Taurine attenuates valproic acid‑induced hepatotoxicity via modulation of RIPK1/RIPK3/MLKL‑mediated necroptosis signaling in mice

Mohammad Javad Khodayar1,2 · Heibatullah Kalantari2,3 · Layasadat Khorsandi4,5 · Nematollah Ahangar6 · Azin Samimi7 · Hadis Alidadi2,3

Received: 11 January 2021 / Accepted: 20 May 2021
© The Author(s), under exclusive licence to Springer Nature B.V. 2021
Hadis Alidadi [email protected]
1 Toxicology Research Center, Medical Basic Sciences Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
2 Department of Toxicology, School of Pharmacy, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
3 Medicinal Plant Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
4 Department of Anatomical Sciences, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
5 Cellular and Molecular Research Center, Medical Basic Sciences Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
6 Department of Pharmacology, School of Medicine, Guilan University of Medical Sciences, Rasht, Iran
7 Legal Medicine Research Center, Legal Medicine Organization, Legal Medicine Office of Khuzestan, Ahvaz, Iran

Abstract

Valproic acid (VPA) is known as a common drug in seizure and bipolar disorders treatment. Hepatotoxicity is the most important complication of VPA. Taurine (Tau), an amino acid, has antioxidant effects. The present research was conducted to evaluate the protective mechanisms of Tau on VPA-induced liver injury, especially focusing on the necroptosis signal- ing pathway. The sixty-four male NMRI mice were divided into eight groups with eight animals per each. The experiment groups pretreated with Tau (250, 500, 1000 mg/kg) and necrostatine-1 (Nec-1, 1.8 mg/kg) and then VPA (500 mg/kg) was administered for 14 consecutive days. The extent of VPA-induced hepatotoxicity was confirmed by elevated ALP (alkaline phosphatase), AST (aspartate aminotransferase), ALT (alanine aminotransferase) levels, and histological changes as steatosis, accumulation of erythrocytes, and inflammation. Additionally, VPA significantly induced oxidative stress in the hepatic tissue by increasing ROS (reactive oxygen species) production and lipid peroxidation level along with decreasing GSH (glutathione). Hepatic TNF-α (tumor necrosis factor) level, mRNA and protein expression of RIPK1 (receptor-interacting protein kinase 1), RIPK3, and MLKL (mixed lineage kinase domain-like pseudokinase) were upregulated. Also, the phosphorylation of MLKL and RIPK3 increased in the VPA group. Tau could effectively reverse these events. Our data suggest which necroptosis has a key role in the toxicity of VPA through TNF-α–mediated RIPK1/RIPK3/MLKL signaling and oxidative stress. Our find- ings suggest that Tau protects the liver tissue against VPA toxicity via inhibiting necroptosis signaling pathway mediated by RIPK1/RIPK3/MLKL and suppressing oxidative stress, and apoptosis.

Keywords Valproic acid · Oxidative stress · Inflammation · Necroptosis · Taurine

Abbreviations
VPA Valproic acid
TNF-α Tumor necrosis factor-alpha
MLKL Mixed lineage kinase domain-like
pseudokinase
ROS Reactive oxygen species
RIPK1 Receptor-interacting protein kinase 1 TNFR1 TNF receptor 1
Tau Taurine
Nec-1 Necrostatin-1
GAPDH Glyceraldehyde 3-phosphate dehydrogenase
AST Aspartate aminotransferase
ALP Alkaline phosphatase
ALT Alanine aminotransferase
MDA Malondialdehyde
GSH Glutathione
DCFH-DA Dichlorodihydrofluorescein diacetate
DCF Dichlorofluorescein
H&E Hematoxylin and eosin FADD
Fas-associated death domain

