Homeodomain-interacting protein kinase 2 regulates NLRP3 inflammasome activation through endoplasmic reticulum stress in septic liver injury

Abstract

Objective

Septic liver injury is a major burden for the clinical management of sepsis. Hepatocyte cell death plays a crucial pathophysiological role in sepsis. A recent study proposed that NLRP3 inflammasome-mediated pyroptosis participates in septic liver injury. Therefore, investigating the mechanism controlling this process may help manage sepsis.

Methods

We investigated the role of homeodomain-interacting protein kinase 2 (HIPK2) in regulating the NLRP3 inflammasome in vivo using mouse models and in vitro in primary hepatocytes.

Results

HIPK2 could improve liver injury and survival in a mouse model of sepsis. Overexpression of HIPK2 could suppress NLRP3 and caspase-1-p20 expression, while HIPK2 knockdown led to higher levels of these two molecules. Importantly, HIPK2 could suppress endoplasmic reticulum (ER) stress. Pharmacologically inhibiting ER stress could abolish activation of the NLRP3 inflammasome in hepatocytes with HIPK2 knockdown.

Conclusion

HIPK2 can regulate ER stress and NLRP3 inflammasome activation in the liver during sepsis, and HIPK2-mediated suppression of ER stress participates in regulating NLRP3 inflammasome activation. The present study highlights the role of HIPK2 in regulating the inflammasome in septic liver injury, which may serve as a target for managing sepsis.

Introduction

Hepatic homeostasis and metabolism play a crucial role during sepsis. ¹ Inflammatory factors, pathogens, and pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS), can impair the function of hepatocytes and induce massive levels of cell death, including apoptosis, necrosis, autophagy, and pyroptosis.^1,2 Clinical management of septic liver injury is focused on decreasing hepatocyte cell death and restoring liver function. ³ Therefore, an in-depth understanding of the specific cell death mechanisms in hepatocytes may help with the development of therapeutic molecules to improve liver function.

Certain triggers induce systematic inflammation, while sepsis can activate multiple adaptive mechanisms that protect the body from organ damage. However, uncontrolled and exacerbated responses could lead to excessive amounts of cell death. ⁴ Unfolded protein response (UPR)-induced endoplasmic reticulum (ER) stress has been proposed as a crucial mechanism in sepsis-related organ injury, which activates damage in mitochondria and other organelles and promotes multiple processes of cell death. ⁵ Recent studies have highlighted inflammasome-mediated pyroptosis as a novel type of cell death during sepsis.^6–9 Hepatocytes stimulated with LPS or other PAMPs can activate Toll-like receptor (TLR) signaling and induce the assembly of inflammasomes, among which the NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome was the main one reported. ⁹ Activation of the NLRP3 inflammasome leads to the cleavage of caspase-1 and subsequent maturation of interleukin (IL)-1β secreted from hepatocytes through pyroptosis. This causes cell membrane rupturing and ultimately cell death. ¹⁰ Investigating the pyroptosis mechanism in hepatocytes may help with the development of targeted therapy and improve liver function during sepsis.

Homeodomain-interacting protein kinase 2 (HIPK2) regulates homeostasis in stress-related cellular injury.^11,12 Previous research reported its crucial roles in DNA damage repair, inflammatory cytokine transcription, and apoptosis.^13–15 One study also found that HIPK2 could interact with nuclear factor E2-related factor 2 (NRF2) and regulate hepatocyte inflammatory responses, thus protecting the liver from toxins. ¹⁶ However, the mechanism of HIPK2 in septic liver injury remains unknown and it is unclear if HIPK2 can influence inflammasome-related pyroptosis.

In the present study, we investigated the potential role of HIPK2 in septic and inflammatory liver injury in vitro and in vivo. We demonstrated its function in regulating the NLRP3 inflammasome and possible mechanisms through ER stress. Our study highlights HIPK2 as an essential regulator of pyroptosis in hepatocytes during sepsis and implicates it as a potential target for the management of septic liver injury.

