Protective effects of Althaea officinalis L. extract against N‐diethylnitrosamine‐induced hepatocellular carcinoma in male Wistar rats through antioxidative, anti‐inflammatory, mitochondrial apoptosis and PI3K/Akt/mTOR signaling pathways

Abstract Hepatocellular carcinoma is the fourth cause of death due to cancer and includes 90% of liver tumors. Therefore, in this study, it was tried to show that Althaea officinalis L. flower extract (ALOF) can protect hepatocytes against N‐diethylnitrosamine (DEN)‐induced hepatocellular carcinoma. Totally, 70 Wistar rats were divided into seven groups (n = 10/group) of sham, DEN, treatment with silymarin (SIL; DEN + SIL), treatment with ALOF (DEN + 250 and 500 ALOF), and cotreatment with SIL and ALOF (DEN + SIL + 250 and 500 ALOF). At the end of the study, the serum levels of liver indices (albumin, total protein, bilirubin, C‐reactive protein, ALT, AST, and ALP), inflammatory cytokines (IL‐6, IL‐1β, IL‐10, and TNF‐α), and oxidants parameters (glutathione peroxidase [GPx], superoxide dismutase [SOD], catalase [CAT] activity along with nitric oxide [NO] levels) were evaluated. The level of Bax, Bcl‐2, Caspase‐3, p53, PI3K, mTOR, and AKT genes were measured. ALOF in cotreatment with SIL was able to regulate liver biochemical parameters, improve serum antioxidant indices, and decrease the level of proinflammatory cytokines significantly (p < .05). ALOF extract in both doses of 250 and 500 mg/kg in cotreatment with SIL caused a significant (p < .05) decrease in the p53‐positive cells and a significant (p < .05) increase in Bcl‐2‐positive cells. Therefore, ALOF was able to modulate the proliferation of cancer cells and protect normal cells through the regulation of Bax/Bcl‐2/p53 and PI3K/Akt/mTOR signaling pathways. It seems that ALOF can be used as a prodrug or complementary treatment in the protection of hepatocytes in induced damages caused by carcinogens.

