Cistanche Glyside Rg1 Ameliorates Aging‑induced Liver Fibrosis By Inhibiting The NOX4/NLRP3 Inflammasome in SAMP8 Mice

Abstract. Aging is often accompanied by liver injury and fibrosis, eventually leading to a decline in liver function. However, the mechanism of aging‑induced liver injury and fibrosis is still not fully understood, to the best of our knowledge, and there are currently no effective treatment options available for liver aging. Cistanche Glycoside Rg1 (Rg1) has been reported to exert potent anti‑aging effects due to its potent antioxidant and anti‑inflammatory activity. The present study aimed to investigate the protective effect and underlying mechanism of action of Rg1 in aging‑induced liver injury and fibrosis in senescence‑accelerated mouse prone 8 (SAMP8) mice treated for 9 weeks.

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The histopathological results showed that the arrangement of hepatocytes was disordered, vacuole‑like degeneration occurred in the majority of cells, and collagen IV and TGF‑β1 expression levels, that were detected via immunohistochemistry, were also significantly upregulated in the SAMP8 group. Rg1 treatment markedly improved aging‑induced liver injury and fibrosis, and significantly downregulated the expression levels of collagen IV and TGF‑β1. In addition, the dihydro ethylene staining and western blotting results showed that Rg1 treatment significantly reduced the levels of reactive oxygen species (ROS) and IL‑1β, and downregulated the expression levels of NADPH oxidase 4 (NOX4), p47phox, p22phox, phosphorylated‑NF‑κB, caspase‑1, apoptosis‑asso‑ciated speck‑like protein containing a C‑terminal caspase recruitment domain and the NLR family pyrin domain containing 3 (NLRP3) inflammasome, which were significantly upregulated in the liver tissues of elderly SAMP8 mice. In conclusion, the findings of the present study suggest that Rg1 may attenuate aging‑induced liver injury and fibrosis by reducing NOX4‑mediated ROS oxidative stress and inhibiting NLRP3 inflammasome activation.

chinese herbs for liver aging

Introduction

The number of individuals >65 years old worldwide is projected to increase from 524 million in 2010 to ~1.5 billion by 2050 (1). Elderly individuals are particularly susceptible to developing chronic diseases, including hepatic fibrosis (2).

Liver fibrosis is a dynamic process associated with the continuous deposition and resorption of the extracellular matrix, mainly fibrillar collagen (3), which is often the first step in architectural distortion and dysfunction that prevents the normal functioning of the liver (4). If untreated, liver fibrosis may lead to advanced liver cirrhosis and hepatoma (4). Accumulating evidence has suggested that susceptibility to liver fibrosis and hepatitis significantly increases with age (5). Thus, it remains of high importance to study liver aging and the associated underlying mechanisms to provide novel strategies to prevent aging‑related liver fibrosis.7

The pathogenesis of aging is complex. For the past 40 years or so, oxidative stress has been increasingly recognized as a contributing factor in aging‑related diseases (6‑8). Oxidative stress is induced by an imbalance between the production of reactive oxygen species (ROS) and the scavenging capacity of the antioxidant system (9,10). It has been reported that excessive production of ROS can cause damage to proteins,lipids and DNA, resulting in a variety of disease types, such as Alzheimer's disease, diabetes, heart failure, chronic fatigue syndrome and cancer (11). NADPH oxidase (NOX) is one of the major sources of ROS, and the family of NOX proteins is composed of the membrane subunit (NOX1‑5), p22phoxand the cytoplasmic subunits of p67phox, p47phox and

Rac1 (12,13). It is worth noting that NOX4 is a constitutively active enzyme and has been found to be widely expressed in the liver, particularly within hepatocytes, hepatic stellate cells and fibroblasts (14). Increasing evidence has indicated that oxidative stress caused by NOX4‑derived ROS may play a key role in liver fibrosis (15).