Introduction

Valproic acid (VPA) is an efficient anti-convulsant drug over the past 50 years. It is also commonly administered in various neurological and bipolar disorders [1, 2]. Although VPA is well tolerated by patients, its prolonged use causes serious life-threatening complications, such as hepatotox- icity, especially in children less than ten years old [3–5]. It has been revealed that excessive production of reactive oxygen species (ROS) possesses a key role in VPA toxicity through antioxidants depletion and damage to hepatocytes [6, 7]. Besides, it has been demonstrated that liver injury induced by VPA leads to increase of inflammatory media- tors, including TNF-α (tumor necrosis factor- α), which can cause cell death [8]. Different researches have shown which hepatocellular damages of VPA are caused by the induction of necrosis [6].
Necroptosis, a mode of regulated cell death, is regulated by activating receptor-interacting protein kinase 1 (RIPK1), RIPK3 and mixed lineage kinase domain-like pseudokinase (MLKL) [9, 10]. Necroptosis signaling can be promoted by stimulating cytokines, chemicals and activating death recep- tors such as TNF-R1 [11].
Taurine (Tau), a sulfur-containing amino acid, is found in the human dairy diet [12]. Tau is synthesized via cysteine and methionine metabolism in hepatic cells and released to the bloodstream [13]. Despite its non-protein structure, Tau possesses important physiological and pharmacological functions such as cell membrane stabilizer, osmoregulator, calcium homeostasis, and detoxification [14]. Numerous researches have that indicated the antioxidant properties of Tau improve cellular defense mechanisms by reducing oxi- dative stress status [15, 16]. Therefore, Tau can be a poten- tial therapeutic agent against xenobiotics-induced hepato- toxicity. However, the protective mechanisms of Tau on VPA-intoxication have not been examined. Accordingly, we investigated the protective effect of Tau on oxidative stress, apoptosis, and RIPK1/RIPK3/MLKL-mediated necroptosis signaling in VPA-intoxicated mice. To assess the role of necroptosis in VPA-induced toxicity, we used necrostatin-1 (Nec-1) as a selective blocker of necroptosis.

Materials and methods

Chemicals
Nec-1 (Cat No: N9037) and Tau (Cat No: T0625) were bought from Sigma Co. (St Louise, USA). VPA (Cat No: 98SV039) was obtained from Rouz Darou Co. (Tehran, Iran). TNF-α assay kit (Cat No: E0117Mo) was purchased from BT Lab (Shanghai, China). Primary antibodies against RIPK3 (Cat No: ab62344), MLKL (Cat No: ab255747) and
GAPDH (Cat No: ab9485), and secondary antibodies: rabbit anti-rat IgG (ab6734) and goat anti-rabbit IgG (ab150077) were provided from Abcam (Cambridge, UK). RNeasy Plus Mini and cDNA synthesis kits were obtained from Qiagen, Inc. Protease inhibitor cocktail (Cat. No.11836153001) was purchased from Roche, Germany.

Animals
Sixty-four male NMRI mice (28–35 g) were provided from the Animal Center of Jundishapur University of Medical Sciences (Ahvaz, Iran) and were housed in controlled condi- tions at room temperature for one week. They had access to adequate water and food at a light/dark cycle for 12-h. All experiments were approved by the Animal Research Eth- ics Committee of Ahvaz Jundishapur University of Medi- cal Science (IR.AJUMS.ABHC.REC.1397.065) and were conducted in agreement with NIH guidelines.

