Locusta migratoria hydrolysates attenuate lipopolysaccharide (LPS)/D-Galactosamine (D-Gal)-induced cytotoxicity and inflammation in Hep G2 cells via NF-κB signaling suppression

The prolonged state of hepatic inflammation can lead to liver damage, a critical driving force in the progression of liver-related diseases. Locusta migratoria (LM), an edible insect, is recognized for its protein richness and potential to produce a range of bioactive polypeptides, presenting a novel solution for liver disease. This study investigated the hepatoprotective effects of LM hydrolysates in human hepatoma G2 (Hep G2) cells challenged with lipopolysaccharide (LPS)/D-Galactosamine (D-Gal), a model of liver injury. Remarkably, LM hydrolysates significantly ameliorated cell damage, as evidenced by the inhibition of the LPS/D-Gal-induced decrease in cell viability and reduction in lactate dehydrogenase (LDH) release. Furthermore, LM hydrolysates alleviated the release of aspartate aminotransferase (AST) from cells exposed to LPS/D-Gal and lowered the secretion of inflammatory cytokines while suppressing the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), a key pathway in inflammation. In particular, LM-N hydrolysate mitigated hepatotoxicity by attenuation of inflammatory responses to reduce interleukin 6 (IL-6) levels, and NF-κB nuclear translocation. These findings suggest that LM hydrolysates could potentially offer hepatoprotective effects by mitigating the inflammatory responses induced by LPS/D-Gal.


Introduction
Acute liver failure (ALF) manifests as a sudden and severe clinical presentation of liver damage, potentially resulting in liver encephalopathy, coagulation disorders, and other symptoms following the abrupt loss of liver metabolic and immune functions [1].A variety of stimuli, including drug toxicity, viral infections, and metabolic disorders, can trigger ALF, but the inflammatory response plays a crucial role in the progression of liver disease [2].Currently, liver transplantation is considered the most effective treatment option for acute liver injury [3].However, organ shortages and prolonged disease durations often result in many patients succumbing to the condition while awaiting transplantation [4].Administering antioxidants and anti-apoptotic drugs present another treatment option for ALF [5].Therefore, dietary or nutraceutical interventions aimed at mitigating hepatic inflammatory responses could effectively preserve hepatic homeostasis and health.
Locusta migratoria (LM) is an insect classified within the family Acrididae and the order Orthoptera.While sometimes considered a pest harmful to crops, LM also has significant potential as a nutritious and sustainable food source.It has been a dietary staple in some African and Arab regions for a long time [6].Additionally, LM is deemed a safe food ingredient, a claim supported by evidence showing no adverse effects in rats fed a high dose (3,000 mg/kg/day) of locust powder [7].Notably, LM boasts a remarkable protein content, approximately 50-65%, and is rich in omega-3 and omega-6 fatty acids as well as minerals [8], making it an excellent candidate for a sustainable protein source.However, the value of LM extends beyond its role as a simple food source.It has been traditionally used in treatments for conditions like hepatitis, jaundice, and liver detoxification.In line with this, an animal study has shown the benefits of LM consumption, including enhanced intestinal protein digestion and improved plasma and liver lipid metabolism in rats [9].Therefore, considering its nutritional profile and the research into its hepatoprotective potential, it is speculated that LM may be effective in improving hepatic inflammation, toxicity, and injury.
Enzymatic hydrolysis of food protein can make bioactive components more industrially applicable [10].A study has demonstrated that salmon byproduct protein hydrolysates attenuate inflammation and oxidative stress in liver cells [11].In addition, camel whey protein hydrolysates are reported to exhibit anti-inflammatory and anti-hepatocarcinogenic effects [12].Furthermore, egg white hydrolysate has been shown to enhance hepatic lipid degradation and metabolism and to exhibit antiinflammatory effects in rats [13].Therefore, we prepared LM hydrolysates using four different proteolytic enzymes and hypothesized that these hydrolysates could prevent ALF by inhibiting inflammation through the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway in lipopolysaccharide (LPS)/D-Galactosamine (D-Gal)-induced human hepatocellular carcinoma G2 (Hep G2) cells, a model for ALF [14].In our study, LM hydrolysates were found to alleviate cytotoxicity and inflammation, alongside inactivating NF-κB.These results support the potential of LM hydrolysates as a preventive or therapeutic agent in the pathological progression of ALF.

