The Effects of Hepatogomax Enteral Formula on Systemic Inflammation, Caecum Short-Chain Fatty Acid Levels, and Liver Histopathology in Thioacetamide-Induced Rats

Liver damage characterized by fibrosis and necrosis can worsen the condition of liver disease. Liver disease is associated with impaired immune response and may affect short-chain fatty acid (SCFA) gut metabolites. Hepatogomax enteral formula was developed, which contains brain-chain amino acids (BCAAs) and middle-chain triglycerides (MCTs), which could repair liver tissue damage, improve the inflammatory status, and modulate SCFA in liver damage. The study aimed to determine the effect of hepatogomax on liver tissue repair, inflammation (TNF-α and IL-6), and SCFA levels in thioacetamide (TAA)-induced rats. The induction of TAA causes liver steatosis, increasing TNF-α and IL-6, and decreasing SCFA levels. Hepatogomax at a dose of 14.6 g/200 gBW significantly reduces TNF-α and IL-6 levels and increases SCFA levels (p < 0.05). The number of steatosis between groups P2 and P3 was lower as compared to a group of negative control [K2] (p < 0.05). Hepatogomax, in a dose-dependent manner, may repair liver tissue and improve inflammatory response and SCFA levels in TAA-induced rats.


Introduction
Liver is a vital organ that plays a role in human metabolism.Liver disease has become one of the major health problems worldwide, with mortality reaching 2 million per year [1,2].Liver disease can be caused by hepatotoxic agents that damage the liver's cell tissue.Tioacetamide (TAA) is one of the hepatotoxic chemical compounds used to induce fbrosis that resembles liver cirrhosis in humans in experimental studies [3].Liver cirrhosis is liver disease's end stage, characterized by increased dead cells and induced liver fbrosis [4].Hepatic fbrosis has been suggested as one of the hallmarks of nonalcoholic steatohepatitis (NASH), characterized by extensive accumulation of connective tissue following extensive tissue damage [5].Of note, multiple cells play a role in the progression of liver disease, including hepatocytes, sinusoidal endothelial cells (SECs), and Kupfer cells (KCs) [6].Furthermore, these cells are involved in cirrhosis development by releasing antigen-presenting cells for viruses, reactive oxygen species (ROS), and infammatory mediators [7].It has been shown that the increase in infammatory response in the tissue of liver disease is associated with systemic infammation [8].
Furthermore, it has been indicated that a hallmark of liver steatosis is characterized by the accumulation of bone marrow-derived macrophages and neutrophils of hepatocytes, Kupfer cells, and liver sinusoidal endothelial cells.Tis immune cell activation induces harmful infammation and leads to NASH progression to cirrhosis [9].Of interest, under NASH progression, there is a shift in the infammatory state from the production of the antiinfammatory cytokines to the production of the proinfammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), which play an important role in the liver regeneration process [10,11].More interestingly, recent studies have suggested a cross-talk between liver infammation, systemic infammation, and gut microbiota dysbiosis [12,13].Te most recent human study demonstrated that an altered gut microbiome parallelly occurred during an acute-on-chronic liver failure (ACLF) stage.It has been determined that ACLF is the most severe clinical stage of cirrhosis [14].
Altered gut microbiome in liver disease may determine changes in the gut microbiota metabolites such as shortchain fatty acid (SCFA).It has been widely investigated that SCFA production is not only observed in the stool but also can bypass the systemic level (i.e., in the blood).A crosssectional study in cirrhosis patients showed that low serum butyrate level was negatively associated with increased metabolic endotoxemia and systemic infammation [15].More interestingly, not only SCFA is decreased in cirrhosis but also another gut metabolite, brain-chain amino acid (BCAA), is decreased.Te BCAA concentrations are low in the end stage of liver disease and may be associated with liver fbrosis, oxidative stress, and the development of a proinfammatory state in liver disease [16,17].BCAA administration in patients with liver disease can prevent progressive liver damage, stimulate protein and cell regeneration, and improve liver function and body immunity [10,11].BCAA can increase protein synthesis related to the immune system.An increased immune response requires the formation of new cells, antigens, immunoglobulins, cytokines, cytokines response, and acute-phase proteins.In addition, BCAA can increase the number of intrahepatic lymphocytes and stimulate natural killer (NK) activity and lectin-dependent cytotoxic activity in the liver [12].Treatment for liver disease using a formula feeding containing the BCAAs may be benefcial in reducing tissue damage in the liver, decreasing systemic infammation, and modulating the SCFA levels.
Te present study uses the hepatogomax enteral formula, made from goat's milk four and soybean four for liver diseases.Tis formula was developed by Rahmadanti et al. [18] in 2020 and has met the European Society for Clinical Nutrition and Metabolism (ESPEN) guideline for the composition of the enteral diet for liver disease.We hypothesized that hepatogomax might reduce the progression of liver tissue damage, decrease infammation (TNF-α and IL-6), and modulate the caecum SCFA level.

