], especially parkin-mediated mitophagy [24]. Therefore, it can be speculated that targeting mitophagy-mediated apoptosis is bene cial of relieving acetaminophen hepatotoxicity. In the present study, an obvious inhibition by PINK1 and Parkin expressions were found in APAP mice, which is consistent with previous reports that suppression of Pink1/Parkin augments acetaminophen hepatotoxicity via impairment of hepatic mitophagy [22,23].
Conversely, CGA signi cantly activated mitophagy, which was manifested by the increase of PINK1 and Parkin levels. Given all this, these ndings provide evidences for the hepatoprotection effects of CGA in activating Pink1/Parkin-mediated mitophagy and inhibiting hepatic apoptosis. In future follow-up studies, we will focus on vitro studies, by administering autophagy inhibitors, and use siRNA to interfere with PINK1 and Parkin, in order to deepen the research on the mechanism of CGA to prevent drug-induced liver injury.
Abbreviations NAPQI, N-acetyl-p-benzoquinone imine; ROS, reactive oxygen species; GSH, glutathione; APAP, acetaminophen; CGA, Chlorogenic acid; AG, Ammonium glycyrrhizinate; HE, hematoxylin and eosin; ALT, alanine transaminase; AST, aspartate transaminase; LDH, lactate dehydrogenase; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling; Cyt-c, cytochrome c. 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 the study and critically reviewed and revised the paper; Li-hong Wan designed the study and critically edited and revised the paper.

Declarations
Con ict of interest All authors have no con ict of interest to disclose.