Inhibition of excessive mitophagy by N-acetyl-L-tryptophan confers hepatoprotection against Ischemia-Reperfusion injury in rats

Chemicals

L-NAT, Beclin1, microtubule- associated protein 1 light chain 3-II (LC3-II), autophagy related protein 7 (ATG-7), SQSTM1 (P62) antibodies and 3-Methyladenine (3-MA) were purchased from Sigma-Aldrich (St.Louis, MO, USA) and GAPDH antibody was obtained from Proteintech Group (Chicago, USA). Secondary anti-rabbit antibody was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). The enhanced chemiluminescence (ECL) system was obtained from Amersham Pharmacia Biotech (Piscataway, NJ). RIPA lysis was purchased from Solarbio (Beijing, China). The cell counting kit-8 (CCK-8) was purchased from 7sea-Biotech (Shanghai, China). ATP assay kit was purchased from Beyotime Biotechnology (Shanghai, China) and MitoTracker Red kit came from Yeasen Biotech (Shanghai, China) and DAPI came from Life Technologies.

Animals

Healthy male Sprague-Dawley (SD) rats weighing 200-220 g were purchased from Pengyue experimental animal center in Jinan, China (Weifang Medical University Medical Ethics Committee provided full approval for this research (No. 2017253)). They were randomly divided into sham group, I/R group, and I/R + L-NAT group, with 6 rats in each group. Rats were fasted for 12 h before surgery, and were given free access to water. In I/R + L-NAT group, L-NAT (10 mg/kg) was intraperitoneal injected 30 min before modeling (Wang et al., 2019). The rats were anesthetized by intraperitoneal injection of ketamine, the left and middle branches of the hepatic pedicle were occluded with a non-traumatic vascular clamp in I/R + L-NAT group and I/R group. The success of the model was apparent once the liver color turned from red to dark purple. After 45 min of ischemia, the clip was removed to allow hepatic reperfusion. In sham group, the rats underwent the same surgery but no vessel clamps were placed. According to the previous literature of our laboratory, the most obvious liver function damage was after 6 h of reperfusion, so we took the liver tissue after 6 h of reperfusion. The Animal Ethics Committee of the University approved all working protocols.

Cell culture and treatment

The rat hepatocyte BRL cell line was purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). BRL cells were cultured in a 37 °C incubator which is a humidified environment of 95% air/5% CO₂. The cells were divided into three groups: control group, H₂O₂ group and H₂O₂+ L-NAT group. Referring to the previous literature in our laboratory, oxidative damage model of BRL cells were prepared by pre-treatment with 200 µM H₂O₂ for 6 h. And H₂O₂+ L-NAT group was pre-treated with 10 µM L-NAT for 2 h before adding H₂O₂ (Wang et al., 2019).

Cell viability assay

Cell viability was determined using CCK-8 colorimetric kit. BRL cells were adjusted as 2 × 10⁵ cells/mL, then were inoculated in 96-well plate with 100 µL/well and were cultured overnight, when a partial monolayer was formed. Then, cells were exposed to 200 µM H₂O₂ with or without L-NAT for 6 h. Subsequently, cells were cultured in a solution of 100 µL CCK-8/mL DMEM for 1 h in a CO₂ incubator in darkness. The absorbance of the sample was measured at 450 nm using a microplate reader (Thermo, Massachusetts, USA). The experiments were performed in 5 replicate holes and repeated three times.

Enhanced ATP assay

The level of intracellular ATP was determined according to the instructions using an ATP assay kit. A same amount of BRL cells in 6-well plates were lysed and centrifuged at 12,000 g for 5 min at 4 °C. Subsequently, 20 µL of the supernatant was mixed with 100 µL of luciferase reagent in an opaque 96-well plate and was measured using the chemiluminometer.

Transmission election microscopy (TEM)

The perfused liver tissue was fixed in PBS (pH = 7.4) containing 2.5% glutaraldehyde for at least 2 h, which was then fixed in 1% osmium tetroxide for 1 h at 4 °C temperature. Tissues were embedded in an epoxy resin and made into 100 nm ultrathin slices. The ultrastructure of the sample was observed using TEM (JEM. 1010, JEOL, Tokyo, Japan).

Immunofluorescence technique

Liver tissue was perfused and fixed, and 10-µm-thick frozen sections were prepared, fixed in 4% paraformaldehyde for 15 min, and washed with PBS. The endogenous peroxidase was inactivated by blocking with 1% goat serum for 30 min at room temperature and then incubated overnight at 4 °C with diluted primary antibody (LC3-II, 1:200, Sigma; Beclin1, 1:200, Sigma; ATG-7, 1:200, Sigma; P62, 1:200, Sigma). The following day, it was washed three times with PBS, incubated with the secondary antibody for 60 min at room temperature, washed three times with PBS, and nucleus were stained by DAPI (1 mg/mL), then observed under the fluorescence microscope (Olympus, Japan).

MitoTracker staining mitochondria

After seeding cells on coverslips and treating with the drug, according to the instructions of MitoTracker Red CMXRos kit (Yeasen, Shanghai, China), 200 nM H-DMEM medium was added onto each slide, and then incubated in 37 °C incubator for 30 min. Cells were washed with PBS, and fixed with paraformaldehyde and permeabilized with cold acetone, then observed cells under the fluorescence microscope (Olympus, Japan).