Hepatogomax Enteral Formula.
Te hepatogomax enteral formula was made at the Food Technology Science Laboratory, Diponegoro University, and was made from soybean four "Kusuka Ubiku," goat's milk four "Skygoat," virgin coconut oil (VCO) "Al Alfat," maltodextrin, and granulated sugar.Te dry ingredients were mixed and stirred using a mixer for 3 minutes, and then, VCO oil was added to it and stirred for 2 minutes.All the ingredients were stirred manually and then stirred using a mixer for 8 minutes until they became homogeneous.Te formula that has been homogenized is sifted to form a smoother formula.Te formula is put into an airtight plastic container and stored at room temperature [18].Te hepatogomax enteral formula contains an energy density of 1.17 kkal/ml, low fat <27.33%, medium protein digestibility 53.44%, BCAAs 2.09%, and medium-chain triglycerides (MCTs) 29.26% [18][19][20][21].

Experimental Animals.
Tis study was a true experimental pre-post test with a control group design conducted at the Experimental Animal Laboratory, Center for Food and Nutrition Studies, Gadjah Mada University, Yogyakarta.Te subject used in this study was male adult Sprague-Dawley rats aged 8-12 weeks with a body weight of 180-250 g.Te rats were then randomly assigned into six groups consisting of six rats (n � 6) for each group.All groups received 20 g/d/ rats of AD-II Comfeed as standard feed and drank water freely.Te K1 group is healthy rats as control negative while the K2 group is TAA-induced rats as control positive.Te groups of K2, K3, P1, P2, and P3 were induced with TAA at 400 mg/kg BW for two weeks to make liver damage condition.Te group of K3 was given a commercial formula at a dose of 4.32 g/200 gBW.In contrast, the groups of P1, P2, and P3 were given hepatogomax enteral formula at a dose of 4.97 g/200 gBW, 9.73 g/200 gBW, and 14.6 g/200 gBW for 28 days.
Blood sampling was carried out twice: (1) preintervention and (2) postintervention.All rats were fasted for 12 hours before taking blood through the retro-orbital plexus.TNF-α and Il-6 levels were determined using the ELISA methods.After the study was completed, the rats were terminated and taken out of the caecum for SCFA analysis.After that, the liver tissue was collected and immediately stored in formalin 10% for further histopathology analysis.

SCFA Analysis.
Te caecum of the rats taken was cleaned using phosphate-bufered saline or sodium chloride (NaCl).After that, the caecum was stored at a temperature (of −80 °C).Prior to analysis, the caecum samples were homogenized using Tissue Lyser (Qiagen ® ) and were centrifuged at 14000 rpm for 15 minutes to collect the supernatant and added 25% metaphosphoric acid to maintain the pH of the bufer in a ratio of 4 : 1. Te supernatant was centrifuged again at 14000 rpm for 30 minutes [22,23].Te 2 Journal of Nutrition and Metabolism supernatant samples were analyzed using gas chromatography to obtain SCFA levels.SCFA levels were analyzed at the Food Technology and Agricultural Products Testing Laboratory, Gadjah Mada University, Yogyakarta.