Preparation of LM hydrolysates
The LM used in this study were obtained in a frozen state from three domestic farms in South Korea in June 2022.A 10% (w/v) suspension was prepared by adding distilled water to the LM powder.The pH of the suspension was adjusted to the optimal level for each enzyme: pH 8.0 for Alcalase®, pH 7.0 for Flavourzyme®, Neutrase®, and Prota-mex®.Subsequently, 1% (v/v) of Alcalase® (Novozymes, Bagsvaerd, Denmark), Flavourzyme® (Novozymes), and Neutrase® (Novozymes), or 1% (w/v) of Protamex® (Novozymes) was added to their respective suspensions.These suspensions were then incubated at 60 °C for Alcalase® and at 50 °C for Flavourzyme®, Neutrase®, and Protamex® for 8 h.After the enzymatic reaction, the enzymes were inactivated by heating the mixture to 90 °C for 30 min.The resulting enzymatic hydrolysates were then filtered through a 100-mesh sieve, freeze-dried, and stored at −70 °C.In subsequent experiments, the LM hydrolysates treated with Alcalase®, Flavourzyme®, Neutrase®, and Protamex® were labeled as LM-A, LM-F, LM-N, and LM-P, respectively.

Amino acids analysis of LM hydrolysates
The content of free amino acids in the LM hydrolysates was analyzed using a high-performance liquid chromatography with a Photodiode Array detector (HPLC-PDA), following methods previously published by Kim et al. [15].Briefly, a homogenous sample was diluted with water at a 1:10 (w/v) ratio, then solidified through heating and subsequently filtered to collect the aqueous layer.The residue was washed three times with a small quantity of water, and the wash solution was combined with the initial aqueous layer.This combined aqueous layer, once concentrated under reduced pressure, was dissolved in a 0.2 N sodium citrate buffer at a pH of 2.2.Sample separations were carried out using a 5 μm Capcell Pak® C18 UG120 column (250 mm × 4.6 mm; Shiseido Co., Tokyo, Japan).The separation employed solvent A (40 mM NaH2PO4 at pH 7.8) and solvent B (acetonitrile: methanol: water in a 45:45:10, v/v/v, ratio).The gradient elution program was set as follows: from 0 to 31 min, 5% solvent B; from 31 to 33 min, 56% solvent B; from 33 to 34 min, maintaining 56% solvent B; from 34 to 38 min, 100% solvent B. Calibration curves were established using an amino acid standard mix ampoule (Agilent Technologies, Santa Clara, CA, USA) and L-Tryptophan (Sigma-Aldrich, St. Louis, MO, USA).

Cell culture
Hep G2 cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA).The cells were cultured in Modified Eagle Medium (MEM; Welgene, Gyeongsan, Korea) supplemented with 10% fetal bovine serum (FBS; Welgene) and 1% antibioticantimycotic solution (Gibco, Grand Island, USA) at 37 °C in a 5% CO 2 environment.Initially, Hep G2 cells were seeded at a density of 1.5 × 10 5 cells/cm 2 and cultured for 24 h.The medium was then replaced with MEM containing 1% FBS, and the cells were incubated for an additional 24 h.Subsequently, the cells were pretreated with various concentrations of LM hydrolysates.After 24 h of pretreatment, the medium was replaced with fresh culture medium containing 5 µg/mL of lipopolysaccharide (LPS; Sigma-Aldrich) and 40 mM D-galactosamine (D-Gal; Sigma-Aldrich) for a final incubation period of 24 h.