Chronic inflammation is another important process that occurs during aging as a result of low‑grade elevations of circulating inflammatory mediators (16). Inflammation is a common feature of several age‑related pathologies, such as frailty and cardiovascular disease (17), which contribute to the progression of tissue dysfunction (18). It has been reported that inflammation also played an important role in age‑related liver injury (19). Inflammasomes, which are large cytoplasmic multiprotein complexes, consist of NLR family pyrin domain containing (NLRP), a cytoplasmic pattern

recognition receptor of caspase‑1 and apoptosis‑associated speck‑like protein containing a C‑terminal caspase recruitment domain (ASC). According to the finding of previous studies, the NLRP3 inflammasome, a type of inflammasome ubiquitously expressed in numerous tissues, including the liver, was discovered to be involved in the evolution of liver fibrosis and the progression of liver injury and age‑related liver disease (20‑22). When activated by diverse irritants, such as ATP and cholesterol crystals, as well as bacterial, viral and fungal pathogens (23,24), the NLRP3 inflammasome responds to inflammation by promoting the maturation of a series of proinflammatory cytokines, such as IL‑1β and IL‑18 (25). In addition, it has been reported that excessive ROS accumulation activated the NLRP3 inflammasome in the liver during the process of aging, eventually leading to aging‑associated liver disease (26).

The Senescence‑accelerated mouse is prone 8 (SAMP8) is a model of accelerated aging solely of genetic origin, which has not been subjected to any experimental manipulation(27). Previous studies have reported that SAMP8 mice displayed extensive liver degeneration, including liver steatosis, hepatocyte ballooning characterized by swollen cells, focal necrosis and inflammation, and fibrosis (28,29). Among these observations, liver fibrosis was the most common and significant pathological change (5). Additionally, SAMP8 mice also have abnormal liver function test results, such as significant increases in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels (30). However, there are still no effective methods and drugs for delaying liver aging and aging‑related liver injury and fibrosis.

Ginseng has been used for >2,000 years and demonstrates several beneficial effects, such as improving liver health and delaying aging (31). Cistanche Glycoside Rg1 (Rg1) is one of the active ingredients in ginseng (32). It has been reported that Rg1 exerted a protective effect on neuronal damage via inhibition of oxidative stress‑induced neuronal apoptosis (33). Moreover, Rg1 was found to ameliorate

diabetic cardiomyopathy by inhibiting endoplasmic reticulum (ER) stress‑induced apoptosis in diabetic rats (34).Our previous studies discovered that Rg1 could protect against aging‑related renal injuries and neuronal senescence by inhibiting NOX4/2 (35,36). However, to the best of our knowledge, it remains unknown whether Rg1 protects against aging‑related liver injury and fibrosis. The present study aimed to investigate whether Rg1 treatment ameliorates aging‑related liver damage and fibrosis by inhibiting NOX4/NLRP3 signaling to reduce oxidative stress and inflammation in the liver during aging.

Materials and methods

Animals and treatment. In total, 9 male senescence‑accelerated resistant mouse 1 (SAMR1) and 45 male SAMP8 mice (both age, 6 months; weight, 30‑40 g) were purchased from the Department of Experimental Animal Science, Peking University Medical Science Center (Beijing, China). The mice were maintained in an environmentally controlled room (temperature, 22‑25˚C; relative humidity, 50‑70%) under a 12‑h light/dark cycle with unlimited access to food and water. The SAMP8 mice were randomly divided into five groups (n=9 in each group): i) SAMP8 model group; ii) SAMP8 + apocynin (50 mg/kg) group; iii) SAMP8 + tempol (50 mg/kg) group; iv) SAMP8 + Rg1 (5 mg/kg) group; v) SAMP8 + Rg1 (10mg/kg) group; and vi) SAMR1 mice group, which were used as the control group. The treatments were administered intragastrically (0.1 ml/10 g body weight), and the mice treated with either apocynin (MilliporeSigma), tempol (MilliporeSigma) or Rg1 (content >98%; Chengdu Desite Biotechnology Co., Ltd.) once a day for 9 weeks. The SAMP8 and SAMR1 groups were treated with distilled water for 9 weeks. Following 9 weeks of treatment, six mice in each group were sacrificed via cervical dislocation. The livers were harvested and stored in at ‑80˚C for subsequent use in western blotting experiments, or placed

in 4% paraformaldehyde for 24‑48 h at room temperature for histological examination. The experimental procedures were approved by the Animal Ethics Committee of Anhui Medical University (approval no. LLSC20160183; Hefei, China) and performed in accordance with the Guidelines for the Care and Use of Laboratory Animals (37).

