Introduction

Aging is a natural process associated with a progressive decline in organ functions and accompanied by the development of age-related disease (Fathi and Farahzadi 2016). Some of the aging hallmarks include genomic and epigenetic alterations, telomere shortening, mitochondrial dysfunction, deregulated nutrient signaling, cellular senescence, and altered intercellular interactions (López-Otín et al. 2013; Fathi, et al. 2019; Ebrahimi 2020; Farahzadi et al. 2016). The aging process increases the risk of inflammatory-related diseases, cancers, and neurodegenerative diseases in humans (Kirkwood 2005; Farahzadi et al. 2020; Fathi et al. 2019). Accordingly, delaying the aging process can hinder the onset and the progression of related disorders (Kaeberlein 2013; Mobarak et al. 2017). According to the overview of the literature, aging is associated with a deterioration of liver function (Mehdizadeh et al. 2017). It has been well documented that the liver has a key role in metabolic homeostasis via regulation of systemic energy metabolism, molecular biosynthesis, and clearance of xenobiotics and endobiotics (Rui 2011). Age-related alteration in liver function is closely linked to systemic susceptibility to age-related diseases (Kim et al. 2015; Basso et al. 1998). It has also been reported that structural and functional alterations in the liver are correlated with a dramatic reduction in mitochondria numbers (Wu et al. 2014). Mitochondria have a fundamental role in producing ATP for cellular demands. Besides, mitochondria decline basal metabolic rate in the aging process (Jang et al. 2018).

It has been well established that the liver has superior role in the redox status by producing 90% of the circulating glutathione (GSH) (Jones 2008). In addition to this, aged hepatocytes are strongly associated with an increased reactive oxygen species (ROS) production (Kroemer et al. 2007; Green et al. 2011; Rezabakhsh, et al. 2017). Noteworthy, ROS can activate/inactivate a variety of signaling pathways and related mediators (Feizy et al. 2016; Saliani et al. 2017). All mentioned events result in progressive mitochondrial dysfunction, inflammatory state, and steatosis in the liver (Bejma et al. 2000). On the other hand, the liver has a crucial role in inflammatory status due to a reservoir of the resident macrophages such as Kupffer cells (Szabo and Csak 2012). According to several studies, aged hepatocytes can produce a higher level of cytokines such as interleukin 6 (IL-6), tumor necrosis factor 1-α (TNFα), and interleukin-1β (IL-1β) (Lasry and Ben-Neriah 2015), which ultimately lead to the development and progression of oxidative stress through ROS-mediated activity. This is mainly accompanied by activation and nucleus translocation of NF-κB as well as regulation of some genes such as COX2 (Iorio, et al. 2003; Wunderlich et al. 2010; Williams et al. 2008). Besides, TNF-α has ability to activate c-Jun amino-terminal kinase (JNKs) as a member of the mitogen-activated protein kinases (MAPKs) family (Kyriakis and Avruch 2001; Roux and Blenis 2004). Activation of JNK can stimulate several pathways which leads to phosphorylation of c-Jun, as a stimulator of inflammatory status (Schwabe and Brenner 2006). Furthermore, evidence declared that the impairment of the autophagosomal–lysosomal networks and β-galactosidase (β-Gal) can be predisposing factor in the onset and progression of many age-related diseases (Rajawat et al. 2009; Carmona-Gutierrez et al. 2016). Therefore, β-Gal has been used as an important biomarker to detect the aging process (Morgunova et al. 2015).

Growing evidence has shown that naturally derived products have benefits on the aging process via modulating biological and cellular processes (Ding et al. 2017). Sericin is a natural protein produced by the silkworm Bombyx mori. It has been demonstrated that sericin has ROS scavenging abilities as well as strong antioxidant capacity that inhibit lipid peroxidation activity(Deori et al. 2016; Seyedaghamiri 2021).

Although the anti-inflammatory, antioxidant, anti-cancer, and anti-tyrosinase activity of sericin has been well documented in many studies (Kunz, et al. 2016; Barajas-Gamboa et al. 2016), its effect on liver aging has not been established. In the present work, we study the possible hepatoprotective effect of sericin in parameters involved in apoptosis, inflammation, oxidative stress, and JNK/p38 signaling networks in aged mice.