Cytotoxicity assay
Hep G2 cells were seeded in a 96-well plate at a density of 1.5 × 10 5 cells/cm 2 and cultured for 24 h.The culture medium was then replaced with MEM containing 1% FBS, and the cells were incubated for an additional 24 h.Subsequently, to assess the impact on cell viability, the cells were treated with various concentrations of LM hydrolysates (0, 0.01, 0.05, 0.1, 0.5, 1, and 5 mg/mL) for 24 h.Additionally, to evaluate whether LM hydrolysates could mitigate the cytotoxicity induced by a combined treatment of LPS and D-Gal, the cells were treated with different concentrations of LM hydrolysates (0, 0.5, and 1 mg/mL) for 24 h, following 24 h of incubation in MEM containing 1% FBS.After this treatment, the cells were incubated for another 24 h in fresh culture medium containing 5 µg/mL LPS and 40 mM D-Gal.Cell viability was determined using the EZ-Cytox cell viability assay kit (DoGenbio, Seoul, Korea), which is based on the watersoluble tetrazolium salt (WST-1).The absorbance was measured at 450 nm using a microplate reader (Molecular Devices, San Jose, USA).The results were expressed as a percentage of absorbance relative to the control group.

Lactate dehydrogenase (LDH) measurements
Hep G2 cells were seeded into 96-well plates at a density of 2.0 × 10 4 cells/cm 2 and incubated for 24 h.The culture medium was then replaced with MEM containing 1% FBS, followed by a further 24-hour incubation.Subsequently, the cells were treated with various concentrations of LM hydrolysate (0, 0.5, and 1 mg/mL) for 24 h.After the treatment, the cells were incubated in a medium containing 5 µg/mL LPS and 40 mM D-Gal for another 24 h.At the end of the incubation period, the cell culture medium was collected through centrifugation at 600×g for 5 min, and the supernatant (10 µL/well) was transferred to a new 96-well plate containing 100 µL of LDH reagent (DoGenBio) in each well.The plates were then incubated for 30 min at room temperature.The concentration of LDH was measured using a microplate reader (Molecular Devices) at 450 nm.The extent of LDH release was calculated using the following formula: % of max value = [(OD_test -OD_blank) / (OD_max -OD_ blank)] × 100.

Aspartate aminotransferase (AST), alanine aminotransferase (ALT) measurements
Hep G2 cells were cultured in 24-well plates at a density of 1.5 × 10 5 cells/cm 2 for 24 h.The culture medium was then replaced with MEM containing 1% FBS and incubated for another 24 h.Subsequently, the cells were treated with various concentrations of LM hydrolysate (0, 0.5, and 1 mg/mL) for 24 h.After this treatment, the cells were exposed to a fresh medium containing LPS and D-Gal.The supernatants were collected 24 h later and stored at 4 °C.AST and ALT levels in the culture medium were determined using commercially available assay kits (Embiel, Gunpo, Korea), according to the manufacturer's instructions.

Enzyme-linked immunoassay (ELISA) analysis
Hep G2 cells were cultured in 24-well plates at a density of 1.5 × 10 5 cells/cm 2 for 24 h.The culture medium was then replaced with MEM containing 1% FBS and the cells were incubated for an additional 24 h.Subsequently, the cells were treated with varying concentrations of LM hydrolysate (0, 0.5, and 1 mg/mL) for 24 h.Following this, the cells were exposed to a medium containing LPS and D-Gal for another 24 h.The cell culture medium was then collected for cytokine analysis.Tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) levels in the supernatants were quantified using the human TNF-α and IL-6 ELISA MAX™ Deluxe set (Biolegend Inc., San Diego, CA, USA), following the manufacturer's instructions.

Western blotting analysis
Hep G2 cells were lysed in ice-cold radioimmunoprecipitation assay buffer (Elpisbio, Daejeon, Korea) containing protease and phosphatase inhibitors (Roche, Mannheim, Germany) after a 30-minute incubation at 4 °C.The hepatocyte lysates were centrifuged at 12,000 × g for 20 min at 4 °C, and the resulting supernatant was collected.The protein concentration in the supernatant was determined using the Pierce™ BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA).Equal amounts of lysate protein (20 µg/lane) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).The proteins were then transferred to a polyvinylidene fluoride membrane (Millipore, Burlington, MA, USA).The membranes were blocked using Everyblot blocking buffer (BIO-RAD, Hercules, CA, USA) and incubated with primary antibodies overnight at 4 °C.β-actin, GAPDH, and Lamin B1 served as endogenous loading controls.The details of the antibodies used for western blot analysis are provided in the Table 1.After incubation with primary antibodies, the membranes were probed with secondary antibodies for 1 h and developed with EZ-Western Lumi Pico Alpha (DoGenBio).Protein bands were visualized using an iBright 750 imaging system (Invitrogen, Waltham, MA, USA).The intensities of the individual protein bands were quantified using ImageJ software (Version 1.8.0,NIH, Bethesda, MD, USA).

