- Email: [email protected]
reperfusion injury in mice
Accepted Manuscript STIM1 deficiency protects the liver from ischemia/reperfusion injury in mice Yanyang Li, Chunyan Lou, Weiying Wang PII:
S0006-291X(18)30006-8
DOI:
10.1016/j.bbrc.2018.01.006
Reference:
YBBRC 39179
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 19 December 2017 Accepted Date: 2 January 2018
Please cite this article as: Y. Li, C. Lou, W. Wang, STIM1 deficiency protects the liver from ischemia/ reperfusion injury in mice, Biochemical and Biophysical Research Communications (2018), doi: 10.1016/ j.bbrc.2018.01.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Title Page Title: STIM1 deficiency protects the liver from ischemia/reperfusion injury in mice
Yanyang Li*, Chunyan Lou, Weiying Wang Address:
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Authors:
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Department of Pediatrics, Huaihe Hospital, Henan University, Kaifeng 475000, China
Dr. Yanyang Li;
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*Corresponding author； ；
Department of Pediatrics, Huaihe Hospital, Henan University, Kaifeng 475000, China
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Email: [email protected]
ACCEPTED MANUSCRIPT Abstract. Hepatic ischemia reperfusion (I/R) injury is unavoidable in various clinical conditions. Despite considerable investigation, the underlying molecular mechanism revealing liver I/R injury remains elusive. Stromal interaction molecule 1 (STIM1) plays essential role in regulating the induction of cellular responses to a number of stress conditions, including temperature changes, elevated ROS, and hypoxia. Here, to
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explore if STIM1 is involved in hepatic injury, wild type (WT) and STIM1-knockout (STIM1-/-) mice were subjected to I/R. Our results indicated that the WT mice with hepatic I/R injury showed higher STIM1 expressions from gene and protein levels in liver tissue samples. Similar results were observed in hypoxia-exposed cells in vitro.
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Significantly, STIM1-/- attenuated hepatic injury compared to the WT mice after I/R, as evidenced by the improved pathological alterations in liver sections. WT mice subjected to liver I/R showed higher serum alanine aminotransferase (ALT) and
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aminotransferase (AST) levels, as well as pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1β, which were significantly reduced by STIM1-/-. In addition, STIM1-/- also decreased the liver mRNA levels of proinflammatory cytokines in mice after I/R injury. Furthermore, significantly decreased oxidative stress was found in STIM1-/- mice after I/R injury compared to the WT
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group of mice, evidenced by the enhanced superoxide dismutase (SOD) activity and the reduced malondialdehyde (MDA) and reactive oxygen species (ROS) levels in liver tissue samples. Moreover, STIM1-/- mice with hepatic I/R injury displayed the down-regulated nuclear factor of activated T cell (NFAT1), Orai1 and cleaved
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Caspase-3 levels in liver, contributing to apoptosis suppression. The results above were confirmed in hypoxia-treated cells lacking of STIM1 expression. Together, the
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findings suggested that STIM1-deletion protects the liver from I/R injury in mice through inhibiting inflammation, oxidative stress and apoptosis. STIM1 could be considered as a potential therapeutic target to ameliorate I/R injury. Keywords: I/R injury, STIM1, inflammation, oxidative stress, apoptosis 1. Introduction Hepatic ischemia reperfusion (I/R) injury is unavoidable during trauma, elective liver resections, shock or liver transplantation, and influences patient adversely [1,2]. It includes the two interrelated phases of local ischemia insult and inflammationregulated reperfusion injury [3]. Liver I/R injury involves the cold and warm ischemia.
ACCEPTED MANUSCRIPT Cold ischemia occurs in liver preservation following by transplantation [4]. Warm ischemia occurs when the organism undergo shock, trauma, as well as elective liver resections that liver blood supply is temporarily disrupted [5]. To improve the life quality of patients suffering from hepatic I/R injury, it is essential to investigate other potential factors and find therapeutic strategies to inhibit liver dysfunction.
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Hepatic I/R injury is complex and multifactorial pathophysiological process that involves the actions of inflammatory cytokines, ROS, and apoptosis [2,6]. Meanwhile, ROS-induced activation of Kupffer cells, which in turn produce large amounts of inflammatory cytokines and oxygen derived free radicals, further aggravates hepatic
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injury [7,8]. Ca2+, as a ubiquitous second messenger, is included in a plethora of cellular functions, such as oxidative stress, inflammation, energy metabolism and cell
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death [9,10]. STIM proteins are Ca2+ store sensors and mediate the induction of cellular responses to a number of stress conditions, including elevated ROS, and hypoxia [11]. Oxidants and Ca2+ are important regulators of vascular signaling in pathophysiological settings, including innate inflammation [12]. However, if STIM1 could act as a regulator involved in hepatic I/R injury is still unknown. Therefore, in the present study the STIM1-/- mice were subjected to 1 h segmental (70%) ischemia
injury.
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to investigate if it could be considered as a therapeutic target for improving liver I/R
In the present study, the findings indicated that the wild type mice with liver I/R
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injury exhibited higher levels of STIM1. And of note, STIM1-knockout significantly reduced liver pathological changes in mice after hepatic I/R injury, accompanied with
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the reduced serum AST and ALT levels. Additionally, STIM1-/- suppressed inflammation and oxidative stress in the liver of mice with I/R injury, evidenced by the down-regulated expression of pro-inflammatory cytokines, as well as the decreased ROS levels. Further, apoptosis occurred in mice underwent I/R injury was also reversed by STIM1-deletion. Moreover, STIM1-knockdown decreased inflammatory response, oxidative stress and apoptosis in hypoxia-exposed cells. Thus, we supposed that STIM1 could be considered as a potential therapeutic target for ameliorating I/R injury. 2. Materials and methods 2.1. Animals and treatments
ACCEPTED MANUSCRIPT Adult male, 8-10 weeks old, C57/BL6 mice (Wild type, WT) and STIM1-deficient mice (C57BL/6 background, STIM1-/-) were purchased from the Jackson Laboratory (Bar Harbor, ME). All mice were acclimatized in a room with 12/12 h light/dark cycle at 25 ± 2°C with 50 ± 5% humidity, and allowed free access to food and water ad libitum. Mice partial (70%) warm ischemia reperfusion (I/R) injury model was
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performed as previously described [13,14]. All animal procedures were performed in line with the Guide for the Care and Use of Laboratory Animals, issued by the National Institutes of Health in 1996 and approved by the Animal Care and Use Committee of Huaihe Hospital, Henan University (Henan, China).
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2.2. Cells and culture
Mice Kupffer cells were isolated following the previous studies [15]. Mice were
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anesthetized by ketamine (100 mg/kg) and xylazine (10 mg/kg), then livers were perfused with 0.05% Collagenase (Invitrogen, USA) in vivo and digested with 0.04% Collagenase for additional 20 minutes at 37°C in vitro and passed through a 250 µm cell strainer. Afer separation by a 25% and 50% Percoll gradient centrifugation for 1 h at 800×g at 4°C, non-parenchymal liver cells were collected from the interface and
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cultured in DMEM with 10% fetal bovine serum. The nonadherent fraction was washed and the adherent Kupffer cells were remained. Small interfering RNA (siRNA) targeting mouse STIM1 was synthesized by GENERAY (Shanghai, China). siRNA were transfected into Kupffer cells using Lipo2000 transfection reagents (Invitrogen,
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USA) following the standard protocol. 2.3. RT-qPCR analysis
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Extraction of total RNA in liver and cells was performed by using TRIzol Reagent
(Takara, Japan). Total RNA was reversed transcribed into cDNA using the SYBR Premix Ex Taq kit (TaKaRa Biotechnology China). SYBR Green quantitative RTPCR was prepared to calculate the expression of the target genes. The PCR primers (GENERAY) used for this study showed in Supplementary Table 1. 2.4. Western blot assays Proteins were extracted from the liver tissues and cells by grinding with protease inhibitors. The proteins were incubated in boiling water for 5 min before the experiment. The protein samples were separated by 10-12% sodium dodecyl sulfate
ACCEPTED MANUSCRIPT polyacrylamide gel electrophoresis and transferred onto a Polyvinylidene Fluoride (PVDF) membrane (Millipore, USA). The membranes were incubated with 5% nonfat milk for 2 h, and then incubated with primary antibodies: rabbit anti-STIM1 (ab1089945), p-NF-κB (ab222494), NF-κB (ab16502), HO1 (ab52947), Nrf2 (ab137550), NFAT1 (ab150330), Orai1 (ab59330), Caspase-3 (ab13586), and
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GAPDH (ab8245). All antibodies were purchased from Abcam (USA) at 1:1000 dilutions. The membranes were visualized via the enhanced chemiluminescence (ECL) detection kit (Millipore). Immunoreactive labeling was analyzed using ImageJ (US
protein level. 2.5. The immunohistochemical analysis
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National Institutes of Health, Bethesda, MD, USA) and standardized against β-actin
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Liver samples were excised from each mouse, fixed in 10% formalin overnight, dehydrated with alcohol, and embedded in wax. Paraffin sections (3 µm thickness) were stained with hematoxylin and eosin (H&E). Liver tissue sections were first deparaffinized by infiltrating in dimethylbenzene twice and rehydrated using graded ethanol. The sections were washed with cold phosphate-buffered saline (PBS) for 3
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times after antigen microwave retrieval and endogenous peroxide blocking. The slides were incubated overnight at 4°C with primary antibody against STIM1, and cleaved Caspase-3 (1:100) after nonspecific antigen was blocked by 10% goat serum. Then, the slides were incubated at 37°C for 30 min with secondary antibody conjugated with
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horseradish peroxidase (HRP), and visualized with diaminobenzidine (DAB) kit (Zsbio, Beijing, China) and nucleuses were counterstained using hematoxylin.
