Forsythiaside A Alleviates Lipopolysaccharide-Induced Acute Liver Injury through Inhibiting Endoplasmic Reticulum Stress and NLRP3 Inflammasome Activation

Abstract

The liver is the primary site of inflammation caused by bacterial endotoxins in sepsis, and septic acute liver injury (SALI) is usually associated with poor outcomes in sepsis. Forsythiaside A (FTA), an active constituent of Forsythia suspensa, has been reported to have anti-inflammatory properties, antioxidant properties, and protective properties against neuroinflammation, sepsis, and edema. Therefore, the purpose of the present study was to examine FTA’s potential effects on lipopolysaccharide (LPS)-induced SALI in mice. Our results indicated that pretreatment with FTA significantly attenuated aspartate aminotransferase (AST) and aminoleucine transferase (ALT) levels in plasma, ameliorated histopathological damage, inhibited hepatocyte apoptosis, diminished the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 in the liver from mice exposed to LPS. Furthermore, our data showed that the administration of LPS resulted in robust endoplasmic reticulum (ER) stress response, as evidenced by glucose-regulated protein 78 (GRP78) upregulation, phosphorylated-protein kinase R-like ER kinase (p-PERK) activation, elF2α phosphorylation, and activating transcription factor 4 (ATF4) and CHOP overexpression in the liver. This, in turn, led to nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) inflammasome activation, including the cleavage of caspase-1, secretion of IL-1β, and pyroptotic cell death in the liver specimens. Importantly, the ER stress response induced by the LPS challenge was blocked by FTA administration. Correspondingly, NLRP3 inflammasome activation was significantly ameliorated by the pretreatment with FTA. Thus, we demonstrated that FTA pretreatment could protect mice from LPS-induced SALI, and its protective effects were possibly mediated by inhibiting ER stress response and subsequent NLRP3 inflammasome activation.

INTRODUCTION

Liver dysfunction often occurs at the early stage of sepsis and is usually associated with a poor prognosis in clinical investigations.^1–3) Actually, septic acute liver injury (SALI) is not only being viewed as a consequence of shock and tissue hypoperfusion during sepsis but also as one of the main actors in the genesis and amplification of multiorgan dysfunction. The inflammatory response depends on the activation of the pattern recognition receptor (PRR) and is crucial to sepsis and SALI pathogenesis.^4,5) Belonging to the recently identified Nod-like receptor (NLR) family, nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) is unique in its ability to recognize various unrelated stimuli, including pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs).⁶⁾ Upon activation, NLRP3 recruits apoptosis-associated speck-like protein containing CARD (ASC) and caspase-1 to form NLRP3 inflammasome, subsequently leading to caspase-1 activation, maturation, and secretion of proinflammatory cytokines, such as interleukin (IL)-1β and IL-18, and initiating a lytic host cell death called pyroptosis.⁷⁾ NLRP3 plays a central role in innate immunity and inflammation; however, the aberrant NLRP3 inflammasome activation contributes to the occurrence and progression of multiple inflammatory diseases, including metabolic diseases, autoimmune diseases, infection, and sepsis.⁸⁾ Specifically, recent reports have indicated that the inflammasome pathway is also implicated in hepatocyte pyroptosis and liver inflammation and dysfunction.⁹⁾ Therefore, it will be extremely important to explore the association of NLRP3 inflammasome with SALI pathogenesis and, meanwhile, refine the upstream signaling that is induced to culminate in NLRP3 activation and inflammasome formation in SALI.

Endoplasmic reticulum (ER) serves as a platform for connecting cellular stress and inflammation.¹⁰⁾ Stress disturbances in redox regulation, inflammatory overload, calcium homeostasis, or the over-expression of protein would disrupt ER homeostatic balance and be perceived by sensors located in the ER membrane.¹¹⁾ There are three sensors, including protein kinase R (PKR)-like ER kinase (PERK), inositol-requiring enzyme 1α (IRE1α), and activating transcription factor (ATF6), which will dissociate with stress chaperon glucose-regulated protein 78 (GRP78) upon stress stimuli and initiate the downstream unfolded protein response (UPR), aiming to re-establish the hemostatic balance.¹²⁾ Additionally, severe ER stress triggers cell dysfunction and usually accompanies inflammation, which has been reported to be responsible for the disruption of the host immune system and the imbalanced inflammatory response in the context of both sepsis and sepsis-related organ injury.¹³⁾ An increasing number of studies have highlighted that the integrated responses of UPR are closely intertwined with NLRP3 activation and subsequent inflammatory response. The PERK signaling pathway, one downstream signal pathway of the UPR, participates in NLRP3 inflammasome activation.¹⁴⁾ In spite of this, no studies have yet explored the interaction between ER stress and NLRP3 inflammasome activation during SALI.