Materials and Methods

Animals

Six-to-eight-week-old mice were purchased from the Shanghai Laboratory Animal Center (SLAC) (China) and raised in a specific pathogen-free (SPF) room at the Research Center of No. 906 Hospital (Approval No. 906202004112). The room was controlled at 18°C to 22°C, 50% to 60% humidity, and a 12-hour light–dark cycle. Food and water were always accessible to the mice. The animal experiments in the present study were reviewed by the Ethics Committee of No. 906 Hospital, People’s Liberation Army (PLA), and all mice subjected to experiments were under anesthesia using 2.5% sevoflurane. All mice received humane care, and experiments were performed according to the relevant guidebooks and regulations.

For HIPK2 transfection in vivo, recombinant adenoviruses expressing murine HIPK2 (labeled as Oe-HIPK2) for overexpression or a short hairpin RNA (shRNAs) targeting HIPK2 (labeled as sh-HIPK2) for knockdown were generated using the pAd-Easy system (Invitrogen, Waltham, MA, USA) and purchased from Obio (Shanghai, China). The murine albumin gene promoter was inserted into the adenovirus system to ensure that HIPK2 would only be overexpressed in the liver. The obtained adenovirus was injected through the mouse tail vein, and the virus was diluted in phosphate-buffered saline (PBS) at a concentration of 2 × 10⁹ plaque-forming units (PFUs).

Animal model

For the LPS-induced sepsis model, 10 mg/kg LPS (Escherichia coli O111:B4, Sigma, Shanghai, China) was diluted in 1 mL of PBS and intraperitoneally injected. For the cecal ligation and puncture (CLP)-induced sepsis model, after the mice were anesthetized with sevoflurane, a middle abdominal incision was made and the cecum was ligated at one-half of the distal terminal. The abdomen was then sutured and 1 mL of 0.9% saline was injected for fluid resuscitation. The sham group received the incision and bowel operation but not ligation. For the LPS-induced sepsis model, each group contained at least three mice, while each group in the CLP-induced sepsis model had at least five mice.

Liver enzyme and cytokine analyses

Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels can indicate liver function. ¹⁷ Blood samples were collected through enucleation and centrifuged at 1000×g for 10 minutes. Then, the serum was collected and analyzed using biochemical kits from Beyotime (P0338L for AST, P0321M for ALT; Shanghai, China). For cytokine analysis, serum was analyzed for mouse interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-1β with enzyme-linked immunosorbent assay (ELISA) kits from R&D Systems (Shanghai, China). All experiments were performed according to the manufacturer’s instructions.

Hematoxylin–Eosin (H&E) staining

Liver samples were harvested 12 hours after the CLP procedure. The liver tissues were fixed with 10% formalin. Then, the fixed samples were processed into paraffin blocks, sectioned at 5 μm, and stained with H&E for histological analysis. Microscopy images are presented at 100× and 200×, and the results are marked with a scale bar at 100 μm and 200 μm.

Isolation of primary hepatocytes

C57 BL/6 mice were anesthetized and underwent surgical dissection. The portal veins were isolated and washed to remove the blood. Collagenase (Sigma-Aldrich, St. Louis, MO, USA) perfusion was then performed, after which the liver was moved to a cell culture dish and minced. Then, hepatocytes were separated by aspiration and filtered through a 70-μm membrane (Millipore, Burlington, MA, USA). Cells were washed with cold Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen), then centrifuged at 50×g for 4 minutes at 4°C. Cells were incubated in 6-cm dishes with DMEM containing 10% fetal bovine serum (FBS, Invitrogen) at a density of 1 × 10⁷ cells/dish. The culture medium was changed every 6 hours.

Hepatocyte stimulation and transfection

Cells were seeded at a density of 5 × 10⁶ cells/dish for hepatocyte stimulation. The cells were stimulated with LPS (1 μg/mL) for 6 hours, then with nigericin (20 μM) for 1 hour. For HIPK2 overexpression in hepatocytes, cells were transfected with adenovirus (oe-HIPK2 or sh-HIPK2) (2 × 10³ PFUs per well) for 16 hours. The medium was changed 16 hours post-transfection, and cells were subjected to further experiments. The ER stress inhibitor, tauroursodeoxycholic acid (TUDCA), was obtained from MCE (Shanghai, China).