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2.6. Measurements of biochemical indicators ALT, AST, SOD, MDA, and ROS levels were measured using the standard
diagnostic kits purchased from Jiancheng Biotechnology Co., Ltd. (Nanjing, China). Serum TNF-α, IL-1β, and IL-6 concentrations were detected using specific ELISA kits purchased from R&D Systems, Inc. (USA). 2.7. Flow cytometry analysis Fluorescent calcium indicator Fura 3-acetoxymethy ester (Fura3-AM) was calculated using flow cytometry. Equal volumes of Fluo-3/AM (5 µM) and pluronic F127 (PF127, 0.1%) were added into HBSS just before incubation as the Fluo-3
ACCEPTED MANUSCRIPT loading solution. After transfection with siSTIM1 for 24 h, the cells were exposed to hypoxia for another 24 h. And then, they were loaded with Fura3/AM for 45 min in the dark at 37°C in a humidified incubator, washed 3 times using PBS, detached with trypsin, resuspended in HBSS, and directly introduced into a Becton Dickinson FACS
2.8. ROS measurements in vitro
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Calibur flow cytometer (USA) for analysis.
Total ROS in cells was quantified using the fluorogenic probe 2’,7’dichlorodihydrofluorescein
diacetate
(DCFH2-DA,
following
the
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manufacturer’s instructions.
Invitrogen)
2.9. Statistical analysis
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Results are expressed as mean±SEM of independent experiments. Group comparisons were performed using one or two-way ANOVA and Turkey’s post hoc test (Graph Pad Prism 6 program). Differences were considered significant at P<0.05. 3. Results
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3.1. Expression of STIM1 was increased in liver tissue of mice after I/R To calculate if STIM1 was implicated in the liver tissue following I/R, RT-qPCR and western blot were performed to measure STIM1 mRNA and protein levels. After warm ischemia for 1 h, mice were sacrificed at 0, 3, 6, 12 and 24 h of reperfusion.
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STIM1 mRNA and protein expression levels were significantly increased at 6, 12 and 24 h in I/R group (Fig. 1A and B). In addition, we detected STIM1 expression in liver
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tissue sections from mice that were subjected to hepatic I/R by IHC. Moreover, STIM1 was highly expressed in mice liver following reperfusion for 6, 12 and 24 h compared to the Con group of 0 h (Fig. 1C and D). Together, these data suggest that STIM1 played an essential role in liver I/R injury. 3.2. STIM1 deletion decreases liver I/R injury Next, the mice underwent 1 h of ischemia, followed by 6 h of reperfusion. Liver pathological alterations were detected using H&E staining in mice following 6 h of reperfusion (Fig. 2A). Of note, STIM1-knockout mice after reperfusion for 6 h exhibited attenuated hepatic pathological changes. Compared to the WT/I/R group of
ACCEPTED MANUSCRIPT mice, the markedly reduced serum AST and ALT levels in STIM1-/- mice after 6 h reperfusion were observed (Fig. 2B). The high levels of serum pro-inflammatory cytokines, TNF-α, IL-6 and IL-1β, in WT/I/R mice were significantly reduced in STIM1-/- mice after hepatic I/R injury through ELISA methods (Fig. 2C). Using RTqPCR analysis, the significantly enhanced expressions of TNF-α, IL-6, IL-1β, iNOS
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and COX2 from mRNA levels were detected compared to the WT/Con group of mice. Notably, the process was ameliorated in STIM1-knockout mice (Fig. 2D). Similar results were detected in phosphorylated NF-κB changes (Fig. 2E). Data from Fig. 2FH indicated that oxidative stress was induced by I/R in WT mice as proved by the reduced SOD activity, whereas the increased MDA and total ROS levels. However,
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STIM1-/- mice showed effective role in attenuating oxidative stress in mice with I/R injury. Consistently, western blot analysis indicated that HO1 and Nrf2 levels were
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decreased in the liver of WT/I/R mice, which were significantly restored by STIM1 deletion (Fig. 2I). The data above demonstrated that the absence of STIM1 could ameliorate hepatic I/R injury through reducing inflammatory response and oxidative stress.
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3.3. STIM1 knockout attenuates hepatic apoptosis after I/R
STIM1 has been reported to be participated in regulating cellular processes, including apoptosis [16]. Here, we found that mice after I/R injury exhibited higher levels of NFAT1 and Orai1, as well as cleaved Caspase-3, which were down-
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regulated by STIM1-knockout (Fig. 3A). Further, compared to the WT/Con group of mice, liver I/R increased the apoptotic levels in the wild type mice; however, mice that were lack of STIM1 showed a significant decrease of apoptotic proportion using
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TUNEL analysis than WT/I/R group of mice (Fig. 3B). Consistently, the wild type mice underwent I/R had greater increase of cleaved Caspase-3 in liver tissue samples, which were down-regulated in STIM1-/-/I/R mice (Fig. 3C). The results above indicated that STIM1-deletion could attenuate apoptosis in mice with hepatic I/R injury. 3.4. Inhibition of STIM1 expression ameliorates inflammatory response, ROS generation and apoptosis in hypoxia-treated Kupffer cells To verify the role of STIM1 in regulating hepatic I/R injury, the in vitro study using Kupffer cells following hypoxia was performed. As shown in Fig. 4A and B, we
ACCEPTED MANUSCRIPT found that exposure of the Kupffer cells to hypoxia (1% oxygen) led to a timedependent up-regulation in STIM1 expression from gene and protein levels in vitro. Following, STIM1 expression was knockdown using STIM1 specific siRNA sequences. Fig. 4C indicated that STIM1 was successfully silenced in Kupffer cells after Hypoxia exposure. Ca2+ is an important messenger in many cell types, with
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pleiotropic effects on cell survival [9,17]. STIM1 is essential Ca2+ store sensor and regulate the induction of cellular responses to a number of stress conditions, including elevated ROS, and hypoxia [18]. Flow cytometry analysis indicated that HO exposure markedly induced cytoplasmic free Ca2+ concentration in Kupffer cells; however, pre-
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transfection using STIM1 siRNA sequences significantly reduced free Ca2+ concentration in cytoplasm compared to the HO group (Fig. 4D). Next, RT-qPCR analysis showed that HO induced higher mRNA levels of TNF-α, IL-6, IL-1β, iNOS
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and COX2, whereas in HO/siSTIM1 group of cells, the expression levels of these genes were reduced (Fig. 4E). In line with the results above, p-NF-κB caused by HO was also down-regulated by STIM1 knockdown (Fig. 4F). In addition, HO exposure resulted in higher ROS generation compared to the Con group using DCF analysis, which was significantly reduced by STIM1 silence (Fig. 4G). Also, HO-induced
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reduction in HO1 and Nrf2 was restored by STIM1-knockdown (Fig. 4H). Moreover, HO exposure led to over-expression of NFAT1, Orai1 and active Caspase-3, while being blocked in STIM1-knockdown group (Fig. 4I). Finally, silencing STIM1
4J). 4. Discussion
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reduced TUNEL-positive cells with HO exposure, suggesting apoptosis blockage (Fig.