Statistical analysis
All data are presented as means ± standard deviation (SD).A one-way analysis of variance (ANOVA) followed by Tukey's post hoc test was used to compare differences between groups [16].A significant difference in means, indicated by non-overlapping letters, was considered statistically significant at p < 0.05.Statistical analyses were performed using SPSS version 26.0 (Statistical Package for the Social Sciences, IBM Corp., Armonk, NY, USA), and all figures were created with GraphPad Prism version 9 (GraphPad Software Inc., San Diego, CA, USA).

Free amino acids of Locusta migratoria (LM) hydrolysates
Based on the known benefits of enzymatic hydrolysis in enhancing functional properties while preserving nutritional value [17], we investigated the impact of four different commercial proteases (Alcalase®, Flavourzyme®, Neutrase®, and Protamex®) on the free amino acid profiles of LM hydrolysates.Using HPLC-PDA analysis, we observed significant variation in amino acid contents among the LM hydrolysates.Notably, LM-F displayed the highest total amino acid level (252.47 mg/g) and the highest ratio of essential amino acids (51.04%), surpassing those of LM-A (139.53 mg/g, 49.13%), LM-N (155.05 mg/g, 49.04%), and LM-P (156.49mg/g, 48.96%) (see Table 2).However, the top four predominant amino acids (Arginine, Alanine, Leucine, and Lysine) were consistently present in LM-A, LM-F, LM-N, and LM-P, constituting 45.57%, 45.1%, 46.99%, and 47.24% of the total amino acids, respectively.These findings suggest that the selection of protease is crucial in determining the functionality and nutritional value of the amino acid profile.

Effects of LM hydrolysates on the cell viability in Hep G2 cells
To assess the potential cytotoxicity of LM hydrolysates, we investigated the cell viability of Hep G2 cells exposed to LM hydrolysates for 24 h using the WST-1 assay.There were no significant differences in cell viability in Hep G2 cells treated with various concentrations (0, 0.01, 0.05, 0.1, 0.5, 1, or 5 mg/mL) of LM-A, LM-F, LM-N, and LM-P (Fig. 1A-D).Interestingly, even the highest test concentration of LM hydrolysates (5 mg/mL) did not induce any significant adverse effects on cell viability, further supporting the non-cytotoxic nature of LM hydrolysates.Therefore, we concluded that LM hydrolysates did not exert adverse effects on human liver cells at the tested concentrations, including the highest level of 5 mg/mL.This concentration can be considered safe for exploring the potential applications of LM hydrolysates in liver cells.

LM hydrolysates attenuated LPS/D-Gal-inducible cytotoxicity in Hep G2 cells
We utilized a cellular model of hepatotoxicity induced by LPS/D-Gal for rapid screening and mechanistic investigation of cytoprotective agents.In this context, we examined whether LPS/D-Gal induces cytotoxicity and if LM hydrolysates can mitigate LPS/D-Gal-induced cytotoxicity in Hep G2 cells.Cells treated with the combination of LPS and D-Gal exhibited significantly lower cell viability compared to the control group (Fig. 2A).However, pretreatment with all four types of LM hydrolysates resulted in improved cell viability compared to the group treated with LPS/D-Gal alone (Fig. 2A).Among these, the LM-N hydrolysate demonstrated the most significant cytoprotective capacity (Fig. 2A).In addition, stimulation with LPS/D-Gal led to a higher release of LDH, a general marker of cell death [18], compared to the control group.Pre-treatment with LM hydrolysates reversed this effect (Fig. 2B).These results suggest that LM hydrolysates have cytoprotective effects against LPS/D-Gal-induced toxicity in human liver cells, with LM-N being the most effective.However, further research is necessary to identify the bioactive compounds and mechanisms responsible for the enhanced efficacy of LM-N.