methylating nitrosamines are compounds that cause damage to the DNA of normal cells. Studies have shown that rats exposed to tobacco-derived nitrosamines developed various tumors in the colon, nose, lung, mouth, liver, esophagus, pancreas, and breast (Hoffmann et al., 2017). DEN is a carcinogen chemical substance used in the induction of various models of respiratory system, skin, gastrointestinal tract, and liver tumors. After the intraperitoneal injection or gavage of 10 mg/kg of body weight, the initial symptoms of liver toxicity (chronic inflammation and fibrosis) appear, and in doses lower than 30 mg/kg of body weight, the initial stages of induction, promotion, and progression of liver tumors are created (Memon et al., 2020). One of the reasons for tumor induction caused by DEN is the increased expression of G1/S-phase regulatory proteins (such as cyclin-dependent kinases) of hepatocytes. In addition, the subsequent biotransformation of DEN and alkylating production metabolites leads to DNA damage of hepatocytes. Following the cellular metabolism of DEN, ROS and RNS are produced. These agents reduce free radical scavenging/production ratio, and besides inhibiting endogenous antioxidant enzymes, they also attack cellular biological macromolecules (proteins, polyunsaturated fatty acids; Arboatti et al., 2018;Tolba et al., 2015).
DEN targets different apoptotic/autophagy/proliferative/differentiation pathways of liver parenchymal cells. PKB/AKT pathway is one of the pathways involved in the proliferation and differentiation of hepatocyte cells, and is inhibited by DEN (Hegazy et al., 2019).
Meanwhile, DEN stimulates Wnt/β-catenin and TGFβ1/Smads tumorigenic pathways of hepatocytes and accelerates their tumorigenesis process . Another tumorigenic pathway of DEN is the stimulation of MEK1, 2/ERK1, 2/cyclin D1, and two signaling pathways along with the stimulation of PI3K/Akt/mTOR signaling pathways in hepatocytes. Studies have also shown that DEN inhibits the proliferation of normal and damaged hepatocytes through the stimulation of Bax/Bcl-2/p53 mitochondrial apoptosis pathway . DEN activates the ROS-and RNS-induced p53-dependent apoptotic pathway associated with cytochrome P450 of hepatocytes and triggers the apoptotic cascade (activating initiator caspases such as caspases 8 and 9 followed by activating the effector caspases such as caspases 3 and 6) by leaking cytochrome c from mitochondria (Mo'men et al., 2020). DEN and its metabolites stimulate Kupffer cells, neutrophils, and liver lymphocytes to increase the panel of proinflammatory cytokines (IL-6, IL-8, and IL-1β) and inhibit the synthesis of anti-inflammatory cytokines (IL-10, IL-4, IL-2, and IL-13; Chen et al., 2015). Therefore, the most suitable strategy for the treatment of HCC caused by DEN is the use of compounds that affect anti-inflammatory, antioxidant, and proliferation/differentiation pathways. Different studies have shown that some plant extracts, including Calendula officinalis L., Cassia fistula L., Salmo gairdneri L., Agaricus blazei L., Tinospora cordifolia L., and Punica granatum L., due to their polyphenolic compounds (flavonoids and isoflavonoids), protect hepatocytes against DENinduced HCC through different pathways. The extracts of these plants contain different compounds such as daidzein, genistein, quercetin, kaempferol, catechin, resveratrol, curcumin, rutin, betulinic acid, and artemisinin, which suppress various tumors including HCC through anti-inflammatory, antiangiogenic, and antiproliferative activities (Dhanasekaran et al., 2009;Dhingra et al., 2022;Kaur et al., 2019;Lee & Hong, 2011).
Since ancient times, ALOF has been used as a seasoning for soups, teas, vegetables, and as an additive in food to treat dry coughs, flatulence, diabetes, hyperlipidemia, diarrhea, relief of high fever, and increase immunity against viruses and bacteria. In modern medicine, ALOF and its effective ingredients are used in the treatment of cardiovascular diseases, respiratory tract infections, and urolithiasis (Kianitalaei et al., 2019). In vitro studies have shown that ALOF extract with antiproliferative activity suppresses the proliferation of breast cancer cell line AMJ13, human lung cancer cell line A549, and prostate cancer cell line PC-3 (Kadhum et al., 2021;Zhang et al., 2016). Studies have shown that the compounds present in this plant, such as kaempferol, genistein, p-coumaric acid, daidzein, and quercetin act through PI3K/AKT/mTOR, MEK-ERK, PI3K-AKT/PKB, and AMPK/p53 signaling pathways, along with anti-inflammatory and antioxidant effects. They suppress various breast, colon, lung, brain, and skin tumors in in vitro and in vivo models (He et al., 2011;Kaushik et al., 2019;Li et al., 2019). In the study of Morovatisharifabad et al. (2020), it was found that ALOF extract in doses of 300 and 600 mg/kg of body weight protected the function and structure of the liver against inflammatory/oxidative damage caused by diazinon toxin in hamsters (Morovatisharifabad et al., 2020). In this study, ALOF extract was able to improve the level of liver enzymes along with antioxidant parameters. For this purpose, in this study, we tried to evaluate the antitumor effects of ALOF against DEN-induced hepatocellular carcinoma in Wistar rats through antioxidative, anti-inflammatory, mitochondrial apoptosis and PI3K/Akt/mTOR signaling pathways.

| DEN and induction of hepatocarcinogenesis
The DEN (Sigma-Aldrich) was purchased in dark bottles as a fresh solution (100 mg/L) and supplied to induce hepatocarcinogenesis following a previous method (Arboatti et al., 2018).