Materials and methods

Animals

Nine male young (2 months) and eighteen aged mice (2 years) were used in the study. All experimental groups were housed in the cages under standard conditions (25 ± 2 °C, 70% humidity, and 12/12 h light/dark photoperiod schedule) and given free access to food and tap water. This study was confirmed by the ethics committee of Tabriz University of Medical Sciences (ethical code No. IR.TBZMED.VCR.REC.1399.334). All experimental procedures were conducted according to the guidelines of the National Institutes of Health (NIH) (publication No. 85–23, revised 1985).

Treatment

The animals were divided into three experimental groups of nine individuals each, including young control (received normal saline, 2 ml/kg/day), aged control (received normal saline, 2 ml/kg/day), and sericin-treated aged mice (received sericin, 100 mg/kg/day). Sericin was purchased from Xian Lyphar Biotech Co. Ltd., Xian, China. Sericin was dissolved in normal saline just before treatment and administered orally for 14 days.

Sampling

At the end of the treatment period, the animals were anesthetized by ketamine and xylazine (90 and 10 mg/kg, i.p., respectively), followed by blood sampling from the heart. Blood samples were centrifuged (3000 rpm for 10 min at 4 °C), and serum was separated and frozen until analyzed. The livers of animals were then cautiously removed, and samples were put into cryo-tubes and were frozen immediately in liquid nitrogen. The samples were kept at − 70 °C for further analysis.

Liver functional assays

To evaluate liver function, alkaline phosphatase (ALP), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels were examined in the serum sample using the commercial kits (Pars Azmoon, Iran) by spectrophotometry method (Olympus AU-600, Tokyo, Japan) based on the previously published study (Bagheri, et al. 2020).

Evaluation of antioxidant capacity and lipid peroxidation

To investigate the antioxidant activity of sericin, the activity of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), the levels of glutathione (GSH), and total antioxidant capacity (TAC) were determined in the liver tissues. Also, malondialdehyde (MDA) levels were assessed in the liver tissues using commercial kits (Zellbio, Biocore, Germany). The procedure was similar to the previous study by this group (Bagheri et al. 2021; Rahimi, et al. 2021).

Liver protein extraction and western blot analysis of selected proteins

The western blotting analysis was used to determine the level of apoptosis (Bcl-2, Bax, and Caspase-3), pro-inflammatory markers (IL-1β, IL-6, TNF-α, and Cox-2), and JNK and P38 in the liver tissues. Briefly, the liver tissues were homogenized with lysis buffer (RIPA with protease inhibitor). Then, protein concentration was measured by the Bradford method, and equal amounts of samples were subjected to SDS polyacrylamide gel electrophoresis (12.5%). After that, the bands were transferred onto PVDF membranes (PVDF) (Roche, UK). After blocking the blots, the membranes were incubated with primary antibodies overnight at 4 °C, according to the manufactures’ instructions. Finally, after being washed with PBS, the membranes were exposed with secondary antibody (rabbit polyclonal) at room temperature for 2 h, visualized by chemiluminescence, an ECL Plus kit (Amesham Life Science Inc., Buckinghamshire, UK). β-Actin was served as a control for normalizing the optical density of the bands. The intensity of bands was calculated using ImageJ software (version 1.62, National Institutes of Health, Bethesda, MD, USA) (Montazersaheb et al. 2019; Tarhriz et al. 2019; Fathi et al. 2021).