Forsythiae Fructus (“Lianqiao” in traditional Chinese medicine), the dried fruit of Forsythia suspensa (Thunb.) Vahl, is a commonly available OTC herbal medicine for the treatment of multiple inflammatory and infectious diseases.¹⁵⁾ Forsythiaside A (FTA, C₂₉H₃₆O₁₅ Fig. 1), which is the major bioactive index ingredient of Forsythiae Fructus, has been reported to possess notable anti-inflammatory and antioxidant activities and exhibit a protective role in neuroinflammation, sepsis, septic lung injury, and liver fibrosis.¹⁶⁾ Mechanically, FTA exhibits anti-inflammatory effects through regulating multiple signaling transduction pathways such as nuclear factor kappa B (NF-κB), mitogen-activated protein kinase (MAPK), Janus kinase (JAK)/signal transducer and activator of transcription (STAT), and nuclear factor-E2-related factor 2 (Nrf2) signaling pathways, as well as ER stress.¹⁷⁾ Additionally, FTA acts as an antioxidant and scavenger of free radicals. The antioxidant and anti-inflammatory properties of FTA make it an attractive therapy for treating liver disease.¹⁸⁾ Thus, in the present study, we first demonstrated that pretreatment with FTA significantly attenuates liver enzyme release and cytokine production and prevents tissue damage in the liver from septic mice, establishing the hepatoprotective effect of FTA. We found that injection of lipopolysaccharide (LPS) results in robust ER stress response and, in turn, leads to the activation of NLRP3 inflammasome in the liver. More importantly, our study enclosed that FTA administration significantly protected the liver from acute septic injury evoked by LPS stimulation via the inhibition of the ER stress response and associated NLRP3 inflammasome activation.

Fig. 1. Chemical Structural Formula of FTA

MATERIALS AND METHODS

Animals Models

C57BL/6 wild-type (WT) mice (8–10 weeks old, male, 22–28 g) were purchased from Beijing Vital River Laboratory Animal Technology Co. (Beijing, China). In order to establish the animal model, all mice were housed in pathogen-free facilities and fed a standard laboratory diet. The mice were randomly divided into three groups:control group, LPS group, and FTA group. Injecting LPS (10 mg/kg) intraperitoneally resulted in the construction of the SALI model, the control group mice were treated with 0.2 mL saline. Mice in FTA group were intraperitoneally injected with FTA (40 mg/kg) 1 h before intraperitoneal injection of LPS. We collected peripheral blood serum and liver samples 24 h after killing mice. We sacrificed mice 72 h after intervention and collected partial liver tissues for further examination by hematoxylin and eosin staining. Experimental manipulations were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals of the National Institute of Health, with the approval of the Scientific Investigation Board at Tianjin Medical University.

Liver Function Measurement

The blood was obtained through the heart, and all serum was individually collected and stored at −20 °C in a refrigerator. A Hitachi 7600-20 automatic biochemical analyzer was used to determine serum aminoleucine transferase (ALT) and aspartate aminotransferase (AST) levels.

Measurement of Cytokines in the Liver

We measured IL-1β, tumor necrosis factor (TNF)-α, and IL-6 levels in liver tissue homogenates using commercial enzyme-linked immunosorbent assay (ELISA) kits (DAKEWE).