| Animals and experimental design
Seventy male Wistar rats weighing 180 ± 20 g randomly were grouped into seven groups (n = 10/group) according to Table 1. The rats were kept for 72 h to adapt to the temperature, food, and water used in the study before starting the study. All rats were kept in propylene cages at a temperature of 25 ± 2°C, relative humidity of 50 ± 5%, and a 12/12-h dark/light cycle. Standard rat pellets and tap drinking water were used for all groups. All procedures of maintenance and sacrifice of rats were carried in accordance with the national standards protocol and guidelines for the care of laboratory animals (NIH Publication 80-23, 1996).
The total duration of the study was 42 days, and in groups 2-7, rats received 100 mg/kg DEN intraperitoneally (i.p.) at 8:00 a.m. on days 7, 14, and 21 of the study, and in groups 3 and 4, in addition to DEN, from days 7 to 42 the animals received 250 and 500 mg/kg of ALOF extract by gavage, respectively, every day at 15:00 p.m. In group 5, in addition to DEN, rats received 0.5% silymarin (SIL) powder by gavage from days 7 to 42 of the study every day at 15:00 p.m., and in groups 6 and 7, in addition to receiving DEN, rats received 250 and 500 mg/kg of ALOF, respectively, and SIL at 15:00 p.m. from days 7 to 42. To select the most appropriate dose of DEN and SIL, previous studies were used along with the pilot study. To choose the most effective and nontoxic dose of ALOF extract, LD 50 (lethal dose) was also considered in addition to previous studies and a pilot study (Hage-Sleiman et al., 2011;Imamoto et al., 2014;Talebi et al., 2014).

| Acute toxicity test (LD 50 ) of ALOF extract
To select a safe dose of ALOF extract, LD 50 was used according to Lork's two-step method. According to this method, first, 12 rats in TA B L E 1 Experimental design and animal grouping.

Group number
Group name Treatment (dose and prescription manner) Treatment period three groups (n = 4/group) of doses of 50, 500, and 5000 mg/kg/i.p.
received ALOF extract, and their general condition was monitored within 24 h. Doses in which any toxic symptoms or possible death were observed in rats were recorded. Then six rats in three other groups (n = 2/group) with doses of 10, 100, and 1000 mg/kg/i.p.
ALOF extract was received, and the procedure was similar to the previous step. Finally, the lowest dose in which toxic symptoms or death were observed (D L ), along with the highest dose (D S ) in which rats did not have any toxic symptoms, were placed in the following formula and LD 50 of ALOF extract was calculated (Feng et al., 2022).
In this study, during the evaluation of LD 50 , only in a dose of 5000 mg/kg ALOF extract caused nausea in rats, while no death occurred in this dose and lower doses. It seems that the nausea in the dose of 5000 mg/kg ALOF is caused by the irritation of the digestive system and this plant has no other toxic side effects.

| ALOF extract total flavonoid content (TFC)
Miliauskas method was used to calculate ALOF extract TFC based on mg/mL. In this method, 0.

| Nitric oxide (NO) assay
The Griess colorimetric method was used to measure serum NO level as one of the most important serum factors indicating lipid peroxidation and oxidative stress. Briefly, 500 μL serum samples were added to 6 mg zinc oxide, and after mixing and centrifugation (10,000g for 15 min), the supernatant was transferred to 500 μL Griess solution.
After incubation at 37°C for 60 min, the absorbance of the resulting mixture was read at a wavelength of 540 and 630 nm by a spec-

| Western blotting assay
To evaluate the effect of ALOF extract on the PI3K/AKT/mTOR pathway in liver tissue, the expression of the proteins of this pathway (PI3K, AKT, and mTOR) was evaluated by western blotting assay. Briefly, after isolating the liver tissues, washing with

| Immunohistochemistry (IHC) assay
In evaluating the apoptosis/survival pathway of Bax/Bcl-2/p53, the level of p53 protein expression in liver cells was evaluated. The rate (%) of p53-and Bcl-2 positive cells/total cells was evaluated with a light microscope (Olympus CH3, Japan) equipped to camera system

| Histopathological assay
Like what was stated in IHC assay, after preparing paraffin blocks from liver samples and 5 μm slices from them, hematoxy-

| Statistical analysis
In designing graphs, version 8 GraphPad Prism software were used.
For statistical analysis, version 16 SPSS software (SPSS, IBM) was used to compare the mean differences of the data between groups by one-way analysis of variance (ANOVA) test followed by Tukey's post hoc test after checking the normality between groups with the help of Kolmogorov-Smirnov test (p > .05). The p < .05 was considered to be significant between data and all data were reported as mean ± standard deviation (SD).