Rhodamine 123 staining

BRL cells were cultured on slide and pretreated with 10 µM L-NAT for 2 h and then treated with 200 µM H₂O₂ for 6 h. Mitochondrial membrane potential was determined using rhodamine 123, a kind of lipophilic cationic fluorescent dye that can pass through the cell membrane of live cells for detecting mitochondrial membrane potential (Jiang et al., 2014). Briefly, the processed cells were directly incubated with 2 µM rhodamine 123 for 40 min in 37 °C CO₂ incubator in dark, followed by rinsing with PBS, and stained nucleus with DAPI (1 mg/mL). Images were taken using a fluorescence microscope.

Flow cytometry

BRL cells were adjusted to 2 × 10⁵ cells/mL, then were inoculated in 6-well plate with 1 mL/well and cultured overnight. After treatment with L-NAT and H₂O₂, then cells were collected into centrifuge tubes and centrifuged at 2,000 rpm for 5 min, the supernatant was discarded and PBS was added. Rhodamine 123 was added to the suspension at a concentration of 2 µg/mL, incubated at 37 °C for 40 min, and then centrifuged at 2,000 rpm for 5 min. Cells were washed twice with PBS, and detected by flow cytometry. The excitation wavelength was 507 nm, and the emission wavelength was 530 nm.

Quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from the treated liver tissue and BRL cells according to the instructions of TRIzol agentia (Life, USA), to reverse transcripted in cDNA and PCR amplification was performed with 20 µL reaction system (Wang et al., 2019).

Western blotting

Tissue or cells were exposed to the mixture of RIPA lysis buffer and phenylmethylsulfonyl (PMSF) fluoride, lysed on ice, and centrifuged at 12,000 g for 15 min at 4 °C to extract total protein and protein concentration was determined by BCA method. Proteins were separated by electrophoresis and then transferred onto a PVDF membrane by electro-blotting. Samples were incubated overnight at 4 °C with antibodies such as LC3-II (1:500), Beclin1 (1:500), ATG-7 (1:200), P62 (1:500) and GAPDH (1:1000). The images were analyzed by ImageJ software. The results were normalized to the ratio of target protein optical density values to those of the internal reference protein GAPDH.

Statistical analysis

All experiments were repeated three times and data were presented as the mean ± SD values. Statistical analyses of the data were performed using a one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) test with Statistical Package for the Social Sciences (SPSS, version 22.0) software, p < 0.05 were considered statistically significant.

Effect of L-NAT on autophagy after hepatic ischemia-reperfusion injury

Given the evidence that excessive autophagy related to hepatocyte injury during HIRI, and the modulation of autophagy may render hepatocyte resistant to I/R injury. Subsequently, the change of autophagy activation was investigated. The times sequential changes of Beclin1 was investigated by qRT-PCR and Western blotting during 0 h, 1 h, 4 h, 6 h, 12 h and 24 h of reperfusion, as shown in Figs. 1A–1C, both the mRNA and protein expression of Beclin1 peaked at 6 h after reperfusion. Accordingly, rats were subjected to 45 min of hepatic ischemia followed by reperfusion 6 h for subsequent experiments.

To understand whether L-NAT could reduce autophagy activation, we next investigated the expression of autophagy markers (LC3-II, Beclin1, ATG-7 and P62) after HIRI present with or without L-NAT using the Rat HIRI model and H₂O₂-induced oxidative damage. Results of qRT-PCR and Western blotting showed that the expression levels of LC3-II, Beclin1, and ATG-7 in I/R group showed an increase than those in sham group, while the P62 level dropped to 0.6-fold, and L-NAT administration could reverse those changes of autophagy markers (Figs. 2A–2I). Immunofluorescence assays showed that LC3-II, Beclin1, and ATG-7 staining (green fluorescence) increased, P62 staining (green fluorescence) significantly decreased in I/R group compared with the sham group (Fig. 2J). As shown in Figs. 3A–3J, results in vitro are consistent with that in vivo. These data indicated that L-NAT could regulate the expression of autophagic protein during HIRI injury.

To confirm the protective effect of L-NAT on HIRI-induced autophagy, the autophagosome in hepatocyte were observed by EM. Results showed that most of autophagosomes in I/R group, showed monolayer membrance, wrapped the degraded mitochondria and other cytosolic component. Meanwhile, in I/R group, autophagosome volume is increased approximately 1.5–2 times larger than that in sham group. And all those morphological changes of autophagosomes were reversed by L-NAT pretreated (Fig. 4). Those data demonstrated that L-NAT could decrease the formation of autophagosome.

Effects of L-NAT on mitochondrial structure

A large body of evidence indicates that mitochondrial dysfunction plays a key role in I/R injury in heart, brain, liver, kidney and other organs. To investigate whether the effect of L-NAT on I/R is related to mitochondrial protection, we investigated the effect of L-NAT on mitochondrial morphology. EM analysis showed that mitochondria of hepatocyte in sham group were evenly distributed in the cytoplasm, with well-arranged cristae structure, while the number of mitochondria in I/R group increased and was centralized distribution around the nucleus, the mitochondrial cristaes were disordered, while the damaged mitochondria were phagocytosed by the vacuolar structure. L-NAT pretreatment improved morphological changes of mitochondria induced by I/R injury (Fig. 5).