Western Blotting Analysis

Liver samples (150 mg) were lysed and homogenized in a 200 µL lysis buffer containing a protease inhibitor cocktail using a sonic dismembrator on ice. We collected the supernatant after centrifuging the samples for 15 min at 4 °C at 14000 rpm. In accordance with the sample loading buffer protocol, the total protein of the sample was separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), followed by transfer to a polyvinylidene difluoride (PVDF) membrane (Merck Millipore, Burlington, MA, U.S.A.). Following one hour of blocking in 5% nonfat dry milk, the membranes were rinsed with tris-buffered saline with tween 20 (TBST) for three times. TBST wash was repeated three times after the membranes were incubated with the antibodies against CHOP (1 : 1000, Cell Signaling Technology (CST), Danvers, MA, U.S.A.; L63F7), phospho-PERK (1 : 1000, Protein tech, Cat.NO: APO886), PERK (1 : 1000, Cell Signaling Technology (CST), C33E10), Bax (1 : 1000, Abcam, Waltham, MA, U.S.A.: ab243140), ATF4 (1 : 1000, Abcam, ab184909), eIF2α (1 : 1000, Beyotime, Shanghai, China; Ser51), P-eIF2α (1 : 1000, Bioworld, S51), GRP78 (1 : 1000, Abcam, ab108613), NLRP3 (1 : 1000, Protein tech, Rosemont, IL, U.S.A.; Cat. No. 19771-1-AP), IL-1β (1 : 1000, Protein tech, Cat. No 66737-1-Ig), caspase-1 (p20) (1 : 1000, AdipoGen, San Diego, CA, U.S.A.; AG-20B-0042-C100), ASC (1 : 1000, Abcam, ab283684), and Gasdermin D (GSDMD, 1 : 1000, Abcam, ab219800) overnight at 4 °C. A shaking bed was used to incubate the membranes with the secondary antibody against peroxidase-labeled anti-mouse immunoglobulin (Ig)G (1 : 3000, Cell Signaling Technology) and anti-rabbit IgG (1 : 3000, Cell Signaling Technology) for 1 h. Image J software was used to analyze the membrane after it had been rinsed thrice with TBST, exposed, and developed with electrochemiluminescence.

Analyses of Real-Time Quantitative PCR

By using an RNA kit (TIANMO BIOTECH, China), liver tissues were extracted for total RNA. An RNA sample was reverse-transcribed into cDNA, which was then amplified using a real-time PCR instrument (Thermo Scientific, Waltham, MA, U.S.A.). For each cycle of amplification, 30 s were spent at 95 °C for denaturation, 40 s at 55 °C for annealing, and 30 s at 72 °C for elongation. In Table 1, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression and primer sequences were shown in this study.

Table 1. Sequences of Primers Used in This Study

Gene Name	Sequence
IL-6	Forward	5′-TAGTCCTTCCTACCCCAATTTCC-3′
	Reverse	5′-TTGGTCCTTAGCCACTCCTTC-3′
IL-1β	Forward	5′-TCGCAGCAGCACATCAACAAGAG-3′
	Reverse	5′-AGGTCCACGGGAAAGACACAGG-3′
TNF-α	Forward	5′-GACGTGGAACTGGCAGAAGAG-3′
	Reverse	5′-TTGGTGGTTTGTGAGTGTGAG-3′
GADPH	Forward	5′-TGGCCTTCCGTGTTCCTAC-3′
	Reverse	5′-GAGTTGCTGTTGAAGTCGCA-3′

Abbreviations: IL-6, interleukin-6; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Histopathological Analysis

Liver tissues were collected 72 h after intervention and fixed with 4% paraformaldehyde for two days, and then embedded in paraffin. All samples were cut into 5 µm paraffin sections and processed with hematoxylin-eosin staining. Finally, all Slides were observed and photographed using Nikon ECLIPSE Ci biological microscope. The pathological scoring criteria were referred to the previous literature.¹⁹⁾

Statistical Analysis

Data were analyzed using GraphPad Prism 9.0 (GraphPad Software Inc., Boston, MA, U.S.A.) and were displayed as mean ± standard deviation. In order to determine statistical significance, one-way ANOVA was used followed by Bonferroni’s post hoc test. A value of p < 0.05 was considered statistically significant.

RESULTS

FTA Attenuates LPS-Induced Liver Injury

The liver sections were examined histopathologically and morphologically using hematoxylin–eosin (H&E) staining for hepatocyte swellings and neutrophil infiltration. As shown in Figs. 2(A) and (B), Upon LPS challenge, the mice’s hepatic architecture was damaged with hemorrhage, necrosis, and neutrophil infiltration. The histological changes related to the LPS challenge, however, markedly improved after FTA pretreatment. According to Figs. 2(C) and (D), serum levels of ALT and AST were analyzed to determine if FTA can prevent LPS-induced liver damage in mice. A significant difference was observed between the LPS and the sham groups when it came to ALT and AST serum levels. Furthermore, FTA pretreatment significantly reduced serum ALT and AST levels compared to LPS pretreatment.