| LD 50 of ALOF extract
After 24-h monitoring of the groups in terms of toxicity and lethality, it was determined that the lowest dose (D L ) in which the rats suffered toxic symptoms or death was in the 5000 mg/kg group and the highest dose (D S ) in which the rats did not show any toxic symptoms was in 1000 mg/kg group. Therefore, according to the formula of Lorke's method, the LD 50 of ALOF extract is 2236 mg/kg (or 2.236 g/ kg) and lower doses can be used in the study.

| Total body and liver weights of rats
Evaluating the total body weight (BW) and liver weight ( (DEN + 500 ALOF + SIL), so that the BW in this group increased significantly compared to both DEN and DEN + SIL groups, and the LW in this group also significantly (p < .05) decreased compared to these two groups ( Figure 1A). One of the reasons for the increase in liver weight in the groups receiving DEN is the high number of nodular inflammatory lesions on the liver, while the sham group did not have these nodular lesions and its weight was less than the other groups ( Figure 1B).

| Liver biochemical parameters
After measuring the activities of serum liver enzymes (ALT, AST, and ALP), it was found that DEN significantly (p < .05) increased the activity of all three liver enzymes compared to the sham group. In the SIL and 500 ALOF treatment groups, the serum activity of all three liver enzymes significantly (p < .05) decreased compared to the DEN group. The maximum reduction in the activity of these enzymes was observed in the cotreatment groups (DEN + 250 and 500 ALOF + SIL), so that both cotreatment groups significantly (p < .05) decreased the activity of all three enzymes in comparison to the DEN and DEN + SIL groups (Figure 2A).
The results of serum BIL and CRP levels also showed that the levels of both factors were significantly (p < .05) higher in the DEN group than in the sham group. SIL was only able to significantly (p < .05) reduce the serum level of CRP compared to DEN group, while 500 ALOF alone was able to significantly (p < .05) reduce the level of both serum factors compared to DEN. In cotreatment groups, especially DEN + 500 ALOF + SIL, it was found that the serum level of BIL and CRP decreased significantly (p < .05) not only in comparison with DEN group but also in comparison with SIL (DEN + SIL) group ( Figure 2B). The evaluation of serum ALB and TP levels between the groups also showed that DEN significantly (p < .05) reduced the levels of both factors. SIL and ALOF extract alone could increase serum ALB and TP levels, but this increase was not significant (p > .05).
While in the DEN + 250 ALOF + SIL cotreatment group, the levels of both factors increased significantly (p < .05) compared to the DEN group. In the DEN + 250 ALOF + SIL cotreatment group, not only the levels of both factors significantly (p < .05) increased compared to the DEN group, but the levels of both factors also increased significantly (p < .05) compared to the DEN + SIL group ( Figure 2B).

| IL-6, IL-10, IL-1β, and TNFα serum levels
After measuring the levels of pro-/anti-inflammatory cytokines in the serum of the studied rats, it was found that DEN significantly (p < .05) increased the levels of all three proinflammatory cytokines (IL-6, IL-1β, and TNFα) compared to the sham group and also significantly suppressed (p < .05) the secretion of anti-inflammatory cytokine IL-10 compared to the sham group. SIL alone was able to significantly (p < .05) decrease the serum levels of IL-6 and TNFα and also significantly (p < .05) increase the level of IL-10 compared to the DEN group. Although in the group that received only ALOF extract, the level of cytokines improved, but these changes were not significant (p > .05). While in ALOF + SIL cotreatment groups, especially DEN + 500 ALOF + SIL, the serum levels of all three proinflammatory cytokines were significantly (p < .05) decreased compared to the DEN group and the SIL group, and the level of the anti-inflammatory cytokine IL-10 increased significantly (p < .05) compared to both groups (Figure 3).