Effects of LM hydrolysates on LPS/D-Gal-inducible aspartate aminotransferase (AST) and alanine aminotransferase (ALT) release from Hep G2 cells
ALT and AST are widely utilized as biomarkers of liver injury for clinical diagnosis and research [19].Therefore, we measured the activities of AST and ALT in the culture medium of Hep G2 cells exhibiting cytotoxicity induced by LPS/D-Gal to determine if LM hydrolysates modulate these biomarkers.As expected, LPS/D-Gal treatment significantly elevated AST activity in the cell culture medium compared to the control group (Fig. 3A).Interestingly, all four enzymatic hydrolysates of LM mitigated the LPS/D-Gal-induced increase in AST activity, even reducing it below the control values (Fig. 3A).In contrast, ALT activity remained unchanged after treatment with LPS/D-Gal and subsequent LM supplementation (Fig. 3B).This pattern is consistent with changes observed in the early stages of liver diseases or in liver injuries related to alcohol and drug use.These findings suggest that LM hydrolysates may alleviate LPS/D-Gal-induced liver damage, potentially by reducing AST leakage.While further research is required, these differential responses could provide valuable insights into the protective mechanisms of LM hydrolysates in hepatocytes.

Effects of LM hydrolysates on LPS/D-Gal-induced secretion of proinflammatory cytokines in Hep G2 cells
The systemic inflammatory response is triggered by proinflammatory cytokines, including TNF-α and IL-6.Elevated levels of these cytokines have consistently been observed in patients with acute liver injury [20].Therefore, we measured the levels of TNF-α and IL-6 in the culture medium of LPS/D-Gal-treated Hep G2 cells to evaluate the anti-inflammatory potential of LM hydrolysates.As expected, LPS/D-Gal treatment significantly elevated TNF-α release compared to the control.In contrast, pre-treatment with LM hydrolysates significantly reduced the LPS/D-Gal-induced increase in TNF-α release (Fig. 4A), suggesting that they partially exert their anti-inflammatory effects by targeting TNF-α production.Additionally, IL-6 levels markedly increased following LPS/D-Gal treatment.However, only LM-N at a concentration of 1 mg/mL significantly reduced the IL-6 level increase induced by LPS/D-Gal (Fig. 4B).In  summary, these results indicate that LPS/D-Gal treatment triggers an inflammatory response through the production of proinflammatory cytokines.LM hydrolysates, especially LM-N, exert anti-inflammatory effects, primarily by reducing the release of TNF-α and potentially targeting other inflammatory pathways.

Effects of LM hydrolysates on LPS/D-Gal-induced NF-κB activation in Hep G2 cells
Upon observing that LM hydrolysates reduced proinflammatory cytokines, we investigated whether they exert anti-inflammatory effects by modulating NF-κB signaling.Indeed, stimulation with LPS/D-Gal increased the phosphorylation of protein kinase B (AKT) and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα), which are upstream activators of NF-κB (Fig. 5A, B, C).Interestingly, while LM hydrolysates did not statistically significantly reverse this effect, a decreasing trend was observed in cells treated with LM hydrolysates (Fig. 5A, B, C).To assess the impact of potential upstream signaling on NF-κB activation, we examined NF-κB translocation, leading to the expression of inflammation-associated target genes.In the presence of LPS/D-Gal stimulation, the level of NF-κB in the cytoplasm was lower compared to the control.However, pre-treatment with 1 mg/mL of LM-F, LM-N, and LM-P resulted in a significant increase in cytoplasmic NF-κB compared to the LPS/D-Gal-treated cells (Fig. 5D, E).Additionally, Hep G2 cells stimulated with LPS/D-Gal exhibited an increase in nuclear NF-κB levels (Fig. 5D,  F).Simultaneously, pre-treatment with 1 mg/mL of LM-F and LM-N showed a relative decrease in nuclear NF-κB protein expression, complementing the observed changes in cytoplasmic proteins (Fig. 5D, E, F).These data suggest that LM-F and LM-N primarily act by inhibiting the NF-κB pathway, thereby decreasing the expression