Evaluation of mRNA level by real-time PCR analysis

In order to evaluate β-Gal mRNA level, the qRT-PCR method was carried out. In brief, RNAs were extracted (Trisol) from all experimental groups and quantified by Picp-Drop (ND-1000, Thermo Scientific, USA). Due to the variation of RNA concentration in each sample, the concentrations were normalized and adjusted before cDNA synthesis. RNAs were subjected to reverse transcription using the RevertAid H Minus First-strand cDNA Synthesis Kit (Takara). After making cDNAs, samples were kept at − 20 °C. The sequences of used primers are listed in Table 1. Real-time PCR was performed in a total volume of 20 μL, consisting of SYBR green master mix 10 μl (Takara), 1 μl of each of the forward and reverse primers, 1 μl of cDNA, and 6 μl of nuclease-free water. The amplification profile was as follows: 94 °C for 4 min followed by 35 cycles of 94 °C for 10 s, 59 °C for 34 s, and 72 °C for 10 min. To quantify gene expression, we normalized the β-Gal gene to GAPDH as a housekeeping gene. Data were analyzed using ABI plus one to determine CT values and then calculated in relation to GAPDH CT by the 2−ΔΔCT formula. The samples were run in triplicates (Tarhriz et al. 2018; Fathi, et al. 2020; Fathi, et al. 2020).

Table 1 Primer sequences used for real-time PCR
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Statistical analysis

Data are presented as mean ± SEM. All results were analyzed by one-way ANOVA followed by a post hoc Tukey using GraphPad Prism 6.01 (GraphPad Software Inc., La Jolla, CA, USA). Statistical significance was considered p value < 0.05.

Results

The impact of sericin administration on liver function

The levels of liver function parameters such as AST, ALT, and ALP were measured in the serum samples of the animals. The results showed that the activity of ALT, AST, and ALP in the aged control was higher than young control (p < 0.05, p < 0.01, and p < 0.05, respectively). Compared to the aged control, sericin treatment significantly reduced ALT and AST levels (p < 0.05 and p < 0.01, respectively). However, no significant alteration was observed in the level of ALP (Fig. 1) (Supplementary file 1).

Fig. 1
figure 1

The effect of sericin on liver function. The level of ALT (A), AST (B), and ALP (C) was determined in all experimental groups. Results are expressed as mean ± SEM. Data were analyzed using one-way ANOVA. *p < 0.05 and **p < 0.01 compared with young controls and #p < 0.05 and ##p < 0.01 compared with age controls

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Sericin administration improved antioxidant capacity in aged mice

As shown in Fig. 2, there was no significant change in MDA level between aged control and young control; however, sericin administration decreased the level of MDA compared to the aged control (p < 0.05). The low expression level of SOD, CAT, GPX, GSH, and TAC was found in the aged controls when compared to the young controls (p < 0.05, p < 0.01, p < 0.05, p < 0.01, and p < 0.05, respectively); also sericin administration significantly increased the activity level of antioxidative markers in comparison to the aged control mice (p < 0.05, p < 0.001, p < 0.001, p < 0.05, and p < 0.01, respectively). These findings imply that sericin treatment could improve the oxidative stress biomarkers to a varying degree in the liver of aged-treated mice (Supplementary file 1).

Fig. 2
figure 2

The effect of sericin on antioxidant capacity in aged mice. The level of MDA (A), SOD (B), CAT (C), GPX (D), GSH (E), and TCA (F) was determined in all experimental groups. Results are expressed as mean ± SEM. Data were analyzed using one-way ANOVA. *p < 0.05 and **p < 0.01 compared with young controls and #p < 0.05 and ##p < 0.01 and ###p < 0.001 compared with age controls

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Administration of sericin improved inflammation-related markers in aged mice

The expression levels of pro-inflammatory markers were analyzed by western blotting analysis in all experimental groups. As depicted in Fig. 3, the expression of Cox-2 as an inflammatory mediator was nearly the same in both young and aged controls; however, the level of this parameter was significantly reduced in sericin-treated mice (p < 0.05). As expected, our analysis revealed a high level of IL-1β, IL-6, and TNF-α in aged controls when compared to young controls. However, sericin administration could markedly suppress the expression level of pro-inflammatory markers compared to the age-matched control (p < 0.01). The results presented here indicate that sericin administration effectively improves inflammatory biomarkers in the liver of aged mice (Supplementary file 2).