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Accordingly, liver ischemia/reperfusion (I/R) is a severe problem when patients undergo elective liver resections, transplantation, or other injuries, which may result in hepatic dysfunction, and even reduce the survival rate in the perioperative period [2,3,19,20]. Hence, attenuating I/R injury is important for patients’ postoperative recovery. To date, various factors have been reported to be involved in the development of liver I/R injury [21]. However, the underlying molecular mechanism is not fully understood. Thus, further research is required to find new target for attenuating hepatic I/R injury. In our present study, we found that mice after liver I/R injury showed higher expression of STIM1 from mRNA and protein levels. Importantly, STIM1-knockout attenuated liver pathological alterations, as well as
ACCEPTED MANUSCRIPT reduced AST and ALT levels in serum, contributing to the improvement of liver damage in mice with I/R damage. Further, STIM1-/- mice reversed inflammation, oxidative stress and apoptosis in the liver tissue of mice with hepatic I/R injury. Moreover, Kupffer cells with STIM1 knockdown following hypoxia exhibited similar results. Therefore, we hypothesized that STIM1 might be also recognized as a
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potential target for alleviating patients suffering from I/R injury. And we attempted to reveal that STIM1 was implicated in hepatic I/R injury both in vivo and in vitro.
STIM1 is a Ca2+ sensor, and upon Ca2+ store depletion, STIM1 translocate into ER/plasma membrane junctions and couples to activate Orai1 [22,23]. Further, it
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could modulate ROS sensor to trigger Ca2+ entry [24]. Ca2+ homeostasis is closely associated with cellular processes, regulating metabolism, signal transduction,
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transcriptional modulation, cell proliferation, as well as cell death [11,12,17]. As previously reported, STIM1 was participated in neutrophil-regulated killing of bacteria and ischemia/reperfusion injury in vivo [25]. Here, knockdown of STIM1 attenuated intracellular Ca2+ overload in hypoxia-treated Kupffer cells in vitro using flow cytometry, which was in agreement with previous studies. In addition, we also found that STIM1 expressed highly in the liver of mice with hepatic I/R injury.
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In a variety of cells, the coordination of Ca2+ release and entry mechanisms gives rise to sustained Ca2+ oscillations important for modulating cellular responses, such as gene expressions and cell fate eventually [10,11,26]. These responses are regulated by
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transcription factors, including NF-κB [27]. NF-κB plays an important role in mediating inflammatory response by potentiating the release of pro-inflammatory cytokines [28]. The NF-κB activation associated with an increase of TNF-α, IL-1β,
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and IL-6 in I/R injury [29]. In our study, mice with liver I/R injury showed higher levels of pro-inflammatory cytokines in serum and in liver tissue samples, including TNF-α, IL-6, IL-1β, iNOS and COX2. Notably, STIM1-deletion markedly reduced the expression of these pro-inflammatory cytokines in comparison to the WT/I/R group, which was along with the decreased p-NF-κB expressions. Also, in vitro, STIM1-knockdown reversed hypoxia-caused over-expression of pro-inflammatory cytokines, as well as p-NF-κB. Therefore, STIM1 played an important role in the development of hepatic I/R injury through modulating NF-κB-linked inflammation. We further verified that STIM1 was implicated in oxidative stress progression, modulating liver I/R injury. Here, we found that liver SOD activity, HO1 and Nrf2
ACCEPTED MANUSCRIPT expressions were markedly down-regulated, while MDA and ROS levels in mice with hepatic I/R injury were up-regulated. Notably, STIM1-/- reduced oxidative stress by reversing the changes of these indicators. As previously reported, ROS can regulate the cytosolic Ca2+ signals, and oxidants are included in the modulation of intracellular Ca2+ release channels [30]. Therefore, we supposed that there might be a possible
reveal the underlying molecular mechanism.
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interrelation between STIM1 and ROS, and further study is still required in future to
Moreover, NFAT1 is another important transcription factor, which is involved in Ca2+ oscillations to regulate inflammatory cytokine expression [31]. NFAT1
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mediates TNF-α up-regulation [32]. In our present study, we found that STIM1deletion significantly reduced NFAT1 expression in the liver of mice with I/R
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damage, indicating that STIM1 could perform its role in mediating inflammation by controlling NFAT1 expressions. STIM1/Orai1 is required for the activation of various cell types, including immune cells [33]. Here, we found that Orai1 was also expressed highly in the liver of I/R mice, which was significantly reduced by STIM1-deficiency. And in STIM1-knockdown Kupffer cells with hypoxia exposure,
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similar results were observed.
Liver I/R injury is also a complex process typified by hepatocyte apoptosis [34]. In hepatic I/R injury, the anti-apoptotic protein decreased and oxidative damage to the enzyme complexes triggered apoptosis. Previous studies suggested that inhibition
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of Caspases significantly decreased I/R damage [35]. Therefore, apoptosis plays a key role in liver damage induced by I/R. In the present study, I/R up-regulated the number of TUNEL-positive cells in vivo, which was inhibited by STIM1 knockout.
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In line with the in vivo study, hypoxia insult increased the number of apoptotic cells, and Caspase-3 cleavage, and STIM1-knockdown significantly suppressed Kupffer cell apoptosis. We speculated that the beneficial effects of STIM1-inhibition in hepatic I/R injury were partially linked to the suppression of apoptosis. The findings here were in accordance with previous studies that STIM1 inhibition prevented cell death through apoptosis-related signals [36]. However, further study in future is required to comprehensively reveal the underlying correlation between oxidative stress and apoptosis modulated by STIM1.
ACCEPTED MANUSCRIPT In conclusion, STIM1-deficiency protected liver against I/R injury through reducing inflammation, oxidative stress and apoptosis, which might be associated with intracellular Ca2+ overload. Herein, STIM1 could be considered as a potential therapeutic target for human suffering from I/R injury.
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References [1] Inoue Y, Shirasuna K, Kimura H, et al. NLRP3 regulates neutrophil functions and contributes
to
hepatic
ischemia–reperfusion
inflammasomes. J Immunol. 192 (2014) 4342-4351.
injury
independently
of
SC
[2] Li CX, Ng KTP, Shao Y, et al. The inhibition of aldose reductase attenuates hepatic ischemia-reperfusion injury through reducing inflammatory response.
M AN U
Ann Surg. 260 (2014) 317-328.
[3] McDonald KA, Huang H, Tohme S, et al. Toll-like receptor 4 (TLR4) antagonist eritoran tetrasodium attenuates liver ischemia and reperfusion injury through inhibition of high-mobility group box protein B1 (HMGB1) signaling. Mol Med. 20 (2014) 639.
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[4] Schlegel A, de Rougemont O, Graf R, et al. Protective mechanisms of endischemic cold machine perfusion in DCD liver grafts. J Hepatol. 58 (2013) 278286.
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[5] Zhai Y, Petrowsky H, Hong JC, et al. Ischaemia–reperfusion injury in liver transplantation—from bench to bedside. Nat Rev Gastro Hepat. 10 (2013) 79-89.
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[6] Sun Y, Pu LY, Lu L, et al. N-acetylcysteine attenuates reactive-oxygen-speciesmediated endoplasmic reticulum stress during liver ischemia-reperfusion injury. World J Gastroenterol. 20 (2014) 15289.
[7] Kalogeris T, Bao Y, Korthuis RJ. Mitochondrial reactive oxygen species: a double edged sword in ischemia/reperfusion vs preconditioning. Redox Bio. 2 (2014) 702-71. [8] Tao X, Wan X, Xu Y, et al. Dioscin attenuates hepatic ischemia-reperfusion injury in rats through inhibition of oxidative-nitrative stress, inflammation and apoptosis. Transplantation. 98 (2014) 604-611.
ACCEPTED MANUSCRIPT [9] Ernst IMA, Fliegert R, Guse AH. Adenine dinucleotide second messengers and T-lymphocyte calcium signaling. Front Immuno. 4 (2013). [10] Chan G, Mooney DJ. Ca 2+ released from calcium alginate gels can promote inflammatory responses in vitro and in vivo. Acta Biomater. 9 (2013) 9281-9291.
RI PT
[11] Zhou Y, Meraner P, Kwon HT, et al. STIM1 gates the store-operated calcium channel ORAI1 in vitro. Nat Struct Mol Biol. 17 (2010) 112-116.
[12] Galan C, Jardín I, Dionisio N, et al. Role of oxidant scavengers in the prevention
SC
of Ca2+ homeostasis disorders. Molecules. 15 (2010) 7167-7187.
[13] Nace GW, Huang H, Klune JR, et al. Cellular‐specific role of toll‐like receptor 4 in hepatic ischemia‐reperfusion injury in mice. Hepatology. 58 (2013)
M AN U
374-387.