Experimental design
Mice were divided into eight groups with eight animal per each group: (1) control; (2) Tau 1000 mg/kg; (3) Nec-1
(1.8 mg/kg); (4) VPA (500 mg/kg); (5–7) Tau+VPA groups: Tau at doses of 250, 500, and 1000 mg/kg were adminis- tered 1 h before VPA treatment; (8) Nec-1+VPA, Nec-1 was administered 1 h before VPA treatment. All administrations were performed once a day for 14 consecutive days, intra- peritoneally. Duration time and the doses of Tau [17, 18], Nec-1 [10, 19] and VPA [20–21] were selected based on the previous studies.
On the 15th day, the animals were anesthetized using xylazine-ketamine (10 mg/kg, 100 mg/kg). The samples of blood were collected from the heart and centrifuged (4000 rpm) for 10 min. The obtained serums were main- tained at -20 °C until use. The liver tissues were removed, washed (with saline), and cut into three pieces. A piece of the tissue was fixed in 10% formalin for evaluation of histological and another piece was homogenized by PBS (phosphate-buffered saline) and centrifuged for 15 min (at 4 °C, 1000×g). The obtained supernatants kept for analyzing biochemical and oxidative stress biomarkers at − 80 °C. The last piece of the liver was maintained at − 80 °C for analysis of gene and protein expression by real-time PCR and west- ern blotting, respectively.

Measurement of the hepatic function biomarkers
The commercial kits were used to assess serum AST (aspar- tate aminotransferase), ALP (alkaline phosphatase), and ALT (alanine aminotransferase) activity based on the manu- facturer’s directions (Pars Azmoon, Tehran, Iran).

Measurement of hepatic glutathione (GSH) content
Bradford assay was used to measure the protein content of the supernatant obtained from the tissue homogenates [23]. The Ellman’s reagent was utilized for assaying the reduced GSH content [24]. The amount of 100 µL of DTNB (dithio- bis-2-nitrobenzoic acid, 0.01 M) solution was blended with 200 µL of the supernatant. Then, the absorbance of the yel- low developed color was read using a spectrophotometer (UV-160A, Shimadzu, Japan) at 412 nm. Finally, the GSH content was represented as µ mol/mg protein.

Measurement of hepatic malondialdehyde (MDA) content
Content of MDA was measured to evaluate the rate of lipid peroxidation in the tissue extracted protein. The amount of 0.5 mL of trichloroacetic acid was blended with 0.5 mL of the homogenized samples and centrifuged (500×g) for 5 min and then thiobarbituric acid (0.5 mL) was added to the supernatant. The samples absorbance was read by a spectro- photometer (UV-160A, Shimadzu, Japan) at 532 nm and the data were represented as µmol/mg protein [25].

Measuring the rate of ROS production
The generation of intracellular ROS was assessed by DCFH- DA (2′, 7′-dichlorodihydrofluorescein diacetate). In brief, the supernatants were mixed with DCFH-DA (3.32 M) and incubated in dark for 30 min. Then, a spectrofluorometer (LS50B PerkinElmer, USA) was used to read the fluores- cence intensity of DCF (dichlorofluorescein), the oxidized form of DCFH, at 500 nm excitation and 520 nm emission.

Histological assessments
For histological evaluation, the paraffin-embedded tissues were cut into 5 µm sections and stained with H&E (hema- toxylin and eosin). Then, six microscopy slides per each animal were blindly analyzed for the histological assess- ments. Moreover, histological features were graded into four groups of intense (3), moderate (2), weak (1), and normal (0). Additionally, the average percentage of the hepatocytes with steatosis was determined.

Measurement of (TNF‑α) content
TNF-α content of liver tissues was determined by a mouse TNF-alpha ELISA Kit (Cat No: E0117Mo; BT LAB, China) according to the manufacturer’s directions. In brief, the sam- ples of tissue were homogenized in phosphate buffer saline by homogenizer and centrifuged for 20 min (3000×g). The amount of 10 µL TNF-α antibody labeled with biotin was added to 40 µL samples. Afterward, the amount of 50 µL streptavidin-HRP was added for forming immune complex and incubated for 60 min at 37 °C. After washing of sample wells, 50 µL chromogen solution A and 50 µL chromogen solution B were added to wells and incubated at room tem- perature for 10 min. To terminate the reaction, a stop solu- tion (50 µL) was added to forming a yellow-colored product. Finally, the absorbance of the samples was read in 450 nm and the resulting values were represented as pg of TNF-α per mg of protein in the liver tissue.