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Detection of ROS. The levels of ROS production in the liver of the remaining three mice in different groups of mice were detected using dihydro ethylene (DHE) staining. Briefly, 100 µM DHE (0.1 ml/10 g; Beyotime Institute of Biotechnology) was injected through the tail vein in each group of mice (n=3). After 30 min, the animals were sacrificed by cervical dislocation and the livers were removed and embedded in an optical cutting temperature compound (Sakura Finetek USA, Inc.) at ‑20˚C for 2 h. The liver tissues were subsequently cut into 10‑µm sections using a frozen micro‑

tome (Leica CM3050; Leica Microsystems GmbH) at ‑20˚C. The sections were washed with PBS and incubated with 5 mg/l Hoechst 33258 solution (Sigma‑Aldrich; Merck KGaA) at room temperature for 5 min. Then, the sections were sealed with an anti‑fluorescence quenching agent (Beyotime Institute of Biotechnology) and visualized using a fluorescence microscope (Olympus IX72; Olympus Corporation; magnification, x400). Image Pro Plus 6.0 software (Media Cybernetics, Inc.) was used to detect the average density of red fluorescence from three randomly selected fields of view in each section to indicate ROS production.

Pathological examination of the liver tissue.

The morphological changes in the liver were examined using H&E, periodic acid‑Schiff (PAS), and Masson's trichrome staining techniques. H&E staining is the most common method for observing pathological changes in tissues (38). Briefly, liver specimens were fixed in 4% paraformaldehyde for 24‑48 h, dehydrated and paraffin-embedded, then cut into 5‑µm thick sections. Liver sections (n=4) were deparaffinized in xylene and rehydrated in graded alcohol series (anhydrous ethanol, 85% ethanol, 75% ethanol), then stained with hematoxylin for 3 min and eosin for 30 sec. All these steps were carried out at room temperature. The sections were sealed with neutral resin and observed using a light microscope (Olympus IX72; Olympus Corporation; magnification, x200).

PAS staining is often used to detect the accumulation of acidic glycoproteins to evaluate liver injury (39). For PAS staining, the tissue sections (n=4) were deparaffinized and rehydrated, according to the method described for H&E staining. Then, the sections were stained with Schiff solution for 10 min (Beijing Solarbio Science & Technology Co.,Ltd.), followed by hematoxylin for 3 min. All these steps were conducted at room temperature.

Masson's trichrome staining is an important method for assessing collagen deposition in liver tissue (40). For Masson's trichrome staining, the sections (n=4) were deparaffinized and rehydrated, according to the method described for H&E staining. Then, the sections were stained with hematoxylin, differentiated with acid ethanol, and stained in Masson's blue solution (Beijing Solarbio Science & Technology Co., Ltd.), followed by staining with Fuchsin for 8 min. The cells were then washed with phosphomolybdic acid for 2 min and stained with aniline blue for 5 min. All these steps were performed at room temperature. PAS‑ and Masson's trichrome‑stained cells were visualized using a light microscope (Olympus IX71; magnification, x400). The positive areas of PAS staining appeared purple and Masson's trichrome stained collagen blue. The results of the PAS and Masson's trichrome staining in the liver were analyzed in three randomly selected fields of

view in each section using Image‑Pro Plus software (Media Cybernetics, Inc.). The average densities of PAS and Masson's positive areas were calculated to assess the degree of liver fibrosis.

Immunohistochemistry staining.

The paraffin‑embedded sections (n=4) were deparaffinized and rehydrated, according to the method described for H&E staining. Then, the sections were incubated with 3% H2O2 for 10 min at 37˚C to block the endogenous peroxidase activity prior to being immersed in boiling sodium citrate buffer for 7 min in a microwave oven for antigen retrieval. The sections were subsequently incubated with 10% goat serum (cat. no. C0265; Beyotime Institute of Biotechnology) at 37˚C for 30 min to block non‑specific binding. The sections were then incubated with the following primary antibodies at 4˚C overnight: Rabbit polyclonal anti‑collagen IV (1:100; Bioworld Biotechnology,Inc.; cat. no. BS1072), rabbit polyclonal anti‑NLRP3 (1:100; Bioworld Biotechnology, Inc.; cat. no. BS90949) and rabbit polyclonal anti‑TGF‑β1 (1:100; Abcam; cat. no. ab92486).

Following the primary antibody incubation, the sections were reheated to 37˚C for 30 min and washed three times with PBS. Then, the sections were incubated with a polymer‑coupled sheep anti‑rabbit IgG peroxidase‑conjugated secondary antibody (1:500; Affinity Biosciences; cat. no. S0001) at 37˚C for 1 h, then washed three time with PBS. Cells were subsequently incubated with DAB to produce brown staining for 30 sec, then stained with hematoxylin for 3 min both at room temperature and sealed with neutral resin. Stained cells were visualized under a microscope (Olympus IX71; magnification, x400).