[14] Bamboat ZM, Balachandran VP, Ocuin LM, et al. Toll‐like receptor 9 inhibition confers protection from liver ischemia–reperfusion injury. Hepatology. 51 (2010) 621-632.
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[15] Franken L, Klein M, Spasova M, et al. Splenic red pulp macrophages are intrinsically superparamagnetic and contaminate magnetic cell isolates. Sci Rep. 5 (2015) 12940.
EP
[16] Li W, Zhang M, Xu L, et al. The apoptosis of non-small cell lung cancer induced by cisplatin through modulation of STIM1. Exp Toxicol Pathol. 65 (2013) 1073-
AC C
1081.
[17] Mason RP. Pleiotropic effects of calcium channel blockers. Curr Hypertens Rep. 14 (2012) 293-303.
[18] Park CY, Shcheglovitov A, Dolmetsch R. The CRAC channel activator STIM1 binds and inhibits L-type voltage-gated calcium channels. Science. 330 (2010) 101-105. [19] Jaeschke H, Woolbright BL. Current strategies to minimize hepatic ischemia– reperfusion injury by targeting reactive oxygen species. Transplant Rev. 26 (2012) 103-114.
ACCEPTED MANUSCRIPT [20] Weigand K, Brost S, Steinebrunner N, et al. Ischemia/Reperfusion injury in liver surgery and transplantation: pathophysiology. HPB Surg. 2012 (2012). [21] Datta G, Fuller BJ, Davidson BR. Molecular mechanisms of liver ischemia reperfusion injury: insights from transgenic knockout models. World J
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Gastroenterol. 19 (2013) 1683. [22] Zhou Y, Srinivasan P, Razavi S, et al. Initial activation of STIM1, the regulator of store-operated calcium entry. Nat Struct Mol Biol. 20 (2013) 973-981.
[23] Lee KP, Yuan JP, Hong JH, et al. An endoplasmic reticulum/plasma membrane
SC
junction: STIM1/Orai1/TRPCs. FEBS letters. 584 (2010) 2022-2027..
[24] Steinckwich N, Schenten V, Melchior C, et al. An essential role of STIM1, Orai1,
M AN U
and S100A8–A9 proteins for Ca2+ signaling and FcγR-mediated phagosomal oxidative activity. J Immuno. 186 (2011) 2182-2191.
[25] Pan LJ, Zhang ZC, Zhang ZY, et al. Effects and mechanisms of store-operated calcium channel blockade on hepatic ischemia-reperfusion injury in rats. World J Gastroenterol. 18 (2012) 356.
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[26] Li W, Llopis J, Whitney M, et al. Cell-permeant caged InsP3 ester shows that Ca2+ spike frequency can optimize gene expression. Nature. 392 (1998) 936-941. [27] Gong G, Waris G, Tanveer R, et al. Human hepatitis C virus NS5A protein alters
EP
intracellular calcium levels, induces oxidative stress, and activates STAT-3 and NF-κB. Proc Natl Acad Sci. 98 (2001) 9599-9604.
AC C
[28] Haddad JJ. Redox regulation of pro-inflammatory cytokines and IκB-α/NF-κB nuclear translocation and activation. Biochem Biophys Res Commun. 296 (2002) 847-856.
[29] Lan L, Tao J, Chen A, et al. Electroacupuncture exerts anti-inflammatory effects in cerebral ischemia-reperfusion injured rats via suppression of the TLR4/NF-κB pathway. Int J Mol Med. 31 (2013) 75-80. [30] Bogeski I, Kappl R, Kummerow C, et al. Redox regulation of calcium ion channels: chemical and physiological aspects. Cell Calcium. 50 (2011) 407-423.
ACCEPTED MANUSCRIPT [31] Dula SB, Jecmenica M, Wu R, et al. Evidence that low-grade systemic inflammation can induce islet dysfunction as measured by impaired calcium handling. Cell Calcium. 48 (2010) 133-142. [32] Ding Y, Huang Y, Song N, et al. NFAT1 mediates placental growth factorinduced myelomonocytic cell recruitment via the induction of TNF-α. J Immuno.
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184 (2010) 2593-2601.
[33] Feske S. ORAI1 and STIM1 deficiency in human and mice: roles of store‐operated Ca2+ entry in the immune system and beyond. Immunol Rev.
SC
231 (2009) 189-209.
[34] Chen Z, Chua CC, Ho YS, et al. Overexpression of Bcl-2 attenuates apoptosis
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and protects against myocardial I/R injury in transgenic mice. Am J Physiol Heart Circ Physiol. 280 (2001) H2313-H2320.
[35] Daemen MARC, de Vries B, Buurman WA. Apoptosis and inflammation in renal reperfusion injury. Transplantation. 73 (2002) 1693-1700. [36] Chiu WT, Tang MJ, Jao HC, et al. Soft substrate up-regulates the interaction of
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STIM1 with store-operated Ca2+ channels that lead to normal epithelial cell apoptosis. Mol Biol Cell. 19 (2008) 2220-2230.
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Figure legends
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Figure 1. Expression of STIM1 is increased in liver tissue from mice afer I/R. Mice underwent 1 h of ischemia and various times of reperfusion. (A) RT-PCR and (B) western blot analysis for liver STIM1 mRNA and protein were performed. (C) IHC analysis of hepatic STIM1 levels. (D) STIM1 positive levels were quantified following IHC analysis. Data represent means ± SEM (n=8). *P<0.05,
**
P<0.01 and
***
P<0.001 vs. Con (0 h after reperfusion) mice.
Figure 2. STIM1 deletion decreases liver I/R injury. The mice underwent 1 h of ischemia, followed by 6 h of reperfusion. (A) H&E-stained liver sections from mice were exhibited. (B) Serum ALT and AST levels were measured. (C) Serum proinflammatory cytokines, including TNF-α, IL-6 and IL-1β, were examined using
ACCEPTED MANUSCRIPT ELISA methods. (D) Hepatic pro-inflammatory cytokines as indicated were calculated by RT-qPCR. (E) Western blot analysis of p-NF-κB in liver of mice. (F) Liver SOD activity, (G) MDA levels and (H) total ROS levels were evaluated. (I) Western blot analysis of HO1 and Nrf2 expression levels in liver of mice. Data represent means ± SEM (n=8). *P<0.05, **P<0.01 and
***
P<0.001 vs. WT/Con group
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of mice. Figure 3. STIM1 knockout attenuates hepatic apoptosis after I/R. The mice underwent 1 h of ischemia, followed by 6 h of reperfusion. (A) Western blot assays of liver NFAT1, Orai1 and cleaved Caspase-3. (B) TUNEL analysis of liver sections for
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apoptosis measurement. (C) IHC analysis of cleaved Caspase-3. The quantification of cleaved Caspase-3 levels was showed. Data represent means ± SEM (n=8). P<0.001 vs. WT/Con group of mice. +P<0.05 vs. WT/I/R group of mice.
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***
Figure 4. Suppressing STIM1 expression ameliorates inflamatory response, ROS generation and apoptosis in hypoxia-treated Kuppfer cells. (A, B) Kuppfer cells were treated with hypoxia at the indicated time points. STIM1 levels in cell lysates were detected using qPCR and western blot. *P<0.05, **P<0.01 and ***P<0.001vs. Con
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(0 h after hypoxia treatment). (C) Kuppfer cells were treated with STIM1-siRNAs (#1 and #2) or negative control (si-Con) for 24 h, and then exposure to hypoxia for 6 h. STIM1 expression levels were measured using western blot analysis. Kuppfer cells were transfected with STIM1#2 siRNA for 24 h, and then treated with hypoxia (HO)
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for another 24 h, followed by further research. (D) Measurement of cytoplasmic free Ca2+ concentration using flow cytometry. (E) RT-qPCR analysis of pro-inflammatory cytokines. (F) Western blot analysis of p-NF-κB. (G) ROSS generation was detected
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via DCF analysis. (H) HO1 and Nrf2 protein levels were measured using western blot analysis. (I) NFAT1, Orai1 and cleaved Caspase-3 protein levels were examined using western blot analysis. (J) TUNEL analysis for apoptosis calculation in Kuppfer cells treated as described. Data represent means ± SEM (n=6). group. +P<0.05 and ++P<0.01 vs. HO group.
***
P<0.001 vs. Con
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ACCEPTED MANUSCRIPT Highlights STIM1 expression was up regulated in liver tissue of mice following I/R. STIM1 deletion ameliorates hepatic I/R injury by reducing inflammatory
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response and oxidative stress. STIM1 knockout attenuates hepatic apoptosis after I/R.