Quantitative real‑time PCR
Total RNA was extracted from the mouse liver tissue with RNeasy Plus Mini kit (Qiagen, Inc. Cat No. 74134) based on the company’s instruction. RNA concentration and purity were monitored at an absorbance ratio of 260 to 280 nm. Complementary DNA was synthesized using a cDNA syn- thesis kit from 6 µg of total RNA regarding the manufac- turer’s instruction. Then, the RT-PCR amplified using SYBR Green PCR Master Mix (Qiagen, Inc. Cat No.180840) in a total volume of 20 µL with regard to the following plan: 95 °C (10 min), followed by 40 cycles at 95 °C (15 s), 57 °C (30 s), and 1 min at 60 °C. The PCR product purity was monitored by a melting curve analysis and each sample was repeated three times. The sequence of used primers has been shown in the supplementary file (S1). Moreover, the GAPDH gene as the housekeeping gene was used to normalize the relative gene expression. Then, the data were analyzed by the 2−△△Ct procedure and the results were rep- resented as the fold changes corresponding to the control.

Western blotting analysis
The protein expression of RIPK3 and MLKL was assessed using a western blot assay. The homogenized tissues were in RIPA lysis buffer and centrifuged for 20 min (12,000 rpm). After determining the protein concentration of each tis- sue lysate by BCA (bicinchoninc acid, protein assay kit), extracted proteins (25 µg) were separated using sodium dodecyl sulfate- polyacrylamide gel electrophoresis (10%) and moved to polyvinylidene difluoride membranes. The blocked membranes in 5% skim milk in PBS were immuno- blotted overnight at 4 °C with primary antibodies of MLKL (ab255747), p-MLKL (ab205421), RIPK3 (ab62344), p-RIPK3 (ab196436), and GAPDH (ab9485). The next day, secondary antibodies: rabbit anti-rat IgG (ab6734) and goat anti-rabbit IgG (ab150077) were added and incubated for 2 h. The band density of proteins was analyzed using ImageJ software (version 1.51). GAPDH protein was used to stand- ardize the bands.

Statistical analysis
The Graph Pad Prism 7.03 was used to analyze the statistical data. Data have shown as the mean ± SD. Finally, the statisti- cal significance was determined by the ANOVA test and post hoc Tukey’s test. The level of significance was considered as P < 0.05.

Results

Effects of Tau on serum biomarkers of VPA‑induced hepatocellular damage
The serum activity of ALP, AST, and ALT as biomarkers of hepatocellular damage was measured. As displayed in the supplementary file (S.2), serum ALT, AST, and ALP activ- ity were considerably elevated in the VPA group compared with the control (P < 0.01). Tau pretreatment with doses of 500 and 1000 mg/kg could markedly alleviate ALT, AST, and ALP activity in the VPA-intoxicated mice. The activity of ALT, AST, and ALP was also considerably reduced in the Nec-1+VPA group compared to the VPA-intoxicated animals (all P < 0.01).

Effects of Tau on VPA‑induced oxidative stress parameters
As demonstrated in Fig. 1A, B, both MDA (lipid peroxi- dation) and ROS contents were significantly enhanced following VPA administration (both P < 0.001), whereas hepatic GSH level was reduced in comparison to the control (Fig. 1C, P < 0.01). Nec-1 and Tau pretreatment displayed hepatoprotective effects by a significant reduction in MDA content and ROS (both P < 0.01) levels as well as an increas- ing in GSH level (P < 0.01) in the VPA-treated mice.

Effects of Tau on the increase of VPA‑induced TNF‑α level
TNF- α level was determined by ELISA kit. VPA-treated mice demonstrated an obvious enhancement in the TNF-α level compared with the control (P < 0.01). The hepatic TNF-α content was significantly declined in Tau+VPA and Nec-1+VPA (P < 0.01) groups compared with the VPA- intoxicated mice (Fig. 1D).