Fig. 2. Effect of FTA on Experimental LPS

(A) Liver samples were obtained 72 h after the LPS exposure and stained for H&E histopathology (original magnification, ×100 and ×200, Scale bars = 100 µm) (n = 4). (B) The slides were histopathologically evaluated as described in Materials and Methods (n = 5). (C, D) Effect of FTA on the levels of aminoleucine transferase (ALT) and aspartate aminotransferase (AST) in the serum for 24h after the LPS exposure (n = 5). All values are shown as means ± standard error of the mean (S.E.M.) from 3 separate experiments. *** p < 0.001 for the LPS group vs. the sham group; ^### p < 0.001 for the LPS group vs. the LPS + FTA group.

FTA Suppresses Cytokine Release after ALI

Acute liver injury is increasingly attributed to proinflammatory cytokines such as TNF-α, IL-6, and IL-1β. We were therefore able to profile the levels of these cytokines in liver homogenates by ELISA and their mRNA content in liver tissue by real-time PCR. As a result of LPS induction, these cytokines increased in the LPS-treated group. As expected, In the FTA-pretreatment group, TNF-α, IL-6, and IL-1β levels were reduced (Fig. 3A). In Addition, Levels of TNF-α, IL-6, and IL-1β mRNA in the liver were increased 24 h after the LPS exposure. A significant attenuation of LPS-induced inflammatory cytokines was observed following FTA pretreatment (Fig. 3B).

Fig. 3. Forsythiaside A (FTA) Inhibits Cytokine Release in Lipopolysaccharide (LPS)-Induced Acute Liver Injury

(A) Liver samples were obtained 24h after the LPS exposure and the levels of TNF-α, IL-1β and IL-6 were measured by enzyme-linked immunosorbent assay (n = 6). (B) Liver samples were obtained 24h after the LPS exposure and the mRNA levels of TNF-α, IL-1β and IL-6 were detected by real-time PCR (n = 6). Data are expressed as the mean ± S.E.M. (** p < 0.01, * p < 0.05 for the LPS group vs. the sham group; ^## p < 0.01, ^# p < 0.05 for the LPS group vs. the LPS + FTA group.

FTA Inhibits LPS-Induced ER Stress Response

Acute liver injury has been shown to induce ER stress and activation of UPR. Therefore, we further examined ER stress by Western blotting (Fig. 4) following FTA preconditioning. In our study, We found that LPS-treated mice produced significantly higher levels of GRP78 and phosphorylated PERK (p-PERK) proteins, whereas LPS + FTA-treated mice produced lower levels. Additionally, LPS slightly increased hepatic levels of phosphorylated eIF2α (p-eIF2α) and eIF2α proteins expression in the liver, while FTA pretreatment prevented LPS-induced eIFα activation and further decreased the p-eIF2α/eIF2α ratio. Furthermore, our result showed that the protein expression of ATF4 and CHOP in the liver was dramatically raised in the LPS group compared to the sham group while being significantly reduced in the LPS + FTA group compared to the LPS group.

Fig. 4. Forsythiaside A (FTA) Suppressed Endoplasmic Reticulum Stress during LPS-Induced Acute Liver Injury

(A) Liver samples were obtained 24h after the LPS exposure and the levels of the ER stress-related proteins GRP78, p-PERK (phosphorylated PERK), PERK, p-eIF2α (phosphorylated eIF2α), eIF2α, ATF4 and CHOP were measured using the Western blot method (n = 6). (B–F) Relative protein levels of GRP78, p-PERK/PERK, p-eIF2α/eIF2α, ATF4, and CHOP (n = 6). Relative protein abundance was semi-quantified by densitometry. Data were expressed as mean ± standard deviation (S.D.) values. ** p < 0.01, * p < 0.05 for the LPS group vs. the sham group; ^## p < 0.01, ^# p < 0.05 for the LPS group vs. the LPS + FTA group.