Inhibition of STIM1 expression ameliorates inflammatory response, ROS
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generation and apoptosis in hypoxia-treated Kupffer cells
S0006-291X(18)30006-8
DOI:
10.1016/j.bbrc.2018.01.006
Reference:
YBBRC 39179
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 19 December 2017 Accepted Date: 2 January 2018
Please cite this article as: Y. Li, C. Lou, W. Wang, STIM1 deficiency protects the liver from ischemia/ reperfusion injury in mice, Biochemical and Biophysical Research Communications (2018), doi: 10.1016/ j.bbrc.2018.01.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Title Page Title: STIM1 deficiency protects the liver from ischemia/reperfusion injury in mice
Yanyang Li*, Chunyan Lou, Weiying Wang Address:
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Authors:
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Department of Pediatrics, Huaihe Hospital, Henan University, Kaifeng 475000, China
Dr. Yanyang Li;
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*Corresponding author； ；
Department of Pediatrics, Huaihe Hospital, Henan University, Kaifeng 475000, China
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Email: [email protected]
ACCEPTED MANUSCRIPT Abstract. Hepatic ischemia reperfusion (I/R) injury is unavoidable in various clinical conditions. Despite considerable investigation, the underlying molecular mechanism revealing liver I/R injury remains elusive. Stromal interaction molecule 1 (STIM1) plays essential role in regulating the induction of cellular responses to a number of stress conditions, including temperature changes, elevated ROS, and hypoxia. Here, to
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explore if STIM1 is involved in hepatic injury, wild type (WT) and STIM1-knockout (STIM1-/-) mice were subjected to I/R. Our results indicated that the WT mice with hepatic I/R injury showed higher STIM1 expressions from gene and protein levels in liver tissue samples. Similar results were observed in hypoxia-exposed cells in vitro.
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Significantly, STIM1-/- attenuated hepatic injury compared to the WT mice after I/R, as evidenced by the improved pathological alterations in liver sections. WT mice subjected to liver I/R showed higher serum alanine aminotransferase (ALT) and
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aminotransferase (AST) levels, as well as pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1β, which were significantly reduced by STIM1-/-. In addition, STIM1-/- also decreased the liver mRNA levels of proinflammatory cytokines in mice after I/R injury. Furthermore, significantly decreased oxidative stress was found in STIM1-/- mice after I/R injury compared to the WT
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group of mice, evidenced by the enhanced superoxide dismutase (SOD) activity and the reduced malondialdehyde (MDA) and reactive oxygen species (ROS) levels in liver tissue samples. Moreover, STIM1-/- mice with hepatic I/R injury displayed the down-regulated nuclear factor of activated T cell (NFAT1), Orai1 and cleaved
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Caspase-3 levels in liver, contributing to apoptosis suppression. The results above were confirmed in hypoxia-treated cells lacking of STIM1 expression. Together, the
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findings suggested that STIM1-deletion protects the liver from I/R injury in mice through inhibiting inflammation, oxidative stress and apoptosis. STIM1 could be considered as a potential therapeutic target to ameliorate I/R injury. Keywords: I/R injury, STIM1, inflammation, oxidative stress, apoptosis 1. Introduction Hepatic ischemia reperfusion (I/R) injury is unavoidable during trauma, elective liver resections, shock or liver transplantation, and influences patient adversely [1,2]. It includes the two interrelated phases of local ischemia insult and inflammationregulated reperfusion injury [3]. Liver I/R injury involves the cold and warm ischemia.
ACCEPTED MANUSCRIPT Cold ischemia occurs in liver preservation following by transplantation [4]. Warm ischemia occurs when the organism undergo shock, trauma, as well as elective liver resections that liver blood supply is temporarily disrupted [5]. To improve the life quality of patients suffering from hepatic I/R injury, it is essential to investigate other potential factors and find therapeutic strategies to inhibit liver dysfunction.
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Hepatic I/R injury is complex and multifactorial pathophysiological process that involves the actions of inflammatory cytokines, ROS, and apoptosis [2,6]. Meanwhile, ROS-induced activation of Kupffer cells, which in turn produce large amounts of inflammatory cytokines and oxygen derived free radicals, further aggravates hepatic
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injury [7,8]. Ca2+, as a ubiquitous second messenger, is included in a plethora of cellular functions, such as oxidative stress, inflammation, energy metabolism and cell
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death [9,10]. STIM proteins are Ca2+ store sensors and mediate the induction of cellular responses to a number of stress conditions, including elevated ROS, and hypoxia [11]. Oxidants and Ca2+ are important regulators of vascular signaling in pathophysiological settings, including innate inflammation [12]. However, if STIM1 could act as a regulator involved in hepatic I/R injury is still unknown. Therefore, in the present study the STIM1-/- mice were subjected to 1 h segmental (70%) ischemia
injury.
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to investigate if it could be considered as a therapeutic target for improving liver I/R
In the present study, the findings indicated that the wild type mice with liver I/R
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injury exhibited higher levels of STIM1. And of note, STIM1-knockout significantly reduced liver pathological changes in mice after hepatic I/R injury, accompanied with
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the reduced serum AST and ALT levels. Additionally, STIM1-/- suppressed inflammation and oxidative stress in the liver of mice with I/R injury, evidenced by the down-regulated expression of pro-inflammatory cytokines, as well as the decreased ROS levels. Further, apoptosis occurred in mice underwent I/R injury was also reversed by STIM1-deletion. Moreover, STIM1-knockdown decreased inflammatory response, oxidative stress and apoptosis in hypoxia-exposed cells. Thus, we supposed that STIM1 could be considered as a potential therapeutic target for ameliorating I/R injury. 2. Materials and methods 2.1. Animals and treatments
ACCEPTED MANUSCRIPT Adult male, 8-10 weeks old, C57/BL6 mice (Wild type, WT) and STIM1-deficient mice (C57BL/6 background, STIM1-/-) were purchased from the Jackson Laboratory (Bar Harbor, ME). All mice were acclimatized in a room with 12/12 h light/dark cycle at 25 ± 2°C with 50 ± 5% humidity, and allowed free access to food and water ad libitum. Mice partial (70%) warm ischemia reperfusion (I/R) injury model was
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performed as previously described [13,14]. All animal procedures were performed in line with the Guide for the Care and Use of Laboratory Animals, issued by the National Institutes of Health in 1996 and approved by the Animal Care and Use Committee of Huaihe Hospital, Henan University (Henan, China).
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2.2. Cells and culture
Mice Kupffer cells were isolated following the previous studies [15]. Mice were
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anesthetized by ketamine (100 mg/kg) and xylazine (10 mg/kg), then livers were perfused with 0.05% Collagenase (Invitrogen, USA) in vivo and digested with 0.04% Collagenase for additional 20 minutes at 37°C in vitro and passed through a 250 µm cell strainer. Afer separation by a 25% and 50% Percoll gradient centrifugation for 1 h at 800×g at 4°C, non-parenchymal liver cells were collected from the interface and
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cultured in DMEM with 10% fetal bovine serum. The nonadherent fraction was washed and the adherent Kupffer cells were remained. Small interfering RNA (siRNA) targeting mouse STIM1 was synthesized by GENERAY (Shanghai, China). siRNA were transfected into Kupffer cells using Lipo2000 transfection reagents (Invitrogen,
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USA) following the standard protocol. 2.3. RT-qPCR analysis
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Extraction of total RNA in liver and cells was performed by using TRIzol Reagent
(Takara, Japan). Total RNA was reversed transcribed into cDNA using the SYBR Premix Ex Taq kit (TaKaRa Biotechnology China). SYBR Green quantitative RTPCR was prepared to calculate the expression of the target genes. The PCR primers (GENERAY) used for this study showed in Supplementary Table 1. 2.4. Western blot assays Proteins were extracted from the liver tissues and cells by grinding with protease inhibitors. The proteins were incubated in boiling water for 5 min before the experiment. The protein samples were separated by 10-12% sodium dodecyl sulfate
ACCEPTED MANUSCRIPT polyacrylamide gel electrophoresis and transferred onto a Polyvinylidene Fluoride (PVDF) membrane (Millipore, USA). The membranes were incubated with 5% nonfat milk for 2 h, and then incubated with primary antibodies: rabbit anti-STIM1 (ab1089945), p-NF-κB (ab222494), NF-κB (ab16502), HO1 (ab52947), Nrf2 (ab137550), NFAT1 (ab150330), Orai1 (ab59330), Caspase-3 (ab13586), and
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GAPDH (ab8245). All antibodies were purchased from Abcam (USA) at 1:1000 dilutions. The membranes were visualized via the enhanced chemiluminescence (ECL) detection kit (Millipore). Immunoreactive labeling was analyzed using ImageJ (US
protein level. 2.5. The immunohistochemical analysis
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National Institutes of Health, Bethesda, MD, USA) and standardized against β-actin
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Liver samples were excised from each mouse, fixed in 10% formalin overnight, dehydrated with alcohol, and embedded in wax. Paraffin sections (3 µm thickness) were stained with hematoxylin and eosin (H&E). Liver tissue sections were first deparaffinized by infiltrating in dimethylbenzene twice and rehydrated using graded ethanol. The sections were washed with cold phosphate-buffered saline (PBS) for 3
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times after antigen microwave retrieval and endogenous peroxide blocking. The slides were incubated overnight at 4°C with primary antibody against STIM1, and cleaved Caspase-3 (1:100) after nonspecific antigen was blocked by 10% goat serum. Then, the slides were incubated at 37°C for 30 min with secondary antibody conjugated with
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horseradish peroxidase (HRP), and visualized with diaminobenzidine (DAB) kit (Zsbio, Beijing, China) and nucleuses were counterstained using hematoxylin.