Effects of Tau on VPA‑induced histological alterations
The liver structure in the control group had a normal appear- ance. VPA administration considerably resulted in altera- tions of histological in mice liver tissue such as steatosis, RBCs congestion, and inflammatory cell infiltration. Tau significantly improved histological criteria in the liver of the VPA-exposed mice. In the Nec-1+VPA group, the histological criteria were also significantly lower than the VPA-intoxicated mice. The histological characteristics are demonstrated in Fig. 2 and Table 1.

Tau attenuated the gene expressions of necroptosis signaling pathway
As demonstrated in Fig. 3, mRNA expressions of caspase-8 (S.3), FADD, RIPK1, RIPK3, and MLKL genes were considerably increased by 2.40, 2.44, 2.27, and 2.29-fold in the VPA group compared to the control, respectively (P < 0.01). In the Tau+VPA and Nec-1+VPA groups, the mRNA expressions of FADD, caspase-8, MLKL, RIPK3, and RIPK1 were considerably reduced compared to the VPA group. Western blotting results of the liver of VPA-exposed mice showed a marked enhancement in the protein expres- sion of MLKL and RIPK3 by 2.35 and 1.96-fold compared to the control. VPA groups treated by Nec-1 and Tau showed a considerable reduction in the protein levels of RIPK3 and MLKL compared to the VPA group. Since phosphorylation of RIPK3 and MLKL is essential for triggering necroptosis, we also investigated the p-RIPK3 and p-MLKL by western blotting. Based on our results, VPA administration signifi- cantly increased the levels of RIPK3 and MLKL phosphoryl- ation by 2.33 and 2.73-fold compared to the normal group. These increases were attenuated by Nec-1 and Tau (Fig. 4).
Tau attenuated the expression of apoptotic markers Bcl-2 and Bax expression evaluated as the main markers of the intrinsic apoptosis pathway. Bax expression, a pro- apoptotic gene, was enhanced significantly in VPA-exposed mice compared with the control while the expression of this gene was considerably declined in treated mice with Tau and Nec-1 compared to the VPA group. In contrast, Bcl-2 expression, as an anti-apoptotic gene was declined in VPA- exposed mice compared to the control. VPA groups treated by Nec-1 and Tau showed a considerable enhancement in the level of Bcl-2 expression (S.3).
Fig. 1 The level of oxidative stress markers and TNF-α in various groups. ROS formation (A), MDA levels (B), GSH levels (C) and hepatic TNF-α content (D). Values represent as mean ± SD (n = 8). *and #p < 0.05, ** and ##p < 0.01, ***p < 0.001; * and # symbols show compared to control and VPA-intoxicated mice, respectively