Fig. 3
figure 3

Sericin improved the inflammation-related proteins in the age group. Representative western blotting is shown in A. The expression level of Cox2 (B), IL-1β (C), IL-6 (D), and TNF-α (E) in the liver tissue of all experimental mice. **p < 0.01 compared with young controls and ##p < 0.01 compared with age control. Data were analyzed using one-way ANOVA. The data were presented as mean ± SEM from three independent experiments

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Administration of sericin changed apoptotic-related proteins in aged mice

The impact of sericin administration on pro-apoptotic and anti-apoptotic proteins was identified by western blotting analysis. Compared with the young control, the expression level of Bax (Bcl-2-associated × protein) and cleaved caspase-3 was higher in aged control (p < 0.05), while the expression level of Bcl-2 showed a slight decrease in the aged control. Following the administration of sericin in aged mice, the protein levels of Bax and cleaved caspase-3 declined (p < 0.05 and p < 0.01, respectively), and the protein level of Bcl-2 went up (p < 0.05) in comparison with age-matched controls (Fig. 4). The present results infer that sericin treatment declines Bax and cleaved caspase-3 expression and upregulates Bcl-2 expression (Supplementary file 3).

Fig. 4
figure 4

Evaluation of pro- and anti-apoptotic proteins in mice subjected to sericin administration. Representative western blotting is shown in A. The expression level of Bax (B), Bcl-2 (C), and cleaved caspase-3 (D) was detected by western blotting. *p < 0.05 compared with young controls, #p < 0.05 and ##p < 0.01 compared with age control. Data were analyzed using one-way ANOVA. The data were presented as mean ± SEM from three independent experiments

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Administration of sericin affected JNK activation in aged mice

Compared with the young control, the JNK level showed a tendency to increase in the aged control. There was no significant change in aged-treated mice when compared to the age-matched control. However, there was an enhancement in the level of p-JNK expression in aged control compared to young control (p < 0.001). Comparing the relative density of p-JNK/JNK between aged control and young control showed this ratio is significantly higher in the aged control (p < 0.001). Collectively, sericin treatment could effectively reduce p-JNK/JNK ratio in the aged-treated group as compared to the age control (p < 0.001) (Fig. 5) (Supplementary file 4).

Fig. 5
figure 5

Evaluation of JNK protein expression in mice subjected to sericin administration. Representative western blotting is shown in A. The expression levels JNK (B), p-JNK (C), and p-JNK/JNK (D) was detected by western blotting. ***p < 0.001 compared with young controls and ###p < 0.001 compared with age control. Data were analyzed using one-way ANOVA. The data were presented as mean ± SEM from three independent experiments

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Effect of sericin treatment on p-38 protein expression

p-38 expression was evaluated by western blot analysis. As presented in Fig. 6, the expression level of p-38 did not differ between both young and aged control. Moreover, there was no difference in the protein expression of p-38 in mice treated with sericin and aged control. There was no significant variation in the expression level of phospho p-38 in both controls. Treatment with sericin decreased the expression level of phospho p-38; however, it was not statistically significant in comparison to aged control. The ratio level of phospho p-38/p-38 notably decreased in sericin-treated mice compared to aged control (p < 0.001) (Supplementary file 5).

Fig. 6
figure 6

Evaluation of p-38 protein expression in mice subjected to sericin administration. Representative western blotting is shown in A. The expression levels p-38 (B), phospho p-38 (C), and phospho p-38/p-38 (D) were detected by western blotting. ##p < 0.01 compared with aged control. Data were analyzed using one-way ANOVA. The data presented as mean ± SEM from three independent experiments

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Effect of sericin on mRNA level of β-Gal

As presented in Fig. 7, the results exhibited no statistically significant alterations in the mRNA level of the β-Gal in all experimental groups. In other words, sericin treatment did not affect β-Gal mRNA level in treated mice (Supplementary file 6).

Fig. 7
figure 7

Impact of sericin administration on the mRNA level of beta-galactosidase. Data were analyzed using one-way ANOVA. The data were presented as mean ± SEM from three independent experiments