Cell viability assay

Cell count kit-8 (CCK-8) (Dojindo, Tokyo, Japan) was used to measure the viability of hepatocytes in the experiments. Primary hepatocytes were seeded into 96-well plates at 1 × 10⁴ cells per well in medium. Cell stimulation included LPS (1 μg/mL) for 6 hours and nigericin (20 μM), poly(dA:dT) (1 μg/mL), flagellin (10 μM), or MDP (200 ng/mL) for 1 hour. The medium was removed, then 100 mL of DMEM containing CCK-8 (10%) was added to each well. The cells were then incubated for 2 hours at 37°C, and the absorbance of each well at 450 nm was measured using a Microtiter Plate Reader (TECAN, Bern, Switzerland). The average absorbance of 10 independent wells for each group was obtained. Seeded cells without stimulation were measured following the same procedure and regarded as 100% viability.

Quantitative real-time polymerase chain reaction (qPCR)

Total RNA was extracted from liver tissues or cells using TRIZOL (Thermo Fisher Scientific, Shanghai, China). Then, reverse transcription was performed in a 10-μL reaction containing 1 μg of total RNA, a reverse transcription premix, and oligo (dT) primers. qPCR was performed with the SYBR Green PCR system using an ABI 7500 thermal cycler (Thermo Fisher Scientific, Waltham, MA, USA). Cycling conditions were as follows: 95°C for 3 minutes, followed by 40 cycles of 95°C for 10 s, 60°C for 5 s, and 72°C for 10 s. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was used for internal reference. The primers were: GAPDH, sense, 5′-CATTCAAGACCGGACAGAGG-3′, antisense, 5′-ACATACTGCACACCAGCATCACC-3′; HIPK2, sense, 5′-CCAGGCCTGCTTGCTCAG-3′, antisense, 5′-TGTACAGATGTGTGGGTGGC-3′. Relative mRNA expression levels were determined by the 2^−ΔΔCt method.

Western blot analysis

Liver tissue or cell samples were lysed in radioimmunoprecipitation assay (RIPA) buffer (Beyotime), and protein concentrations were determined using a BCA assay (Thermo Fisher Scientific). The protein samples (about 30 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride (PVDF) membranes (Merck, Frankfurt, Germany), and blocked with 5% non-fat dry milk in PBS with Tween (PBST), pH 7.5. Membranes were incubated with primary antibodies for 4 hours at room temperature or overnight at 4°C, then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 3 hours at room temperature. An enhanced chemiluminescence kit (Pierce, Waltham, MA, USA) was used for analysis. The anti-HIPK2 (#5091), anti-NLRP3 (#15101), anti-β-actin (#3700), and anti-pro-IL-1β (#12242) primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) and diluted to 1:1000.

Statistical analysis

All data are presented as mean ± standard error of the mean (SEM). A student’s t-test (comparisons between two groups) and one-way analysis of variance test (comparisons between multiple groups) followed by a post hoc analysis using Tukey’s honestly significant difference test were used for comparisons and analyzed with SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). Statistical significance was indicated by a P-value <0.05.

Results

Elevated HIPK2 attenuates liver injury in sepsis

We used LPS- and CLP-induced sepsis models and measured HIPK2 expression levels in the liver. In these models, the serum AST and ALT levels were highly elevated 6 hours after the operation (Figure 1a, b; P < 0.05). In the CLP model, HIPK2 expression levels were decreased at 3 hours and 6 hours post-operation (Figure 1c), while HIPK2 mRNA levels in the LPS model were reduced at 3 hours and reached the lowest level at 6 hours after the LPS injection (Figure 1d). Interestingly, HIPK2 expression remained reduced at the 24-hour timepoint in both the LPS and CLP models. Our data also show that the HIPK2 protein levels were decreased 12 hours after the CLP procedure (Figure 1e). These results indicate that HIPK2 expression was reduced during the septic liver injury.

Figure 1.