Discussion

This work has indicated the useful effects of Tau on hepa- totoxicity caused by VPA. Our findings indicated that VPA treatment increased significantly the AST, ALT, and ALP levels, which are considered indicators of hepatocellular damage. The obtained data were consistent with the results reported in previous researches [8, 26]. The increased activ- ity of these enzymes might be owing to the plasma mem- brane rupture and cellular damages induced by VPA and rapid release of the enzymes from the cytoplasm into the blood circulation [27]. The histological results also con- firmed VPA-induced liver injury as evidenced by the stea- tosis, RBCs congestion, and inflammatory cell infiltration. These alterations were in parallel with a previous study, which reported that VPA-intoxicated liver showed conges- tion and inflammatory cell infiltration [28]. In the present research, Tau pretreatment could significantly attenuate the elevation of liver biomarkers level and improve these his- tological changes caused by VPA. Recent studies showed that Tau pretreatment effectively alleviated the increase of hepatic biomarkers and histology changes of liver tis- sue caused by Propylthiouracil and doxorubicin [22, 29]. A previous study showed that Tau declined the levels of ALP, AST, and ALT in CCl4-induced liver injury [30].
The increased liver biomarkers and histological changes were accompanied by inducing oxidative stress by VPA. Oxidative stress is a main suggested mechanism of VPA- induced liver damage. It is indicated that VPA induces lipid peroxidation through a reaction with the polyunsaturated fatty acids of lipid membranes which leads to depletion of antioxidant enzymes such as GSH [7, 17]. In this regard, Tong et al. revealed that treatment with VPA (500 mg/kg) for 14 consecutive days elevates lipid peroxidation levels
Fig. 2 Histological findings of liver sections stained with H&E under a light microscope in different groups (magnifications: ×250). C conges- tion of RBCs, I infiltration of inflammatory cells, S steatosis Values represent as mean ± SEM (n = 8). ** and ##p < 0.01, ***and ### p < 0.001; * and # symbols show compared to control and VPA-intox- icated mice, respectively and induces liver tissue steatosis and necrosis [21]. Our data have indicated that VPA treatment increases ROS and MDA levels and reduces GSH levels. Tau could increase GSH level and decrease ROS and MDA levels in the VPA- exposed mice. It has been reported that Tau treatment has antioxidant effects on alloxan-induced hepatic injury in rats by reducing ROS and MDA levels [31].
Oxidative stress can promote massive tissue damages through increasing TNF-α, which leads to necroptosis or apoptosis [32]. The present research displayed TNF-α level significantly increased by VPA, which was in accordance with previous studies [33, 34]. While pretreatment with Tau could reverse the increase of TNF-α level and reduce VPA-induced liver damage. Based on the study of Zhang et al., Tau pretreatment reduced TNF-α levels in the liver of lipopolysaccharide-exposed rats and inhibited the hepato- cytes death [35]. Several studies have shown that TNF-α stimulation causes excessive ROS production by the JNK pathway as a positive feedback cycle [36, 37]. The excessive ROS generation also leads to RIPK1 autophosphorylation and ultimately accelerates RIPK3 phosphorylation to form necrosome [38].
To understand the role of necroptosis signaling path- way in VPA-induced liver damage, we investigated mRNA expression of FADD, caspase-8, MLKL, RIPK3, and RIP- K1genes. It has been known that FADD activates caspase-8, in which both caspase-8 and FADD form a complex with RIPK1, leading to TNF-induced apoptosis [39]. Our results indicated that VPA treatment causes a marked increase in FADD, caspase-8, and RIPK1 expressions in mouse liver tissue compared to the control group. These findings suggest FADD-caspase-8-RIPK1-mediated apoptosis could induce in hepatocytes by VPA. As shown in our results, Tau could decrease FADD, caspase-8 and RIPK1 expression levels in VPA-treated mice. It is well known that ROS overproduction is a driving force for the executioner of necrotic cell death in various cells owing to its ability to form more necrosome [37]. Besides, under excessive stimuli such as excessive ROS production, FADD can directly form a complex with RIPK1 and RIPK3 in the present caspase and shift apoptosis to necroptosis [40, 41]. FADD not only acts as a regulator for TNF-induced apoptosis, but it is essential for TRAIL or Fas
Fig. 3 The mRNA expression of FADD, RIPK1, RIPK3 and MLKL genes in various groups. Values represent as mean ± SD (n = 8). *and