Histopathological Examination.
Te resected liver organs were fxed using 10% neutral bufered formalin and were made into parafn blocks.Tese blocks were then cut 3-4 micrometres.After that, staining was performed using hematoxylin-eosin (H&E).A microscope (Olympus CX33, Evident ® ) with a magnifcation of 400 times was used to examine the liver structure histopathologically.Histopathological analysis was carried out at the Department of Anatomical Pathology, Gadjah Mada University, Yogyakarta.

Efect of Hepatogomax Enteral Formula on Body Weight.
Te results of the average body weight of all groups increased during the study (see Table 1).Te K2, K3, P1, P2, and P3 groups had lower body weight after being induced by TAA.
Tere was a signifcant increase in body weight in all groups after TAA induction (p < 0.05).Te mean weight change before and after TAA induction showed that all groups experienced a signifcant increase in body weight (p � 0.003).In addition, only K1 group had a signifcant diference in the increase in all groups (p < 0.05).After the intervention, it showed a signifcant increase in body weight in all groups (p < 0.001).Te P3 group experiences more weight gain than the K3 group after the intervention.Tere was a signifcant diference in the mean increase in weight change between groups after the intervention (p < 0.001).Te P3 group did not have a signifcant diference from the K1 and K3 groups (p > 0.05).

Efect of Hepatogomax Enteral Formula on Infammatory
Biomarkers.Tere was a signifcant diference in infammatory status levels in all groups after the intervention (p < 0.05).Te TAA-induced groups had higher TNF-α and IL-6 serum levels than the K1 group.After the intervention, the P3 group had lower levels of TNF-α and IL-6 serum compared to the K3 group and almost the same as the K1 group.Te changes in the mean levels of TNF-α and IL-6 serum showed signifcant diferences between groups (p < 0.05).Changes in the mean decrease in TNF-α and IL-6 serum levels, which were greatest in the P3 group, are 13.49pg/ml and 72.05 pg/ml.Te groups of P1, P2, and P3, which were given hepatogomax intervention, had signifcant diferences from all control groups (p < 0.05) (see Table 2).

Efect of Hepatogomax
Enteral Formula on SCFA.Te results of acetic acid, propionic acid, and butyric acid levels between groups had signifcant diferences (p < 0.05).Groups K3, P1, P2, and P3 had signifcant diferences in SCFA levels compared to groups K1 and K2 (p < 0.05).Te P3 group had the highest levels of acetic acid, propionic acid, and butyric acid than the TAA-induced groups (see Table 3).Te result of histopathological analysis showed no diference in the number of necrosis, ballooning, and infammatory cells in all groups.However, there was a signifcant diference in the amount of steatosis (p < 0.05).Groups P1 and P2 signifcantly difered from group K2 (see Table 4).

Discussion
In this study, we demonstrated that hepatogomax formula feeding containing BCAAs and medium-chain triglycerides (MCTs) increases body weight, reduces systemic infammations (markedly by a decrease in TNF-α and IL-6), modulates the caecum SCFA levels (acetate, butyrate, and propionate), and prevents progression of tissue damage in the liver of TAA-induced rats model.Tus, the results of this study suggest that hepatogomax formula feeding may infuence the repairing of infammation and tissue cell damage in the liver by modulating the production of SCFA by the gut microbiota associated with BCAAs.Body weight before TAA-induced in all groups of rats was homogeneous and corresponded to the research inclusion criteria, 180-250 g.Te results showed a lower increase in body weight before intervention in the K2, K3, P1, P2, and P3 groups compared to the K1 group.A lower increase in that group was due to TAA induction.Tis is due to a previous study showing that the group of rats induced by TAA 400 mg/kgBW for two weeks had a lower body weight (191.93 g) compared to the healthy rat group (244.86 g) [3].TAA is a hepatotoxic compound that can cause centrilobular necrosis with a regenerative response of the liver that leads to the formation of liver cirrhosis [24].Cirrhosis conditions in the liver cause a decrease in liver function, so malnutrition can occur, characterized by weight loss or low weight gain.Malnutrition in liver cirrhosis patients is caused by various factors, such as hypermetabolism due to infection, fat malabsorption, and nutritional metabolic disorders related to increased gluconeogenesis, protein catabolism, and decreased glycogenolysis [25].