Elevated HIPK2 attenuates liver injury in sepsis. (a) Serum AST and (b) ALT concentrations in control, LPS-injected, sham surgery, and CLP model mice. HIPK2 mRNA levels in the liver of mice with (c) CLP or (d) LPS injection (n = 5). (e) HIPK2 protein levels in mice livers 12 hours after the CLP procedure (n = 5). (f) HIPK2 mRNA levels in the organs of mice 6 hours after CLP (n = 5). (g) HIPK2 protein levels in the livers of mice transfected with an HIPK2-overexpression or vector adenovirus (n = 5 per group). (h) Hematoxylin and eosin staining of the livers of mice transfected with an HIPK2-overexpression or vector adenovirus that underwent the CLP procedure. The upper bar shows 200 μm and the lower bar shows 100 μm. (i–l) Serum AST and ALT concentrations in mice with HIPK2 overexpression or vector that underwent the CLP procedure (i, j) or LPS injection (k, l) (n = 3). (m–o) Serum (m) IL-6, (n) TNF-α, and (o) IL-1β concentrations in mice with HIPK2 overexpression or vector that underwent the CLP procedure (n = 3). Data are presented as mean ± SEM; *P < 0.05 compared with the sham or control group.

We then investigated whether elevated expression of HIPK2 could be protective in liver injury. We overexpressed HIPK2 (oe-HIPK2) with adenovirus injection through the tail vein and investigated any altered HIPK2 expression in the liver. These experiments indicate that oe-HIPK2 could successfully enhance both HIPK2 mRNA and protein expression in the liver compared with the ad-vector. Moreover, HIPK2 expression levels in other organs remained unaffected (Figure 1f, g; P < 0.05). We then evaluated the effects of HIPK2 overexpression in the LPS and CLP-induced septic liver injury models. Overexpressed HIPK2 in the liver could attenuate CLP-induced liver injury (Figure 1h; P < 0.05) and decrease the serum levels of AST and ALT, as well as of IL-6, TNF-α, and IL-1β, compared with the vector group in the grinding fluids of the liver. However, HIPK2 knockdown using sh-HIPK2 increased the AST, ALT, IL-6, TNF-α, and IL-1β levels (Figure 1i–o; P < 0.05). IL-1β showed the most significant reduction after HIPK2 overexpression. Thus, these results indicate that HIPK2 can show protective effects in septic liver injury.

HIPK2 suppressed activation of the NLRP3 inflammasome in septic liver injury

Because IL-1β was the most significantly reduced factor following HIPK2 overexpression, we focused on if HIPK2 could regulate inflammasome activation. We investigated the effects of HIPK2 overexpression on the NLRP3, melanoma 2 (AIM2), and NLR family CARD domain-containing protein 4 (NLRC4) inflammasomes, which LPS could induce with nigericin, poly(dA:dT), flagellin, and MDP treatment, respectively. We found that HIPK2 overexpression could only decrease IL-1β levels in the NLRP3 inflammasome, while showing limited effects in the AIM2, NLRC4, and NALP3 inflammasomes (Figure 2a; P < 0.05). We also found that cell viability was increased in hepatocytes stimulated with LPS and nigericin, while decreased in those stimulated with LPS and poly(dA:dT), flagellin, or MDP (Figure 2b; P < 0.05).

Figure 2.

HIPK2 suppresses activation of the NLRP3 inflammasome in septic liver injury. (a) Supernatant IL-1β and (b) cell viability levels of primary hepatocytes with HIPK2 overexpression or vector stimulated with LPS and nigericin, poly(dA:dT), flagellin, or MDP (n = 3). (c) Western blot analysis and (d) quantitative analysis of hepatocytes with HIPK2 overexpression or vector stimulated with LPS and nigericin (n = 3). Data are presented as mean ± SEM; *P < 0.05 compared with the control group.

We further investigated NLRP3 inflammasome activation and found that HIPK2 overexpression led to NLRP3 suppression and decreased caspase-1-p20. Additionally, oe-HIPK2 also decreased gasdermin D (GSDMD) levels. However, overexpression of HIPK2 did not alter the pro-IL-1β or pro-caspase-1 expression levels in cell lysates (Figure 2c, d; P < 0.05). These results suggest that HIPK2 may regulate NLRP3 inflammasome activation in septic liver injury.