#p < 0.05, ** and ##p < 0.01; * and # symbols show compared to control and VPA -intoxicated mice, respectively receptor-mediated necroptosis. Also, the study of Duprez et al. demonstrated that inhibition of caspase-8 is always no essential for the execution of necroptosis [42].
In our findings, the expression levels of mRNA and pro- tein of RIPK3 and MLKL were enhanced after VPA treat- ment. Furthermore, the phosphorylation level of MLKL and RIPK3 was considerably higher in VPA-exposed mice than the control. RIPK1 and RIPK3 are bind to and phosphorylate MLKL, which phosphorylated MLKL disrupts the plasma membrane and results in necroptosis. Ramachandran et al. have reported that liver damage induced by acetaminophen increases RIPK3 expression level as well as ALT levels [43]. Furthermore, hepatic steatosis can lead to elevated protein levels of RIPK3 in hepatocytes via defects in proteasomal degradation of RIPK3, which eventually induces necrop- tosis [44]. It is reported that VPA can lead to neuronal cell death via enhanced expression of RIPK1 and inducing the necroptosis pathway [45]. The obtained results suggest that progressing liver injury under VPA-induced oxidative stress can cause necroptotic cell death in hepatocytes. Treatment of Tau indicated a significant decrease in mRNA and pro- tein expression level of RIPK3 and MLKL compared to the VPA group. A possible mechanism for decreased expression of MLKL, RIPK3, and RIPK1 by Tau may be due to its inhibitory effect on ROS generation. In the present study, Nec-1 and Tau have demonstrated similar inhibitory effects on the increased RIPK3 and MLKL protein expression and attenuated VPA-induced hepatic injury through inhibiting necroptotic cell death. Furthermore, Tau has a potent cell membranes stabilizer effect as well as antioxidant properties. Therefore, the suppressing effect of Tau on the necroptosis pathway induced by VPA may be to prevent MLKL-medi- ated cell membrane rupture via its cell membrane stabilizing effects, as well as suppression of oxidative stress.
To understand the role of the necroptosis signaling path- way in VPA-induced liver injury, we used Nec-1 to suppress necroptosis and then evaluated the toxicity of VPA in pres- ence of Nec-1. Despite Nec-1 has no antioxidant properties,
Fig. 4 The protein level of MLKL and RIPK3 genes in various groups. Values represent as mean ± SD (n = 8). ** and ##p < 0.01, *** and
###p < .001; * and # symbols show compared to control and VPA-intoxicated mice, respectively it could reduce serum levels of ALT, AST, and ALP. This result was parallel to a previous report, which showed Nec-1 effectively could reduce serum levels of aminotransferases in concanavalin-exposed mice [46]. Nec-1 may prevent the release of ALT, AST by necroptosis inhibition. Decreased expression of RIPK1, RIPK3 and MLKL by Nec-1 is due to the fact that Nec-1 acts as a potent inhibitor of necroptosis and directly regulates the expression of necroptosis mol- ecules [47]. Takemoto et al. reported that Nec-1 had inhibi- tory effects against acetaminophen-induced liver damage [48].
Surprisingly, we observed that VPA treatment could also increase the apoptotic marker expression, Bax, and decline the antiapoptotic marker expression, Bcl-2, com- pared to the control. It seems that both intrinsic apoptosis and necroptosis occur simultaneously in VPA-induced liver damage. A study done by Lin et al. [49] showed that Tanshinone IIA could induce simultaneously apoptosis and necroptosis in human hepatocellular cancer cells. In contrast, treated mice with Tau and Nec-1 indicated a significant downregulation of Bax expression and a considerable upregulation of Bcl-2 expression compared to the VPA group. Li et al. indicated that Tau pretreatment downregulated the level of Bax expression and upregu- lated Bcl-2 level in the liver of arsenic-exposed mice [50].

Conclusions

Our results demonstrated that VPA effectively proceeds hepatotoxicity by inducing oxidative stress and simultane- ously stimulating intrinsic apoptosis and necroptosis sign- aling pathway in mice. It is possible that VPA-induced excessive ROS generation leads to activation of TNF-α mediated necroptotic signaling pathway. Tau can amelio- rate liver damages via suppression of different signaling pathways including RIPK1-RIPK3-MLKL signaling and apoptosis (S.4). According to our knowledge, this is the first report to reveal necroptosis has a key role in hepato- cyte cell death after exposure to VPA.

Declarations
Conflict of interest The authors declare that there is no conflict of in- terest regarding the publication of this article.

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