FTA Attenuates LPS-Induced NLRP3 Inflammasome Activation

Previous reports have shown that LPS-induced inflammatory response in the liver might be caused by NLRP3 inflammasome activation.^20,21) Inflammasome activation has been shown to magnify hepatocyte injury and inflammation in the liver. In our study, Western blotting was used to evaluate the protective effect of FTA against acute liver injury caused by LPS on the expression of NLRP3 inflammasome-associated proteins, including NLRP3, ASC, caspase-1 (p20), IL-1β, and Gasdermin D. As shown in Fig. 5, There was a significant increase in these proteins in the LPS group compared to the control. However, in the LPS + FTA group, FTA significantly inhibited the expression of these proteins.

Fig. 5. Forsythiaside A (FTA) Attenuated NLRP3 Inflammasome during LPS-Induced Liver Injury

(A) Liver samples were obtained 24h after the LPS exposure and the levels of the NLRP3 inflammasome-associated proteins NLRP3, ASC, caspase-1 (p20), IL-1β, and Gasdermin D were measured using the Western blot method (n = 6). (B–F) Relative protein levels of NLRP3, ASC, caspase-1 (p20), IL-1β and Gasdermin D (n = 6). Relative protein abundance was semi-quantified by densitometry. Data were expressed as mean ± S.D. values. *** p < 0.001, ** p < 0.01, * p < 0.05 for the LPS group vs. the sham group; ^### p < 0.001, ^## p < 0.01, ^# p < 0.05 for the LPS group vs. the LPS + FTA group.

DISCUSSION

Hepatic dysfunction and acute liver injury developing in sepsis pose a serious threat to clinically ill patients. Because of its rapid course, poor prognosis, and high morbidity, clinically effective drugs for the treatment of SALI are urgently needed.²²⁾ In the present study, we first verified the protective effect of FTA delivery in LPS-induced SALI. We showed that the PERK branch of the UPR and NLRP3 inflammasome, which both are activated in the liver upon LPS stimulation, are interrelated and closely involved in SALI pathogenesis. Our further data demonstrated that pretreatment with FTA significantly inhibited the PERK-mediated UPR signaling and subsequent NLRP3 inflammasome activation in the liver specimens, offering a mechanistic basis for the therapeutic potential of FTA in SALI.

NLRP3 is a unique intracellular sensor in its ability to recognize multiple molecular patterns, being able to detect a broad range of microbial motifs, endogenous danger signals, and interact with the adapter molecule apoptosis-associated speck-like protein containing a CARD (ASC) in the cytosol, and environmental irritants and resulting in the formation and activation of NLRP3 inflammasome.²³⁾ An assembly of NLRP3 inflammasome leads to the caspase-1-dependent release of the proinflammatory cytokines, IL-1β and IL-18, as well as to gasdermin D-mediated pyroptotic cell death.²⁴⁾ The activation of NLRP3 inflammasome plays a role as a critical innate immune component orchestrating host immune homeostasis; however, excessive activation of the inflammasome is associated with various diseases.²⁵⁾ The importance of the inflammasome pathways in sepsis has been highlighted in animal and human studies.²⁶⁾ For example, in humans, NLRP3, the protein mutated in familial Mediterranean fever, has been shown to regulate the production of mature IL-1β.²⁷⁾ In a murine model of sepsis, Lee et al. demonstrated that genetic deletion of NLRP3 contributes to the amelioration of inflammation and mortality in sepsis.²⁸⁾ Similarly, in our study, we showed that significant upregulation of NLRP3 and ASC, activation of caspase-1, and excessive release of inflammatory cytokines, such as IL-1β, were verified in liver samples from septic mice. Notably, we characteristically observed gasdermin D-mediated cell pyroptosis in the liver specimens from septic mice. Pyroptosis eventually causing an excessive inflammatory response. Thus, consistent with previous reports, our data indicated that NLRP3 inflammasome, which instigates the inflammatory response, could be a central player in SALI pathogenesis.