Discussion
ALF is a clinical syndrome primarily characterized by acute massive necrosis of hepatic cells, leading to impaired coagulation function and the development of hepatic encephalopathy [21].ALF has a variable definition and pathogenesis, which makes it challenging to treat.ALF is defined in over 40 ways, with considerations including whether there is coagulation dysfunction, the presence of pre-existing liver disease, the existence and grading of hepatic encephalopathy, and other factors [22].While paracetamol toxicity is the primary cause of ALF in developed countries, various viral diseases are the main etiologies in developing countries [23].In this study, we used a combination of LPS and D-Gal on Hep G2 cells to create a cellular model of ALF, mimicking the inflammatory response of certain ALF causes, and investigated the potential hepatoprotective effects of LM hydrolysates.
Liver cells play a crucial role in organismal metabolism; however, the inherent challenges of expanding primary hepatocytes in vitro while preserving both their proliferative capability and metabolic functionality have constrained the scope of hepatocyte research [24].Although Hep G2 cells originate from hepatic carcinoma cells, their retention of differentiated liver cell characteristics makes them a popular tool for studying liver function, including research on in vitro hepatotoxicity [25].Additionally, it has been reported that LPS/D-GalN-treated Hep G2 cells, used as a model to study acute liver failure (ALF) in vitro, exhibited elevated levels of AST, LDH, and TNF-α [26].Therefore, we hypothesized that LM hydrolysates could generally alleviate ALF-related indicators in LPS/D-Gal-induced Hep G2 cells.Meanwhile, we also acknowledge that the concentrations used in this pilot study, as well as the yet-to-be-discovered key bioactive components, may not provide conclusive evidence for future clinical research.Therefore, further research is essential to pinpoint the precise and effective concentrations and to identify the key bioactive components of LM hydrolysates for clinical application.
ALF is known to increase LDH production due to hepatic anaerobic conditions and leakage through damaged hepatocyte membranes [27].AST and ALT are predominant clinical indicators for liver disease assessment, as damage to hepatocytes results in their release, potentially exhibiting different release patterns during injury [28].Intriguingly, pre-treatment with four LM hydrolysates enhanced Hep G2 cell viability (Fig. 2A) and reduced AST (Fig. 3A) and LDH (Fig. 2B) release after LPS and D-Gal challenge, while effects on ALT were not statistically significant (Fig. 3B).This suggests potential differences in AST and ALT release patterns during hepatic injury.Previous animal studies have shown that diets containing LM components lowered serum levels of AST and ALT compared to control groups without additional drug interventions [9], indicating LM's potential benefits in liver health improvement, not just mitigating liver damage.LPS triggers inflammation and oxidative stress, mediating liver injury progression to septic endotoxemia, shock, or death [29].D-Gal co-administration enhances liver cells' sensitivity to endotoxins, triggering an inflammatory storm and hepatocellular death [30].It has been shown that ALF induced by LPS and D-Gal involves the expression of proinflammatory factors, while some natural extracts alleviate proinflammatory cytokine release [31,32].In addition, a study showed that cytokines such as TNF-α and IL-6 were involved in the process of hepatocyte necrosis and enhanced the extent of D-Galinduced hepatic necrosis [33].Our study demonstrated that all four LM hydrolysates significantly reduced TNF-α levels (Fig. 4A), with 1 mg/mL of LM-N particularly effective in lowering IL-6 concentration (Fig. 4B), suggesting their potential to alleviate ALF through antiinflammatory activity.Besides, the liver is a complex organ composed of many different types of cells, each of which has a specific location and function in the liver and works together to coordinate and accomplish the physiological activities of the liver [34].Particularly in liver injury and activation of hepatocyte necrosis, Kupffer cells are a major source of cytokines, which would exhibit increased cytotoxicity [35].We therefore suggest that further investigation of the anti-inflammatory and immunologic effects of LM hydrolysates in hepatic immune cells may provide potential therapeutic targets for ALF management.