| Serum GPx, SOD, and CAT activity and also NO levels
After measuring the activity of antioxidant enzymes (GPx, SOD, and CAT), it was found that DEN significantly (p < .05) reduced the activity of all three enzymes compared to the sham group. Although SIL alone was able to increase the activity of all three enzymes compared to the sham group, these changes were not significant (p > .05). While 500 ALOF alone could significantly (p < .05) increase the activity of GPx and SOD compared to the DEN group. The most changes in the activity level of all three endogenous antioxidant enzymes were observed in the cotreatment groups, especially DEN + 500 ALOF + SIL, so that in these groups, the activity level of all three enzymes compared to both DEN and SIL groups significantly (p < .05) increased ( Figure 4A). In the evaluation of the results related to the serum NO level in different groups, it was also found that DEN caused a significant (p < .05) increase in its serum level compared to the sham group.
ALOF in both 250 and 500 mg/kg groups (DEN + 250 and 500 ALOF) alone was able to significantly (p < .05) reduce the level of NO compared to the DEN group. Serum NO levels in both DEN + 250 and 500 ALOF + SIL cotreatment groups significantly (p < .05) decreased compared to both DEN and SIL groups ( Figure 4A).

| Liver tissue TAC, TBARS, and thiol levels
DEN caused a significant decrease in the antioxidant power and eventually significantly (p < .05) decreased the tissue level of FRAP, TBARS, and thiol compared to the sham group. Although SIL alone was able to increase the level of FRAP, TBARS, and thiol compared to the sham group, these changes were not significant (p > .05).
While 500 ALOF alone could significantly (p < .05) increase the levels of all three tissue antioxidative parameters compared to the DEN group. The most changes in the level of all examined antioxidative parameters were observed in cotreatment groups (DEN + 250 and 500 ALOF + SIL), so that in these groups, the level of all three antioxidative parameters compared to both DEN and SIL groups significantly (p < .05) increased ( Figure 4B). After analyzing the results related to the expression of PI3K/AKT/ mTOR signaling pathway genes, it was found that DEN stimulated this pathway and significantly (p < .05) increased all three genes of this pathway compared to the sham group. SIL was able to decrease the expression level of all three genes compared to the DEN group, but this decrease was significant (p < .05) only in the PI3K gene. The results showed that 500 mg/kg ALOF (in DEN + 500 ALOF group) alone could significantly (p < .05) reduce the expression level of all three genes of this pathway compared to DEN group. ALOF (250 mg/ kg) was also able to significantly (p < .05) reduce the expression of all F I G U R E 3 Serum levels of interleukin 10 (IL-10; ng/mL), IL-1β, IL-6, and tumor necrosis factor alpha (TNFα; pg/mL; n = 10 rat/group, data are mean ± SD) in sham (500 μL PBS), diethylnitrosamine (DEN), treatment with silymarin (SIL; DEN + SIL), treatment with 250/500 mg/ kg Althaea officinalis L. (ALOF; DEN + 250 and 500 ALOF), and SIL cotreatment with 250/500 mg/kg ALOF (DEN + SIL + 250 and 500 ALOF) groups. a (p < .01) comparison of DEN with sham, b (p < .01) comparison of all treatment with DEN, and c (p < .01) comparison of ALOF treatment with SIL.

Cas-3, Bcl-2, and Bax genes
three genes of this pathway in the SIL cotreatment group compared to the DEN group (in DEN + 250 ALOF + SIL group). Meanwhile, 500 mg/ kg ALOF also significantly (p < .05) reduced the expression of all three genes in the SIL cotreatment group (in DEN + 500 ALOF + SIL group) compared to both DEN and SIL groups ( Figure 6).