ER is a vital intracellular organelle responsible for protein quality control in eukaryotic cells.¹⁰⁾ ER dysfunction caused by many stress factors, such as increased oxidative stress, hypoxia, and Ca²⁺ level changes, would trigger UPR, an evolutionary, highly conserved mechanism aiming to restore homeostatic balance. However, once the stress is beyond the compensatory capacity of UPR, cell dysfunction and accompanied inflammation would be initiated.²⁹⁾ Recent studies from our laboratory and others have demonstrated that components of the ER stress signal pathway were upregulated in cecal ligation and puncture (CLP)- or LPS-induced animal models.^30,31) Importantly, compared to non-septic control, Li et al. reported there was a significantly high level of secreted GRP78 in sepsis patients.³²⁾ In the present study, we observed that LPS injection resulted in PERK signaling pathway activation, as evidenced by enhanced GRP78 expression, phosphorylation of PERK and eIF2α, and overexpression of CHOP in the liver from septic mice. These data are in line with previous reports showing that the expression of hepatic canonical ER stress markers, such as GRP78, p-elF2α, ATF-4, and CHOP, was significantly increased in LPS-treated mice and led to the suggestion that administration of LPS elicited robust ER stress response in the liver, reinforcing the notion that enhanced ER stress contributes to SALI development. More importantly, ER is currently considered a platform for linking cellular stress and inflammation, and several recent studies have addressed the role of ER stress in the regulation of NLRP3 inflammasome activation.³³⁾ The PERK signal pathway especially contributes to the activation of NLRP3 inflammasome through a CHOP-dependent mechanism.³⁴⁾ Therefore, combined with these above-described findings, our current data suggested the tight link between the PERK branch of UPR, NLRP3, and inflammatory response in SALI pathogenesis.

There are several compound preparations of the medicinal herb Forsythia suspensa, including Yinqiao Jiedu tablets and Lianhuaqingwen capsules, which treat inflammation, fever, and infectious diseases.³⁵⁾ As the principal bioactive index constitute extracted from Forsythiae Fructus, FTA exerts prominent bioactivities in treating various diseases. Interestingly, Gong et al. have reported that FTA inhibited the NF-κB or ER stress signal pathway to suppress the release of proinflammatory cytokines, such as TNF-α, IL-6, and IL-1β, and exhibited hepatoprotective activity in response to overdose acetaminophen-induced liver injury in zebrafish larvae.³⁶⁾ Similarly, Pan et al. showed that pretreatment with FTA alleviated liver pathological scores and reduced serum ALT and AST levels in mice with LPS/D-galactosamine-induced hepatic injury.³⁷⁾ Besides, FTA also has a remarkable free radical scavenging ability and could enhance the endogenous antioxidant defense system by Nrf2-dependent mechanism. Notably, both excessive inflammatory response and oxidative stress are hallmarks of sepsis.³⁸⁾ Then, the present study was designated to evaluate the therapeutic potential of FTA in sepsis-induced liver injury. We demonstrated that serum ALT and AST level elevation, along with liver histopathological scores, caused by septic insults were attenuated by FTA pretreatment. Moreover, the expression of proinflammatory cytokines, such as hepatic TNF-α, IL-6, and IL-1β, upon LPS stimulation was effectively diminished by FTA pretreatment. Mechanistically, our data showed that FTA pretreatment suppressed inflammation via the strong inhibition of NLRP3-dependent caspase-1 activation, IL-1β secretion, and pyroptosis. Additionally, this protection following FTA administration was paralleled by the inhibition of PERK and elF2α and the down-regulation of CHOP expression, indicating the concurrent inhibition of the PERK branch of UPR.

It should be noted that there are still limitations in the present study. First, our model that recapitulate sepsis induced by intraperitoneal injection of LPS is only a subtype of sepsis. Then, results from different models, for example cecal ligation and puncture model, should be evaluated to confirm the hepatoprotective effect of FTA. Furthermore, it is essential in the clinical setting to begin treating septic patients as soon as they become infected. Therefore, it will be more valuable to investigate the efficacy of posttreatment with FTA on SALI.

In conclusion, our data demonstrated that prophylactic administration of FTA was effective at attenuating hepatic pro-inflammatory cytokines production and liver damage induced by LPS-induced SALI. The beneficial effects of FTA administration are likely due to a potential mechanism involving the inhibition of the PERK branch of ER stress pathway and subsequent NLRP3 inflammasome activation. A greater understanding of the molecular basis of the FTA-mediated effects is needed to harness its actions as a potential tool for counteracting SALI.

Acknowledgments

Funding Sources: The current study was supported by the National Natural Science Foundation of China (82172122 282 to MT) and Tianjin Municipal Health Commission (RC20145 to ZT). A portion of this work was also funded by the Tianjin Key Medical Discipline (Specialty) Construction Project.

Conflict of Interest

The authors declare no conflict of interest.

Data Availability

All data generated or analyzed during this study are included in this paper.

Supplementary Materials

This article contains supplementary materials.

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