Journal of Nutrition and Metabolism
Te increase in body weight during intervention showed that giving hepatogomax formula enteral which contained macronutrients, 4.59 g of BCAA, and 76.66 g of MCT was able to repair damage to the liver so that there was an increase in body weight [21].Te condition of liver cirrhosis is related to insulin resistance, which causes the inability of the body to inhibit the gluconeogenesis process resulting in weight loss [26].BCAA can improve insulin resistance in the liver by increasing sterol regulatory elements that bind to the protein-1c pathway, activating liver-type glucokinase (L-GK), and glucose transporters.Furthermore, BCAA suppresses liver expression of glucose-6-phosphatase (G6P) and increases peroxisome proliferator activator receptor (PPARc) and uncoupling protein 2 (UCP2), which can stimulate free fatty acids oxidation [16,27].L-GK expression plays a role in reducing blood glucose by increasing glycogen synthesis or reducing gluconeogenesis which causes an increase in muscle mass and protein tissue to increase body weight [27][28][29].Besides BCAA, MCT plays a role in improving muscle function and strength through the activation of protein kinase B (Akt) and signaling of the adenosine monophosphate protein kinase (AMPK) pathway, inhibiting, transforming growth factor-β (TGF-β) signaling, and increased metabolism in skeletal muscle [30].
Increased serum levels of TNF-α and IL-6 after TAA induction are associated with infammation, proliferation, apoptosis, and fbrosis in the liver [31].TAA is capable of causing liver damage by forming reactive oxidative metabolites, namely, S-oxide and SS-dioxide [32].Tese result in fatty acid accumulation, protein and DNA damage, and the formation of reactive oxygen species (ROS), followed by the synthesis of a cascade of proinfammatory cytokines such as TNF-α and IL-6 [33].Hepatogomax, which contains BCAAs and MCTs, can reduce TNF-α and IL-6 by decreasing ROS production by activating the antioxidant mechanism, thereby reducing oxidative stress and infammation in the liver.Improvement of infammation in the liver allows a decrease in TNF-α and IL-6 levels in liver damage conditions [34].
Patients with liver damage had a lower abundance of SCFA-producing bacterial species [35].Decreased SCFA content can impair intestinal barrier function and increase circulating permeability.Tis study showed that hepatogomax formula feeding increased SCFA levels in TAA-induced rats with liver damage.Te increase in SCFA levels was associated   [36,37].Tis study demonstrated that elevated SCFA levels were inversely related to TAA-induced systemic infammation.Tis is similar to LPS in liver infammation, which is related  6 Journal of Nutrition and Metabolism to the activation of infammasome nod-like receptor protein 3 (NLRP3) and the production of interleukin (IL)-1β through activation of the nuclear factor-κB (NF-κB) pathway [38].NF-κB is a key transcription factor of M1 macrophages.It is required to induce the number of infammatory genes such as TNF-α and IL-6 [39].SCFA can inhibit lipopolysaccharide (LPS)-induced infammation [40].SCFA reduces systemic infammation by suppressing LPS production and maintaining intestinal integrity so that circulating LPS levels decrease.In addition, SCFA decreases the liver's infammatory response by inhibiting the activity of histone acetyltransferases reducing the generation of regulatory T cells and the expression of cytokines in T cells.Propionate increased the inhibitory activity of Treg cells, while butyrate inhibited the activity of the NLRP3 infammasome and increased the diferentiation of regulatory Tcells.Ten, SCFA butyrate and propionate suppress NF-κB activation resulting in a decrease in the production of proinfammatory cytokines [36,41,42].Apart from that, based on other studies, it was explained that the production of various metabolites of the gut microbiota, such as SCFA (acetate, propionate, and butyrate), which is produced the most in the caecum, shows anti-infammatory properties [43].Te content of MCTs and BCAAs can reduce the accumulation of fat in the liver by preventing fat malabsorption.Decreased fat malabsorption causes an increase in bile acid secretion into the intestinal lumen, which is related to an increased composition of SCFA-producing bacteria [44,45].BCAAs also contribute to energy homeostasis and lipid metabolism and have an anti-infammatory efect that can suppress infammatory reactions in the intestine, so that gut microbiota of SCFA production increases [45].Also, BCAAs are precursors of microbial-derived SCFA in the gut.It causes an increase in the bioavailability of SCFA [46].Of interest, it has been suggested that the BCAAs from exogen (i.e., from the diet) may shape the gut microbiota Ruminococcus favefaciens.Tis leads to an increase in the bioavailability of acetate, where acetate will decrease the expression of lipogenesis genes, resulting in reduced fat accumulation and steatosis in the liver [45].
In addition, lower necrosis and ballooning cells in the P1 and P2 groups suggest that BCAAs-enabled repair of liver tissue damage is possible.BCAAs could attenuate liver infammation, including IL-6, by suppressing the activation of the LPS-binding protein, toll-like receptor 4, and signal transduction and activator of transcription-3 (LBP-TLR4-STAT3) pathway and reducing the translocation microbiota of Enterococcus faecalis into the liver.Suppressing LBP expression and STAT3 activation reduced hepatocyte death in liver damage conditions [47].However, this study showed severe ballooning cells in the P3 group.Tere may be an unfavorable efect on the tissues when given high doses.
Tis study has certain limitations.First, we did not perform an analysis of the gut microbiome, serum LBP or LPS levels, other infammation markers, and markers related to liver damage and steatosis, which could beneft from observing the underlying mechanism.Furthermore, we did not analyze serum BCAA levels to determine whether the improvement in all variables was based on the increase in serum BCAAs.Parameters in our study are used for hypotheses and mechanism bridges, but further research is needed with other parameters to be able to explain the improvement of the pathophysiological mechanism of the efect of the hepatogomax formula.Finally, we need further research on the mechanism of side efects of high-dose BCAAs and MCTs in the hepatogomax enteral formula against damaged liver tissue.