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2.6. Measurements of biochemical indicators ALT, AST, SOD, MDA, and ROS levels were measured using the standard
diagnostic kits purchased from Jiancheng Biotechnology Co., Ltd. (Nanjing, China). Serum TNF-α, IL-1β, and IL-6 concentrations were detected using specific ELISA kits purchased from R&D Systems, Inc. (USA). 2.7. Flow cytometry analysis Fluorescent calcium indicator Fura 3-acetoxymethy ester (Fura3-AM) was calculated using flow cytometry. Equal volumes of Fluo-3/AM (5 µM) and pluronic F127 (PF127, 0.1%) were added into HBSS just before incubation as the Fluo-3
ACCEPTED MANUSCRIPT loading solution. After transfection with siSTIM1 for 24 h, the cells were exposed to hypoxia for another 24 h. And then, they were loaded with Fura3/AM for 45 min in the dark at 37°C in a humidified incubator, washed 3 times using PBS, detached with trypsin, resuspended in HBSS, and directly introduced into a Becton Dickinson FACS
2.8. ROS measurements in vitro
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Calibur flow cytometer (USA) for analysis.
Total ROS in cells was quantified using the fluorogenic probe 2’,7’dichlorodihydrofluorescein
diacetate
(DCFH2-DA,
following
the
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manufacturer’s instructions.
Invitrogen)
2.9. Statistical analysis
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Results are expressed as mean±SEM of independent experiments. Group comparisons were performed using one or two-way ANOVA and Turkey’s post hoc test (Graph Pad Prism 6 program). Differences were considered significant at P<0.05. 3. Results
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3.1. Expression of STIM1 was increased in liver tissue of mice after I/R To calculate if STIM1 was implicated in the liver tissue following I/R, RT-qPCR and western blot were performed to measure STIM1 mRNA and protein levels. After warm ischemia for 1 h, mice were sacrificed at 0, 3, 6, 12 and 24 h of reperfusion.
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STIM1 mRNA and protein expression levels were significantly increased at 6, 12 and 24 h in I/R group (Fig. 1A and B). In addition, we detected STIM1 expression in liver
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tissue sections from mice that were subjected to hepatic I/R by IHC. Moreover, STIM1 was highly expressed in mice liver following reperfusion for 6, 12 and 24 h compared to the Con group of 0 h (Fig. 1C and D). Together, these data suggest that STIM1 played an essential role in liver I/R injury. 3.2. STIM1 deletion decreases liver I/R injury Next, the mice underwent 1 h of ischemia, followed by 6 h of reperfusion. Liver pathological alterations were detected using H&E staining in mice following 6 h of reperfusion (Fig. 2A). Of note, STIM1-knockout mice after reperfusion for 6 h exhibited attenuated hepatic pathological changes. Compared to the WT/I/R group of
ACCEPTED MANUSCRIPT mice, the markedly reduced serum AST and ALT levels in STIM1-/- mice after 6 h reperfusion were observed (Fig. 2B). The high levels of serum pro-inflammatory cytokines, TNF-α, IL-6 and IL-1β, in WT/I/R mice were significantly reduced in STIM1-/- mice after hepatic I/R injury through ELISA methods (Fig. 2C). Using RTqPCR analysis, the significantly enhanced expressions of TNF-α, IL-6, IL-1β, iNOS
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and COX2 from mRNA levels were detected compared to the WT/Con group of mice. Notably, the process was ameliorated in STIM1-knockout mice (Fig. 2D). Similar results were detected in phosphorylated NF-κB changes (Fig. 2E). Data from Fig. 2FH indicated that oxidative stress was induced by I/R in WT mice as proved by the reduced SOD activity, whereas the increased MDA and total ROS levels. However,
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STIM1-/- mice showed effective role in attenuating oxidative stress in mice with I/R injury. Consistently, western blot analysis indicated that HO1 and Nrf2 levels were
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decreased in the liver of WT/I/R mice, which were significantly restored by STIM1 deletion (Fig. 2I). The data above demonstrated that the absence of STIM1 could ameliorate hepatic I/R injury through reducing inflammatory response and oxidative stress.
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3.3. STIM1 knockout attenuates hepatic apoptosis after I/R
STIM1 has been reported to be participated in regulating cellular processes, including apoptosis [16]. Here, we found that mice after I/R injury exhibited higher levels of NFAT1 and Orai1, as well as cleaved Caspase-3, which were down-
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regulated by STIM1-knockout (Fig. 3A). Further, compared to the WT/Con group of mice, liver I/R increased the apoptotic levels in the wild type mice; however, mice that were lack of STIM1 showed a significant decrease of apoptotic proportion using
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TUNEL analysis than WT/I/R group of mice (Fig. 3B). Consistently, the wild type mice underwent I/R had greater increase of cleaved Caspase-3 in liver tissue samples, which were down-regulated in STIM1-/-/I/R mice (Fig. 3C). The results above indicated that STIM1-deletion could attenuate apoptosis in mice with hepatic I/R injury. 3.4. Inhibition of STIM1 expression ameliorates inflammatory response, ROS generation and apoptosis in hypoxia-treated Kupffer cells To verify the role of STIM1 in regulating hepatic I/R injury, the in vitro study using Kupffer cells following hypoxia was performed. As shown in Fig. 4A and B, we
ACCEPTED MANUSCRIPT found that exposure of the Kupffer cells to hypoxia (1% oxygen) led to a timedependent up-regulation in STIM1 expression from gene and protein levels in vitro. Following, STIM1 expression was knockdown using STIM1 specific siRNA sequences. Fig. 4C indicated that STIM1 was successfully silenced in Kupffer cells after Hypoxia exposure. Ca2+ is an important messenger in many cell types, with
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pleiotropic effects on cell survival [9,17]. STIM1 is essential Ca2+ store sensor and regulate the induction of cellular responses to a number of stress conditions, including elevated ROS, and hypoxia [18]. Flow cytometry analysis indicated that HO exposure markedly induced cytoplasmic free Ca2+ concentration in Kupffer cells; however, pre-
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transfection using STIM1 siRNA sequences significantly reduced free Ca2+ concentration in cytoplasm compared to the HO group (Fig. 4D). Next, RT-qPCR analysis showed that HO induced higher mRNA levels of TNF-α, IL-6, IL-1β, iNOS
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and COX2, whereas in HO/siSTIM1 group of cells, the expression levels of these genes were reduced (Fig. 4E). In line with the results above, p-NF-κB caused by HO was also down-regulated by STIM1 knockdown (Fig. 4F). In addition, HO exposure resulted in higher ROS generation compared to the Con group using DCF analysis, which was significantly reduced by STIM1 silence (Fig. 4G). Also, HO-induced
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reduction in HO1 and Nrf2 was restored by STIM1-knockdown (Fig. 4H). Moreover, HO exposure led to over-expression of NFAT1, Orai1 and active Caspase-3, while being blocked in STIM1-knockdown group (Fig. 4I). Finally, silencing STIM1
4J). 4. Discussion
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reduced TUNEL-positive cells with HO exposure, suggesting apoptosis blockage (Fig.