NF-κB activation via the PI3K/Akt pathways is a known response to LPS in liver cells [36].In the absence of external stimuli, NF-κB is bound by IκB, but IκB phosphorylation and degradation activate NF-κB [37].NF-κB dimers then translocate to the nucleus to regulate transcription of inflammation-related genes [38].In our study, LPS and D-Gal promoted PI3K and Akt phosphorylation, decreased cytoplasmic NF-κB, and increased its nuclear translocation.While LM hydrolysates did not significantly reduce p-IκBα and p-AKT levels, a trend toward downregulation was observed (Fig. 5A, B, C).Importantly, pre-treatment with LM-F, LM-N, and LM-P was found to increase the expression of NF-κB in the cytoplasm compared to the LPS/D-Galtreated group (Fig. 5D, E).Concurrently, nuclear levels of NF-κB decreased in cells pre-treated with LM-F and LM-N (Fig. 5D, F).Moreover, LM-N specifically exhibited a mitigating effect on the release of IL-6 (Fig. 4B) and demonstrated superior effectiveness in reducing LPS/D-Gal-induced cytotoxicity (Fig. 2A).These observations suggest that LM hydrolysates, especially LM-N, may mitigate the inflammatory response in Hep G2 cells induced by LPS/D-Gal, in part, by inhibiting the nuclear translocation of NF-κB.Notably, previous research has underscored the effectiveness of modulating the NF-κB signaling pathway in alleviating ALF [39,40].Therefore, we propose that LM hydrolysates could potentially prevent or treat ALF by targeting the NF-κB signaling pathway.
Enzymatic hydrolysis of proteins can release different amounts of amino acids and bioactive peptides, depending on the hydrolytic enzymes used [41].Additionally, the hard exoskeleton of LM is rich in chitin, which may limit the bioavailability of nutrients [42]; however, hydrolysis effectively addresses this issue by unlocking bioactive compounds such as retinol, vitamin D, vitamin B12, various carotenoids, and antioxidant peptides [43].Notably, edible insects are reported to contain high levels of polyphenols that play significant roles in specific biological activities, including antioxidant, anti-inflammatory, and anticancer activities [44].In this study, we employed four distinct commercial proteases (Alcalase®, Flavourzyme®, Neutrase®, and Protamex®) to hydrolyze LM and determined the content of free amino acids in the LM hydrolysates.Alcalase® is a commercial enzyme from Bacillus licheniformis, combining several proteases with different specificities, and has been shown to reduce the allergenicity of whey proteins [45].Additionally, Flavourzyme® exhibits a preference for cleaving peptide bonds between Leucine and Proline, while Neutrase® preferentially cleaves bonds between Leucine and Phenylalanine at the C-terminus [46].Moreover, Protamex® is a composite enzyme produced by Bacillus subtilis, known for its broad specificity in enzymatic activity [47].
The functional characteristics of hydrolysates are influenced by their amino acid composition, which depends on the enzyme and protein source used [48].Our results indicated that LM-A, LM-F, LM-N, and LM-P were rich in Leucine, accounting for 13.75%, 13.58%, 12.43%, and 12.46% of the total free amino acids, respectively (Table 2).Dietary Leucine supplementation has been shown to improve lipid metabolism in the liver and alleviate hepatic oxidative stress and inflammatory responses [49].Furthermore, the content of Alanine and Arginine was relatively high in all four LM hydrolysates (Table 2).Notably, a study revealed that Alanine dietary intervention significantly improved hepatic functional parameters in D-Gal-induced SD rats [50].Long-term oral supplementation of L-Arginine has also been shown to alleviate damage to liver structure and function and mitigate high-fat diet-induced inflammation and oxidative stress [51].The four LM hydrolysates used in our study contain essential amino acids constituting over 48% of the total free amino acids (Table 2).Therefore, we speculate that the differing anti-inflammatory activities observed in the LM hydrolysates may be associated with their unique amino acid profiles.Consequently, further investigation into the composition of active polypeptides in LM hydrolysates and the key bioactive components could be a rational and effective strategy for elucidating how LM hydrolysates exert anti-inflammatory and hepatoprotective effects.
This study has highlighted the potential of LM hydrolysates as hepatoprotective agents.We found that LM hydrolysates mitigated LPS/D-Gal-induced cell damage in Hep G2 cells, enhancing cell viability and reducing LDH release.Furthermore, LM hydrolysates counteracted the detrimental effects of LPS/D-Gal by decreasing the release of a liver damage marker (AST) and inflammatory cytokines.The mechanistic investigation suggests that LM hydrolysates' suppression of the NF-κB signaling pathway may contribute to their anti-inflammatory effects.These findings lay a promising groundwork for future research, including animal models and clinical trials.However, additional studies are necessary to identify the specific bioactive components responsible for these effects and to examine their in vivo efficacy for potential clinical application.