Conclusions
Te present study showed that hepatogomax enteral formula administration on TAA-induced may improve systemic infammation, increase SCFA levels, and reduce steatosis in a dose-dependent manner.Ten, we need further research about the mechanism involved.

3. 4 .
Histopathology.Histopathological examination of the liver showed normal liver architecture with minimal infammatory necrosis, ballooning cells, and steatosis (Figure1(a)).TAA induction showed a severe increase in ballooning cells and large steatosis (Figure1(b)).Te administration of hepatogomax enteral formula at a dose of 4.97 g/200 gBW and 9.73 g/200 gBW showed a moderate ballooning cell and slight steatosis (Figures1(d) and 1(e)).However, the administration of high doses of hepatogomax showed a slight increase in infammatory necrosis and severe ballooning cells with barely noticeable steatosis (Figure1(f )).

Table 1 :
Body weight of rats.

Table 3 :
SCFA levels after intervention.witha dose-dependent manner in which high doses of hepatogomax increased SCFA levels the most.Te increase in SCFA levels indicated that the BCAAs and MCTs content in the hepatogomax enteral formula increased the composition of the bacteria-producing SCFA.BCAAs, through glycolysis, are converted into pyruvate, the precursor of three SCFAs.Gut microbiota such as Anaerostipes, Faecalibaterium, and Clostridium use pyruvate to produce SCFA acetate and butyrate via the acetyl-CoA pathway.Ten, propionate is produced via the acetyl-CoA and succinate pathway by the gut microbiota Coprococcus and Dialister