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Discussion

In this study, the administration of sericin showed protective effects on the liver of aged mice in terms of antioxidant and anti-inflammatory activities. Higher levels of antioxidants, as well as a reduction in MDA level, indicated the protective role of sericin against lipid peroxidation and ROS generation in aged animals. A significant reduction in the liver enzymes also indicated improvement of the liver function in comparison with aged controls. It seems that the hepatoprotective effects of sericin were mediated by restoring the normal indices of antioxidant enzymes and preventing ROS generation in the liver. The antioxidant properties of sericin could be related to the high serine and threonine content, whose hydroxyl groups’ act as a chelating agent for trace elements such as copper and iron (Kato et al. 1998). Parsong concluded that the presence of polyphenols and flavonoids in sericin is responsible for its antioxidant activity (Prasong 2011). Furthermore, Kato et al. showed that sericin inhibits lipid peroxidation in rat brain homogenate (Kato et al. 1998). The lipid peroxides, derived from polyunsaturated fatty acids, are unstable and may decompose into malondialdehyde (Yagi 1998; Armstrong and Browne 1994), whose levels are associated with cardiovascular risk factors, hypertension, diabetes, and hyperlipidemia (Walter et al. 2004).

In this study, sericin decreased the expression level of the pro-inflammatory mediator production COX-2 and IL1β, IL6, and TNF-α in aged-treated mice. It can be assumed that the anti-inflammatory effect of sericin is possibly mediated through the suppression of ROS generation and ROS-activated mitochondrial apoptotic pathway. Consistently, Chlapanidas et al. found that sericin has anti-proliferative activities in peripheral blood mononuclear cells stimulated in vitro and reduces the release of IFN-γ without having effects on the release of IL-10 and TNF-α (Chlapanidas et al. 2013). Besides, Aramwit et al. investigated the inflammatory mediators induced by sericin in vitro and in vivo (Aramwit et al. 2009).

In the current study, the determination of Bcl-2 (an anti-apoptosis signal) and other pro-and anti-apoptotic proteins (Bax and cleaved caspase-3) showed that sericin could significantly stimulate the expression of these apoptotic-related proteins in the aged-treated group when aged control. This is supported by the findings by Dash et al. (2008) that pre-treatment with sericin could upregulate the expression level of Bcl-2 and downregulate Bax expression (Dash et al. 2008). These findings imply the anti-aging and anti-apoptotic benefits of sericin in the liver of aged mice. In this regard, the induction of pro-apoptotic agents results in the reduced activity of MAPK. Five groups of MAPK have so far been identified of which JNK and p38 MAPK are involved in the stress-related response of the cell (Pearson et al. 2001). The roles of JNK and p38 MAPK in aging invertebrates are not fully understood. A similar role of these kinases in mammals would open up the possibility of therapeutic prolongation of cell viability by inhibiting these kinases. However, the validity of this hypothesis remains to be verified (Sadler et al. 2004). In the present study, we demonstrated the effect of JNK and p38 MAPK inhibition on aging. Phosphorylation of tyrosine and threonine is necessary for the activation of JNK and p38 MAPK (Tarín et al. 2001). Comparing the results in each group showed that the values of JNK and P38 independently did not change significantly in each group, although the increase in P-JNK and decrease in p-P38 were seen in the aged control. Sericin treatment leads to a significant reduction in P-JNK, while no significant change was observed in the level of P38 in the treated group. Generally, the low ratios of p-JNK to JNK and P-p38 to p-38 in the aged-treated group than aged control indicate an association between sericin and increasing in the activity of P-38 and JNK.

It is worth mentioning that young mice were used for confirmation of aging. In other words, the aging of mice was validated by comparison of corresponding parameters in aged mice with young mice. Accordingly, the comparison of aged-treated mice with young untreated mice has not been related to the aim of the current study.

There are some limitations to this study. We did not evaluate the effect of sericin on the body weight or the liver tissues in all experimental groups; thereby, the impact of sericin on body and/or tissue weight is still unclear. However, according to our literature review, sericin treatment did not affect body weight (Qin, et al. 2020; Kunz 2016; SASAKI, M.,, et al. 2000).

Conclusion

The beneficial effect of sericin is most likely mediated through the modulation of the series of events, including inflammation, antioxidant, and apoptosis pathways in the liver tissue. Based on the current findings, sericin would be a successful hepatoprotective agent for ameliorating liver dysfunctions in aged mice. In fact, antioxidant and anti-apoptotic properties of sericin may be a plausible way to alleviate the detrimental effects of aging in the liver tissues. Further studies are needed to elucidate the exact molecular mechanisms.

Supplementary information