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Accordingly, liver ischemia/reperfusion (I/R) is a severe problem when patients undergo elective liver resections, transplantation, or other injuries, which may result in hepatic dysfunction, and even reduce the survival rate in the perioperative period [2,3,19,20]. Hence, attenuating I/R injury is important for patients’ postoperative recovery. To date, various factors have been reported to be involved in the development of liver I/R injury [21]. However, the underlying molecular mechanism is not fully understood. Thus, further research is required to find new target for attenuating hepatic I/R injury. In our present study, we found that mice after liver I/R injury showed higher expression of STIM1 from mRNA and protein levels. Importantly, STIM1-knockout attenuated liver pathological alterations, as well as
ACCEPTED MANUSCRIPT reduced AST and ALT levels in serum, contributing to the improvement of liver damage in mice with I/R damage. Further, STIM1-/- mice reversed inflammation, oxidative stress and apoptosis in the liver tissue of mice with hepatic I/R injury. Moreover, Kupffer cells with STIM1 knockdown following hypoxia exhibited similar results. Therefore, we hypothesized that STIM1 might be also recognized as a
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potential target for alleviating patients suffering from I/R injury. And we attempted to reveal that STIM1 was implicated in hepatic I/R injury both in vivo and in vitro.
STIM1 is a Ca2+ sensor, and upon Ca2+ store depletion, STIM1 translocate into ER/plasma membrane junctions and couples to activate Orai1 [22,23]. Further, it
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could modulate ROS sensor to trigger Ca2+ entry [24]. Ca2+ homeostasis is closely associated with cellular processes, regulating metabolism, signal transduction,
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transcriptional modulation, cell proliferation, as well as cell death [11,12,17]. As previously reported, STIM1 was participated in neutrophil-regulated killing of bacteria and ischemia/reperfusion injury in vivo [25]. Here, knockdown of STIM1 attenuated intracellular Ca2+ overload in hypoxia-treated Kupffer cells in vitro using flow cytometry, which was in agreement with previous studies. In addition, we also found that STIM1 expressed highly in the liver of mice with hepatic I/R injury.
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In a variety of cells, the coordination of Ca2+ release and entry mechanisms gives rise to sustained Ca2+ oscillations important for modulating cellular responses, such as gene expressions and cell fate eventually [10,11,26]. These responses are regulated by
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transcription factors, including NF-κB [27]. NF-κB plays an important role in mediating inflammatory response by potentiating the release of pro-inflammatory cytokines [28]. The NF-κB activation associated with an increase of TNF-α, IL-1β,
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and IL-6 in I/R injury [29]. In our study, mice with liver I/R injury showed higher levels of pro-inflammatory cytokines in serum and in liver tissue samples, including TNF-α, IL-6, IL-1β, iNOS and COX2. Notably, STIM1-deletion markedly reduced the expression of these pro-inflammatory cytokines in comparison to the WT/I/R group, which was along with the decreased p-NF-κB expressions. Also, in vitro, STIM1-knockdown reversed hypoxia-caused over-expression of pro-inflammatory cytokines, as well as p-NF-κB. Therefore, STIM1 played an important role in the development of hepatic I/R injury through modulating NF-κB-linked inflammation. We further verified that STIM1 was implicated in oxidative stress progression, modulating liver I/R injury. Here, we found that liver SOD activity, HO1 and Nrf2
ACCEPTED MANUSCRIPT expressions were markedly down-regulated, while MDA and ROS levels in mice with hepatic I/R injury were up-regulated. Notably, STIM1-/- reduced oxidative stress by reversing the changes of these indicators. As previously reported, ROS can regulate the cytosolic Ca2+ signals, and oxidants are included in the modulation of intracellular Ca2+ release channels [30]. Therefore, we supposed that there might be a possible
reveal the underlying molecular mechanism.
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interrelation between STIM1 and ROS, and further study is still required in future to
Moreover, NFAT1 is another important transcription factor, which is involved in Ca2+ oscillations to regulate inflammatory cytokine expression [31]. NFAT1
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mediates TNF-α up-regulation [32]. In our present study, we found that STIM1deletion significantly reduced NFAT1 expression in the liver of mice with I/R
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damage, indicating that STIM1 could perform its role in mediating inflammation by controlling NFAT1 expressions. STIM1/Orai1 is required for the activation of various cell types, including immune cells [33]. Here, we found that Orai1 was also expressed highly in the liver of I/R mice, which was significantly reduced by STIM1-deficiency. And in STIM1-knockdown Kupffer cells with hypoxia exposure,
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similar results were observed.
Liver I/R injury is also a complex process typified by hepatocyte apoptosis [34]. In hepatic I/R injury, the anti-apoptotic protein decreased and oxidative damage to the enzyme complexes triggered apoptosis. Previous studies suggested that inhibition
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of Caspases significantly decreased I/R damage [35]. Therefore, apoptosis plays a key role in liver damage induced by I/R. In the present study, I/R up-regulated the number of TUNEL-positive cells in vivo, which was inhibited by STIM1 knockout.
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In line with the in vivo study, hypoxia insult increased the number of apoptotic cells, and Caspase-3 cleavage, and STIM1-knockdown significantly suppressed Kupffer cell apoptosis. We speculated that the beneficial effects of STIM1-inhibition in hepatic I/R injury were partially linked to the suppression of apoptosis. The findings here were in accordance with previous studies that STIM1 inhibition prevented cell death through apoptosis-related signals [36]. However, further study in future is required to comprehensively reveal the underlying correlation between oxidative stress and apoptosis modulated by STIM1.
ACCEPTED MANUSCRIPT In conclusion, STIM1-deficiency protected liver against I/R injury through reducing inflammation, oxidative stress and apoptosis, which might be associated with intracellular Ca2+ overload. Herein, STIM1 could be considered as a potential therapeutic target for human suffering from I/R injury.
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References [1] Inoue Y, Shirasuna K, Kimura H, et al. NLRP3 regulates neutrophil functions and contributes
to
hepatic
ischemia–reperfusion
inflammasomes. J Immunol. 192 (2014) 4342-4351.
injury
independently
of
SC
[2] Li CX, Ng KTP, Shao Y, et al. The inhibition of aldose reductase attenuates hepatic ischemia-reperfusion injury through reducing inflammatory response.
M AN U
Ann Surg. 260 (2014) 317-328.
[3] McDonald KA, Huang H, Tohme S, et al. Toll-like receptor 4 (TLR4) antagonist eritoran tetrasodium attenuates liver ischemia and reperfusion injury through inhibition of high-mobility group box protein B1 (HMGB1) signaling. Mol Med. 20 (2014) 639.
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[4] Schlegel A, de Rougemont O, Graf R, et al. Protective mechanisms of endischemic cold machine perfusion in DCD liver grafts. J Hepatol. 58 (2013) 278286.
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[5] Zhai Y, Petrowsky H, Hong JC, et al. Ischaemia–reperfusion injury in liver transplantation—from bench to bedside. Nat Rev Gastro Hepat. 10 (2013) 79-89.
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[6] Sun Y, Pu LY, Lu L, et al. N-acetylcysteine attenuates reactive-oxygen-speciesmediated endoplasmic reticulum stress during liver ischemia-reperfusion injury. World J Gastroenterol. 20 (2014) 15289.
[7] Kalogeris T, Bao Y, Korthuis RJ. Mitochondrial reactive oxygen species: a double edged sword in ischemia/reperfusion vs preconditioning. Redox Bio. 2 (2014) 702-71. [8] Tao X, Wan X, Xu Y, et al. Dioscin attenuates hepatic ischemia-reperfusion injury in rats through inhibition of oxidative-nitrative stress, inflammation and apoptosis. Transplantation. 98 (2014) 604-611.
ACCEPTED MANUSCRIPT [9] Ernst IMA, Fliegert R, Guse AH. Adenine dinucleotide second messengers and T-lymphocyte calcium signaling. Front Immuno. 4 (2013). [10] Chan G, Mooney DJ. Ca 2+ released from calcium alginate gels can promote inflammatory responses in vitro and in vivo. Acta Biomater. 9 (2013) 9281-9291.
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[11] Zhou Y, Meraner P, Kwon HT, et al. STIM1 gates the store-operated calcium channel ORAI1 in vitro. Nat Struct Mol Biol. 17 (2010) 112-116.
[12] Galan C, Jardín I, Dionisio N, et al. Role of oxidant scavengers in the prevention
SC
of Ca2+ homeostasis disorders. Molecules. 15 (2010) 7167-7187.
[13] Nace GW, Huang H, Klune JR, et al. Cellular‐specific role of toll‐like receptor 4 in hepatic ischemia‐reperfusion injury in mice. Hepatology. 58 (2013)
M AN U
374-387.
[14] Bamboat ZM, Balachandran VP, Ocuin LM, et al. Toll‐like receptor 9 inhibition confers protection from liver ischemia–reperfusion injury. Hepatology. 51 (2010) 621-632.