Effects of L-NAT on mitochondrial function

ATP, produced by mitochondria, is the main form of intracellular energy supply. To investigate whether L-NAT improves mitochondrial function changes caused by HIRI, we measured the ATP content in H₂O₂-induced oxidative damage model with or without L-NAT. Results showed that compared with control group, H₂O₂-mediated BRL cells caused mitochondrial damage, resulting in a ATP decrease of 0.35-fold, which was reversed to 0.8-fold by L-NAT pretreatment. Those changes of ATP indicated that L-NAT can rescue mitochondrial function (Fig. 6A).

The above results showed that L-NAT can protect mitochondrial structure and ATP which is caused by I/R-induced, thereby alleviating mitochondrial dysfunction. A large body of evidence indicated that thirteen kinds of proteins encoded by mitochondrial DNA (mtDNA) are closely related to mitochondrial function, and maintaining stability of the quantity and quality of mtDNA are critical for the maintenance of mitochondrial function. In order to clarify the effect of L-NAT on mtDNA changes after I/R, we designed primers for the coding genes ND1 and COX-1 of mtDNA heavy and light chains to detect copy number of mtDNA. Additionally mtAtp6 is used to represent mtDNA (Codogno & Meijer, 2013), and expression of Rp113 is used to represent cellular total DNA, therefore, the ratio of them represents the content of mitochondria in each cell. Subsequently, qRT-PCR was used to detect the expression of the ND1, COX-1, mtAtp6 and Rp113 in I/R-mediated oxidative stress injury model. We found that the radio of mtAtp6/Rp113 was increased, which is 1.42-fold higher than that in sham group, and the expression of ND1 and COX-1 was attenuated to 0.67-fold and 0.74-fold in I/R group compared with sham group, respectively. The L-NAT pre-treatment group significantly improved these changes (Figs. 6B–6D). Taken together, the experimental results provided strong evidence that maintaining the quantity and quality of mtDNA stability might be closely related to the protective effect of L-NAT.

As an extension of the above results, the mitochondrial transmembrane potential (ΔΨ_m) assay was used as a specific test for the earliest events of mitochondrial injury. The results showed that rhodamine 123 fluorescence in control group cells showed a punctate distribution, which has an appearance of a grainy rhodamine 123 fluorescence in cytoplasm. Upon H₂O₂ exposure, the fluorescent signal from rhodamine 123 becomes diffuses, which was considered a diffused pattern. L-NAT pretreatment reversed the H₂O₂-induced dissipation of rhodamine123 fluorescence, indicating that ΔΨ_m was maintained (Figs. 6E–6F).

In addition to the rhodamine 123 staining pattern, the fluorescence intensity was also investigated. As shown in Fig. 6G, the fluorescence intensity of rhodamine 123 in H₂O₂ group decreased significantly compared to the untreated group, which implied the loss of ΔΨ_m, and L-NAT administration significantly inhibited the decreased fluorescence intensity induced by H₂O₂. Those data provide evidence that L-NAT pretreatment could prevent the dissipation of ΔΨ_m induced by H₂O₂.

Effect of L-NAT on mitophagy

To further explore whether the protective effect of L-NAT on hepatocytes was achieved by acting on mitophagy, we first measured the level of Beclin1 in H₂O₂-mediate BRL cells by immunofluorescence staining, then labeled mitochondrial by MitoTracker to observe the changes of mitophagy in H₂O₂-mediated BRL oxidative damage model. The data indicated that the number of co-localization of Beclin1 and MitoTracker were significantly increased after H₂O₂ induction. L-NAT effectively reduces H₂O₂-induced the number of Beclin1 and MitoTracker double positive cells induced by H₂O₂ (Fig. 7). This observation suggested that L-NAT could decreased the co-location of autophagy protein and mitochondrial.

The autophagy inhibitor on cytoprotecyive effect of L-NAT

The above experiments have clarified that L-NAT can alleviate HIRI by inhibiting mitophagy. Subsequently, we investigated the effect of 3-MA, an autophagy inhibitor, on the hepatoprotective of L-NAT. According to preliminary experiment, the cell viability reached the highest point after 5 mM 3-MA pretreatment, and 5 mM 3-MA was subjected for subsequent experiments. As showed in Fig. 8A, L-NAT administration could elevate the decline of cell viability which was induced by H₂O₂ and 3-MA pretreatment could reverse part of those changes. Interestingly, the results of Western blotting and qRT-PCR revealed that the combination of 3-MA elevated the Beclin1 level of H₂O₂-induced BRL cells, compared with the usage of L-NAT solely (Figs. 8B–8D), implied that the autophagy inhibitor 3-MA partially weaken the protective effect of L-NAT on HIRI. Taken together, these results futher identified that the hepatoprotective effect of L-NAT depended on mitophagy process.