| Expression of liver PI3K, AKT, and mTOR proteins
The analysis of the results related to the expression of PI3K/AKT/ mTOR signaling pathway proteins was also in line with the results related to the expression of the genes of this pathway. DEN strengthened this signaling pathway and significantly (p < .05) increased the expression of all three proteins of this pathway compared to the sham group. SIL alone decreased the expression of all three proteins of this pathway, but this decrease was not significant (p > .05). This was while 500 mg/kg ALOF (in DEN + 500 ALOF group) alone could significantly (p < .05) reduce the protein expression level of AKT and mTOR compared to DEN group. While ALOF, especially in 500 mg/ kg along with SIL, was able to significantly (p < .05) reduce the expression of proteins of this pathway compared to both DEN and SIL groups (Figure 7).

| Histopathologic findings
Histopathological evaluations of the liver tissue in the studied groups showed that DEN caused the formation of hepatic nodules (HN) consisting of degenerated hepatocytes (D) and lymphocytic infiltration (LI) next to the necrotic tissue. In this group, the normal hepatic lobule (HL) and hepatic triad (HT) were lost, and congestion (CO) and central venule (CV) dilatation were evident. In treatment groups with only SIL or ALOF extract, the volume of the formed HN was reduced to some extent fibrotic tissue and LI were visible next to CO. This was despite the fact that in cotreatment groups, especially 500 mg/kg ALOF, HL formation was normalized, and LI was greatly reduced. Also, the HN was not evident in the liver and the arrangement of hepatocytes (H) was clearly visible next to the liver sinusoids (S; Figure 10).

| DISCUSS ION
The results of this study showed that ALOF extract, along with SIL, can have synergistic effects through improving antioxidant  Beer et al., 1982;Hattori et al., 2003;Hotamisligil et al., 1996;Nabata et al., 2008;Xi et al., 2011;Zarubin & Han, 2005).  of ATP pumps of the electron transport chain, synthesis of proteins, and glucose uptake of tumor cells (Miricescu et al., 2020;Yu et al., 2022). In addition, Sahin et al. (2014) showed that DEN strengthens the NF-κB/cyclooxygenase-2 pathway through the stimulation of the PI3K/Akt/mTOR and Nrf-2/HO-1 axis, and after tumorigenesis of hepatocytes and nodule formation, the function of these cells decreased and liver function indices changed (Sahin et al., 2014). In this study, in addition to strengthening the inflammatory pathways (increasing the level of IL-6, IL-1β, and TNFα), stimulating the mitochondrial apoptotic cascade (Bax/Bcl-2/Cas-3/ p53) and PI3K/AKT/mTOR axis also occurred. (ALT, AST, and ALP), BIL, and CRP and increase ALB, TP both alone and together with SIL in a synergistic and dose-dependent manner (especially in DEN + 500 ALOF + SIL group). Also, ALOF extract, especially in cotreatment groups with SIL in a dose-dependent manner, inhibits inflammatory pathways (decrease in the level of IL-6, IL-1β, and TNFα and increase in IL-10) and oxidative stress (increase in the activity of GPx, SOD, and CAT and decrease NO levels) induced by DEN.

| CON CLUS ION
Medicinal plants in pure form or effective substances extracted from them can be used as prodrugs/additives as low-cost, simple, and ecofriendly agents in the pharmaceutical industry in the prevention/treatment of various tumors. According to the results of this study, it seems that ALOF extract can protects the physiological function of hepatocytes and preserve the normal architecture of liver lobules against oxidative/inflammatory damages caused by carcinogens. It is suggested that for complementary and clinical trial studies in humans, other possibly effective signaling pathways of ALOF extract were investigated in other models of hepatic damages. Furthermore, it is possible to use green synthesis or ALOF extract (or effective ingredients) loaded in nanocarriers in various animal tumor models (especially HCC) through signaling pathways related to the growth and metastasis of tumor cells.

ACK N OWLED G M ENTS
The financial support of the 905th Hospital of the Chinese People's Liberation Army Navy is highly appreciated.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that they do not have any conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

E TH I C S S TATEM ENT
This article does not contain any studies with human participants performed by any of the authors. Maintaining, feeding, and sacrificing animals were done according to the national standards protocol and guidelines for the care of laboratory animals (NIH Publication 80-23, 1996)