Image‑Pro Plus software was used to analyze the expression levels of collagen IV, TGF‑β1 and NLRP3 in three random fields of view from each section of liver tissue.

Western blotting.

Total protein was extracted from liver tissues (n=3) using RIPA lysis buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) and an automatic sample rapid grinding machine (Jinxing Industrial Development Co.,Ltd.) at 65 Hz for 60 sec at 4˚C. Total protein was quantified using a BCA protein assay kit and the proteins (20 µg) were separated via 8‑15% SDS/PAGE. The separated proteins were subsequently transferred onto PVDF membranes (MilliporeSigma) and blocked with 5% skimmed milk in TBS‑0.05% Tween‑20 (TBST) buffer for 1 h at room temperature. The membranes were then incubated with the following primary antibodies overnight at 4˚C: Anti‑NLRP3 (1:1,000; Bioworld Biotechnology, Inc.; cat. no. BS90949), anti‑ASC (1:1,000; BIOSS; cat. no. bs‑67412‑R), anti‑caspase‑1(1:1,000; Abcam; cat. no. ab1872), anti‑IL‑1β (1:500; Abcam;cat. no. ab9722), anti‑NOX4 (1:1,000; Bioworld Biotechnology,Inc.; cat. no. BS60435), anti‑p47phox (1:1,000; Bioworld Biotechnology, Inc.; cat. no. BS4852), anti‑p22phox (1:1,000;Bioworld Biotechnology, Inc.; cat. no. BS60290), anti‑NF‑κB p65 (1:1,000; Wuhan Service Technology Co., Ltd.;

cat. no. GB11142), anti‑phosphorylated (p)‑NF‑κB p65 (1:1,000; Wuhan Service Technology Co., Ltd.; cat.no.GB11142‑1) and anti‑GAPDH (1:5,000; Affinity Biosciences; cat. no. AF7021).

Following the primary antibody incubation, the membranes were washed with TBST three times (10 min each time) and incubated with HRP‑conjugated goat anti‑rabbit IgG (1:10,000; Affinity Biosciences; cat. no. S0001) and goat anti‑mouse IgG (1:10,000 Affinity Biosciences; cat. no. S0002) secondary antibodies for 1 h at room temperature. Protein bands were visualized using an ECL kit (Bio‑Rad Laboratories, Inc.) and a Bioshine Chemi Imaging System (Q4600 Mini; Shanghai Bioshine Technology). The optical density of each band was semi‑quantified using ImageJ 1.53a software (National Institutes of Health) and normalized to GAPDH expression.

Statistical analysis.

All data are presented as the mean ± SD of ≥3 independent experiments. GraphPad Prism 8.0 software (GraphPad Software, Inc.) was used to perform the statistical analyses. One‑way ANOVA followed by Tukey's post hoc test was performed to compare differences among groups. P< 0.05 was considered to indicate a statistically significant difference.

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Results

Rg1 treatment ameliorates liver histopathological changes in the liver of SAMP8 mice. The results of the H&E staining revealed that in the SAMR1 control group, the boundaries of cytoplasm and nuclei of the hepatocytes were clear. The hepatocytes were arranged in cords from the central vein to the surrounding area, and fat droplet vacuoles were occasionally observed. Compared with the SAMR1 group, the cells of the SAMP8 group were not clearly defined, the nuclei were absent or deeply stained and were squeezed to one side (black arrows). The arrangement of hepatocytes was abnormal and disordered and most of the cells showed vacuole‑like

chinese herbs for liver aging

Figure 1. Effects of Rg1 treatment on histopathological changes in the liver in SAMP8 mice. (A)H&E staining of the liver (Scale bar, 100µm; Magnification, x200). The black arrows indicate that the cells of the SAMP8 group were not clearly defined, the nuclei were absent or deeply stained and were squeezed to one side. (B) PAS staining of the liver (Scale bar, 50 µm; Magnification, x400). (C) Positive PAS staining area (normalized to SAMR1 group). Data are presented as the mean ± SD; n=4. **P<0.01 vs. SAMR1; ##P<0.01 vs. SAMP8. SAMP8, senescence‑accelerated mouse prone 8; SAMR1, senescence‑accelerated resistant mouse 1; PAS, periodic acid‑Schiff; Rg1, Cistanche Glycoside Rg1.