TE D
[15] Franken L, Klein M, Spasova M, et al. Splenic red pulp macrophages are intrinsically superparamagnetic and contaminate magnetic cell isolates. Sci Rep. 5 (2015) 12940.
EP
[16] Li W, Zhang M, Xu L, et al. The apoptosis of non-small cell lung cancer induced by cisplatin through modulation of STIM1. Exp Toxicol Pathol. 65 (2013) 1073-
AC C
1081.
[17] Mason RP. Pleiotropic effects of calcium channel blockers. Curr Hypertens Rep. 14 (2012) 293-303.
[18] Park CY, Shcheglovitov A, Dolmetsch R. The CRAC channel activator STIM1 binds and inhibits L-type voltage-gated calcium channels. Science. 330 (2010) 101-105. [19] Jaeschke H, Woolbright BL. Current strategies to minimize hepatic ischemia– reperfusion injury by targeting reactive oxygen species. Transplant Rev. 26 (2012) 103-114.
ACCEPTED MANUSCRIPT [20] Weigand K, Brost S, Steinebrunner N, et al. Ischemia/Reperfusion injury in liver surgery and transplantation: pathophysiology. HPB Surg. 2012 (2012). [21] Datta G, Fuller BJ, Davidson BR. Molecular mechanisms of liver ischemia reperfusion injury: insights from transgenic knockout models. World J
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Gastroenterol. 19 (2013) 1683. [22] Zhou Y, Srinivasan P, Razavi S, et al. Initial activation of STIM1, the regulator of store-operated calcium entry. Nat Struct Mol Biol. 20 (2013) 973-981.
[23] Lee KP, Yuan JP, Hong JH, et al. An endoplasmic reticulum/plasma membrane
SC
junction: STIM1/Orai1/TRPCs. FEBS letters. 584 (2010) 2022-2027..
[24] Steinckwich N, Schenten V, Melchior C, et al. An essential role of STIM1, Orai1,
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and S100A8–A9 proteins for Ca2+ signaling and FcγR-mediated phagosomal oxidative activity. J Immuno. 186 (2011) 2182-2191.
[25] Pan LJ, Zhang ZC, Zhang ZY, et al. Effects and mechanisms of store-operated calcium channel blockade on hepatic ischemia-reperfusion injury in rats. World J Gastroenterol. 18 (2012) 356.
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[26] Li W, Llopis J, Whitney M, et al. Cell-permeant caged InsP3 ester shows that Ca2+ spike frequency can optimize gene expression. Nature. 392 (1998) 936-941. [27] Gong G, Waris G, Tanveer R, et al. Human hepatitis C virus NS5A protein alters
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intracellular calcium levels, induces oxidative stress, and activates STAT-3 and NF-κB. Proc Natl Acad Sci. 98 (2001) 9599-9604.
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[28] Haddad JJ. Redox regulation of pro-inflammatory cytokines and IκB-α/NF-κB nuclear translocation and activation. Biochem Biophys Res Commun. 296 (2002) 847-856.
[29] Lan L, Tao J, Chen A, et al. Electroacupuncture exerts anti-inflammatory effects in cerebral ischemia-reperfusion injured rats via suppression of the TLR4/NF-κB pathway. Int J Mol Med. 31 (2013) 75-80. [30] Bogeski I, Kappl R, Kummerow C, et al. Redox regulation of calcium ion channels: chemical and physiological aspects. Cell Calcium. 50 (2011) 407-423.
ACCEPTED MANUSCRIPT [31] Dula SB, Jecmenica M, Wu R, et al. Evidence that low-grade systemic inflammation can induce islet dysfunction as measured by impaired calcium handling. Cell Calcium. 48 (2010) 133-142. [32] Ding Y, Huang Y, Song N, et al. NFAT1 mediates placental growth factorinduced myelomonocytic cell recruitment via the induction of TNF-α. J Immuno.
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184 (2010) 2593-2601.
[33] Feske S. ORAI1 and STIM1 deficiency in human and mice: roles of store‐operated Ca2+ entry in the immune system and beyond. Immunol Rev.
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231 (2009) 189-209.
[34] Chen Z, Chua CC, Ho YS, et al. Overexpression of Bcl-2 attenuates apoptosis
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and protects against myocardial I/R injury in transgenic mice. Am J Physiol Heart Circ Physiol. 280 (2001) H2313-H2320.
[35] Daemen MARC, de Vries B, Buurman WA. Apoptosis and inflammation in renal reperfusion injury. Transplantation. 73 (2002) 1693-1700. [36] Chiu WT, Tang MJ, Jao HC, et al. Soft substrate up-regulates the interaction of
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STIM1 with store-operated Ca2+ channels that lead to normal epithelial cell apoptosis. Mol Biol Cell. 19 (2008) 2220-2230.
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Figure 1. Expression of STIM1 is increased in liver tissue from mice afer I/R. Mice underwent 1 h of ischemia and various times of reperfusion. (A) RT-PCR and (B) western blot analysis for liver STIM1 mRNA and protein were performed. (C) IHC analysis of hepatic STIM1 levels. (D) STIM1 positive levels were quantified following IHC analysis. Data represent means ± SEM (n=8). *P<0.05,
**
P<0.01 and
***
P<0.001 vs. Con (0 h after reperfusion) mice.
Figure 2. STIM1 deletion decreases liver I/R injury. The mice underwent 1 h of ischemia, followed by 6 h of reperfusion. (A) H&E-stained liver sections from mice were exhibited. (B) Serum ALT and AST levels were measured. (C) Serum proinflammatory cytokines, including TNF-α, IL-6 and IL-1β, were examined using
ACCEPTED MANUSCRIPT ELISA methods. (D) Hepatic pro-inflammatory cytokines as indicated were calculated by RT-qPCR. (E) Western blot analysis of p-NF-κB in liver of mice. (F) Liver SOD activity, (G) MDA levels and (H) total ROS levels were evaluated. (I) Western blot analysis of HO1 and Nrf2 expression levels in liver of mice. Data represent means ± SEM (n=8). *P<0.05, **P<0.01 and
***
P<0.001 vs. WT/Con group
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of mice. Figure 3. STIM1 knockout attenuates hepatic apoptosis after I/R. The mice underwent 1 h of ischemia, followed by 6 h of reperfusion. (A) Western blot assays of liver NFAT1, Orai1 and cleaved Caspase-3. (B) TUNEL analysis of liver sections for
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apoptosis measurement. (C) IHC analysis of cleaved Caspase-3. The quantification of cleaved Caspase-3 levels was showed. Data represent means ± SEM (n=8). P<0.001 vs. WT/Con group of mice. +P<0.05 vs. WT/I/R group of mice.
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Figure 4. Suppressing STIM1 expression ameliorates inflamatory response, ROS generation and apoptosis in hypoxia-treated Kuppfer cells. (A, B) Kuppfer cells were treated with hypoxia at the indicated time points. STIM1 levels in cell lysates were detected using qPCR and western blot. *P<0.05, **P<0.01 and ***P<0.001vs. Con
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(0 h after hypoxia treatment). (C) Kuppfer cells were treated with STIM1-siRNAs (#1 and #2) or negative control (si-Con) for 24 h, and then exposure to hypoxia for 6 h. STIM1 expression levels were measured using western blot analysis. Kuppfer cells were transfected with STIM1#2 siRNA for 24 h, and then treated with hypoxia (HO)
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for another 24 h, followed by further research. (D) Measurement of cytoplasmic free Ca2+ concentration using flow cytometry. (E) RT-qPCR analysis of pro-inflammatory cytokines. (F) Western blot analysis of p-NF-κB. (G) ROSS generation was detected
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via DCF analysis. (H) HO1 and Nrf2 protein levels were measured using western blot analysis. (I) NFAT1, Orai1 and cleaved Caspase-3 protein levels were examined using western blot analysis. (J) TUNEL analysis for apoptosis calculation in Kuppfer cells treated as described. Data represent means ± SEM (n=6). group. +P<0.05 and ++P<0.01 vs. HO group.
***
P<0.001 vs. Con
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ACCEPTED MANUSCRIPT Highlights STIM1 expression was up regulated in liver tissue of mice following I/R. STIM1 deletion ameliorates hepatic I/R injury by reducing inflammatory
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response and oxidative stress. STIM1 knockout attenuates hepatic apoptosis after I/R.
Inhibition of STIM1 expression ameliorates inflammatory response, ROS
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generation and apoptosis in hypoxia-treated Kupffer cells