HIPK2 downregulates ER stress in septic liver injury

We then examined how HIPK2 can regulate inflammasome activation. Because ER stress can reportedly influence NLRP3 inflammasome activation, we questioned if HIPK2 could regulate NLRP3 by suppressing ER stress.^18,19 We first found that markers of ER stress, inositol-requiring enzyme (IRE)-1α, double-stranded RNA-dependent protein kinase (PKR)-like ER kinase (PERK), and activating transcription factor 6 (ATF6), were all elevated in the liver during sepsis. Phosphorylation of IRE-1α and PERK and ATF6 protein levels were increased in LPS-induced liver injury (Figure 3a, b; P < 0.05). However, when HIPK2 was overexpressed, IRE-1α and PERK phosphorylation decreased, while ATF6 was downregulated (Figure 3a, b; P < 0.05). A similar phenomenon was also observed in LPS-induced hepatocytes in vitro. Stimulation with LPS and nigericin significantly enhanced the phosphorylation of IRE-1α and PERK and ATF6 protein levels, while overexpression of HIPK2 decreased p-PERK levels (Figure 3c, d; P < 0.05). However, IRE-1α phosphorylation did not decrease in vitro. These results suggest that HIPK2 can decrease phosphorylation of IRE-1α and PERK and may subsequently suppress the ER stress in hepatocytes during septic liver injury.

Figure 3.

HIPK2 downregulates endoplasmic reticulum (ER) stress in septic liver injury. (a) Western blot analysis and (b) quantitative analysis of the livers of mice transfected with HIPK2-overexpression or vector adenovirus and stimulated with LPS peritoneal injection (n = 5). (c) Western blot analysis and (d) quantitative analysis of hepatocytes with HIPK2-overexpression or vector stimulated with LPS and nigericin (n = 5). Data are presented as mean ± SEM; *P < 0.05 compared with the control group.

HIPK2-mediated suppression of ER stress is essential for regulating the NLRP3 inflammasome in septic liver injury

We then investigated whether the effects of HIPK2 on ER stress are essential for NLRP3 inflammasome regulation. Using an inhibitor of ER stress, TUDCA, we found that this inhibition could suppress IL-1β levels in hepatocytes treated with LPS and nigericin (Figure 4a; P < 0.05), suggesting that ER stress positively regulates the NLRP3 inflammasome. Furthermore, after knocking down HIPK2 levels with the shRNA adenovirus, we observed enhanced phosphorylation of IRE-1α and PERK.

Figure 4.

HIPK2-mediated suppression of endoplasmic reticulum (ER) stress is essential for regulating the NLRP3 inflammasome in septic liver injury. (a) Supernatant IL-1β levels in primary hepatocytes stimulated with LPS and nigericin with or without TUDCA treatment (n = 3). (b) Western blot analysis and (c) quantitative analysis of hepatocytes with HIPK2 knockdown (sh-HIPK2) or vector stimulated with or without LPS and nigericin (n = 3). d: Supernatant IL-1β levels in primary hepatocytes with HIPK2 knockdown or vector stimulated with LPS and nigericin with or without TUDCA treatment (n = 3). (e) Western blot analysis and (f) quantitative analysis of hepatocytes with HIPK2 knockdown (sh-HIPK2) or vector stimulated with LPS and nigericin with or without TUDCA treatment (n = 3). Data are presented as mean ± SEM; *P < 0.05 compared with the control group.

HIPK2 knockdown also increased the NLRP3 and caspase-1-p20 levels in LPS and nigericin-treated hepatocytes (Figure 4b, c; P < 0.05). In addition, sh-HIPK2 enhanced IL-1β production from hepatocytes, while TUDCA treatment abolished this increased IL-1β (Figure 4d; P < 0.05). These results suggest the negative regulation of HIPK2 in ER stress and the NLRP3 inflammasome. Additionally, inhibiting ER stress using TUDCA treatment could abolish the activation of the NLRP3 inflammasome in sh-HIPK2 hepatocytes (Figure 4e, f; P < 0.05). These results suggest that HIPK2-mediated suppression of ER stress is essential for regulating the NLRP3 inflammasome in hepatocytes during septic liver injury.

Discussion

Organ dysfunction plays a crucial role in the pathophysiology of sepsis. ³ Though inflammatory responses act as an initiator, managing septic organ dysfunction remains the central therapy of sepsis treatment. ³ The liver plays a vital role in homeostasis and metabolism.^1,2 Impaired liver function during sepsis usually leads to poor prognosis and inevitable death. ¹ Therefore, investigating methods for decreasing septic liver injury would help manage sepsis and improve patient prognosis.