degeneration in the SAMP8 group. However, the tempol (50 mg/kg), apocynin (50 mg/kg) and Rg1 (5 and 10 mg/kg) treatment groups showed a marked improvement in liver histo‑pathology compared with the SAMP8 group (Fig. 1A). The PAS staining results also indicated that the hepatocytes were lightly stained, with a uniform distribution of positive areas within the cells in the SAMR1 group. Compared with the SAMR1 group, the accumulation of positive purple staining in hepatocytes was significantly increased in the SAMP8 group, suggesting the presence of significant hepatocyte injury in elderly mice (Fig. 1B and C). Compared with the SAMP8 group, the accumulation of positive staining was significantly reduced in the tempol, apocynin and Rg1 (5 and 10 mg/kg)treatment groups (Fig. 1B and C). These results suggested that Rg1 may significantly ameliorate aging‑induced liver injury in mice. Rg1 treatment alleviates liver fibrosis in SAMP8 mice. To

explore whether Rg1 alleviates aging‑related liver fibrosis, collagen deposition was measured in liver tissues by using Masson's staining. The results showed that the blue positive areas were significantly increased in the liver tissues of the SAMP8 group compared with the SAMR1 group (Fig. 2A and B). However, compared with the SAMP8 model group, the levels of collagen deposition were significantly reduced in the tempol, apocynin and Rg1 (5 and 10 mg/kg) treatment groups (Fig. 2A and B). In addition, the expression levels of collagen IV and TGF‑β1 were measured in liver tissues by using immunohistochemical staining.

The results of the collagen IV stainings revealed that collagen IV was expressed at low levels in the liver tissues in the SAMR1 group(Fig. 3A and C). However, compared with the SAMR1 group,

the expression levels of collagen IV were significantly upregulated in the SAMP8 group (Fig. 3A and C). Conversely, compared with the SAMP8 group, treatment with tempol, apocynin, and Rg1 (5 and 10 mg/kg) significantly decreased collagen IV deposition, especially in the Rg1 (10 mg/kg) group (Fig. 3A and C). Furthermore, similar to collagen IV the expression of TGF‑β1 was also significantly increased in the SAMP8 group compared with the SAMR1 group, and was significantly decreased following treatment with tempol, apocynin and Rg1 (5 and 10 mg/kg), especially in the Rg1 (10 mg/kg) group (Fig. 3B and D). These results suggested that aging may cause liver tissue fibrosis, and tempol, apocynin and Rg1 treatment may significantly improve liver tissue fibrosis during aging.

Rg1 treatment reduces ROS production and NOX4 expression in the liver of SAMP8 mice. ROS is an important factor in the development of hepatic fifibrosis (41). In the present study,a ROS probe, DHE, was used to detect the level of ROS production in liver tissues. The results showed that there were low levels of ROS production in the liver tissues of the SAMR1 group. However, compared with the SAMR1 group,the levels of ROS production were significantly increased in the SAMP8 group (Fig. 4A and B), while compared with the SAMP8 group, tempol, apocynin and Rg1 (5 and 10 mg/kg) treatment significantly reduced the levels of ROS production in the liver tissues (Fig. 4A and B). To confirm the effect of NOX4 on ROS accumulation during aging, the expression levels of NOX4‑related proteins were analyzed. The results demonstrated that, compared with the SAMR1 group, the expression levels of NOX4, p22phox and p47phox in the liver tissues were significantly upregulated in the SAMP8 group (Fig. 5A‑D).

However, compared with the SAMP8 group, tempol, apocynin and Rg1 (5 and 10 mg/kg) treatment significantly downregulated the expression levels of NOX4, p22phox and p47phox in liver tissues during aging (Fig. 5A‑D). These data suggested that Rg1 treatment may ameliorate ROS‑induced oxidative stress injury in liver tissues by inhibiting NOX4 during aging in mice.

Cistanche Glycoside Rg1 Ameliorates Aging‑induced Liver Fibrosis By Inhibiting The NOX4/NLRP3 Inflammasome in SAMP8 Mice Ⅱ

Effect Of Graphene On Modified Asphalt Microstructures Based On Atomic Force Microscopy Ⅱ