The major burden of managing septic liver injury is hepatocyte cell death, which impairs the capacity for metabolism and toxin removal. ²⁰ During sepsis, cytokines or PAMPs, like LPS, can activate TLR signaling in hepatocytes and initiate apoptosis, autophagy, and other types of cell death. ²¹ Pyroptosis has been reported as a major type of cell death in liver injury, as the ruptured cells can induce subsequent cellular damage in neighboring hepatocytes. ²² Investigating potential regulators of pyroptosis may help improve liver function during sepsis.

In the present study, HIPK2, a stress-induced regulator in hepatocytes, could regulate NLRP3 inflammasome activation and suppress pyroptosis during septic liver injury. Our results suggested that HIPK2 expression levels were decreased in the liver during sepsis and could subsequently enhance hepatocyte pyroptosis. When HIPK2 was overexpressed, liver injury was decreased both in vivo and in vitro, suggesting that HIPK2 played a protective role during sepsis. These findings are in accordance with previous research that showed that HIPK2 could induce autophagy in hepatocytes, thus reducing liver inflammation and improving the survival of septic mice. ²¹ One study reported that autophagy could act as a regulator of the NLRP3 inflammasome, and autophagy- or proteasome-mediated NLRP3 degradation could negatively regulate NLRP3 inflammasome activation. ²³ Therefore, enhanced autophagy and decreased pyroptosis may result from similar upstream regulation.

We also investigated the possible mechanism of HIPK2-mediated NLRP3 inflammasome suppression. Our results showed that HIPK2 could also attenuate the phosphorylation of PERK and IRE-1α, two markers of ER stress.

An interesting phenomenon in the present study was that IRE-1α phosphorylation was reduced in the oe-HIPK2 group in vivo, while the in vitro study showed inconsistent results. This inconsistency possibly results from the disturbance of Kupffer cells that participate in multiple liver injuries through ER stress,^24,25 while the in vitro study may hinder their function. Further investigation for this specific subset is also needed.

Experiments also demonstrated that suppressing ER stress using the inhibitor TUDCA could abolish activation of the NLRP3 inflammasome in the hepatocytes with HIPK2 knockdown.^5,26 Research has reported that ER stress was an early signal in cellular injury that could influence numerous downstream molecular processes, including TLR signaling, apoptosis, and autophagy.^8,27 Here, we report that ER stress may also participate in HIPK2-mediated NLRP3 inflammasome suppression. Previous research showed that HIPK2 could regulate autophagy through calpain signaling, which is also related to calcium regulation through the ER. ²¹ Therefore, our study also suggests that HIPK2 potentially functions as a protective molecule by influencing the activation of ER stress, thus regulating numerous pathways. Though we reported that HIPK2 can regulate the phosphorylation of IRE-1α and PERK in ER stress, there is limited evidence that HIPK2 directly interacts with these molecules. Regulation of HIPK2 may not equally influence the three ER stress markers, as we showed that overexpression or knockdown of HIPK2 led to minor alterations of ATF6, suggesting that the effects on ER stress may be indirect. Thus, further investigation applying specific inhibition of ER stress is required. Moreover, the mechanism controlling how ER stress influences the NLRP3 inflammasome in this scenario should also be examined in more depth. Therefore, more detailed research is still needed.

In addition, we used the LPS and CLP models to evaluate the effects of HIPK2 on septic and inflammatory liver injury. Furthermore, we showed the possible mechanism in the LPS-induced liver injury model. Our work with these two models suggests that HIPK2 crucially regulates the liver injury induced by septic inflammation, though not utterly reflecting septic liver injury. Therefore, further translational studies of HIPK2 in clinical settings are needed.

There are several limitations in the present study. First, the specific mechanism of how HIPK2 influences ER stress is not fully clear. The regulation of IRE-1α, ATF6, and PERK includes several signaling mechanisms. Future studies investigating the interaction of HIPK2 with these signaling pathways should be investigated. Secondly, we only used a pharmaceutical inhibitor to analyze the essential role of ER stress in NLRP3 regulation. Further evaluation, such as a gene knockout model, should be used to verify the results.

The present study demonstrated that HIPK2 could suppress NLRP3 inflammasome activation in hepatocytes during septic liver injury, which is mediated through attenuation of ER stress. We showed that HIPK2 is a possible protective target that could be applied for managing septic liver injury.