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

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Figure 4. Effect of Rg1 treatment on ROS production in the liver of SAMP8 mice. (A) Accumulation of ROS production in the liver was measured via DHE fluorescence and Hoechst 33258 staining (Scale bar, 20 µm; Magnification, x400). (B) Mean density of ROS production (red fluorescence) in the liver. Data are presented as the mean ± SD; n=3. **P<0.01 vs. SAMR1; ##P<0.01 vs. SAMP8. Rg1, Cistanche glycoside Rg1; SAMP8, senescence‑accelerated mouse prone 8; SAMR1, senescence‑accelerated resistant mouse 1; DHE, dichloroethylene.

Rg1 treatment downregulates the expression levels of NLRP3,ASC, caspase‑1 and IL‑1β in the liver of SAMP8 mice. It has been reported that activation of the NLRP3 inflammasome plays an important role in senescence‑related renal fibrosis (42). Therefore, the present study further investigated the expression levels of NLRP3‑related proteins to confirm whether the NLRP3 inflammasome is involved in age‑related liver fibrosis.The results showed that the expression levels of NLRP3,ASC, caspase‑1 and IL‑1β in liver tissues were significantly upregulated in the SAMP8 group compared with the SAMR1 group (Fig. 6A‑E). Compared with the SAMP8 group, the tempol, apocynin and Rg1 (5 and 10 mg/kg) treatment groups had significantly downregulated expression levels of NLRP3, ASC, caspase‑1 and IL‑1β in the liver tissues of SAMP8 mice (Fig. 6A‑E). Immunohistochemical staining was performed to measure the expression levels of NLRP3 in liver tissues. The results were consistent with the expression of NLRP3 measured using western blotting. The expression of NLRP3 was significantly upregulated in the liver of the SAMP8 group compared with the SAMR1 group (Fig. 7A and B), and tempol, apocynin and Rg1 (5 and 10 mg/kg) treatment significantly downregulated the expression levels of NLRP3 (Fig. 7A and B). These results suggested that NLRP3 inflammasome activation may be closely related to liver fibrosis during aging and Rg1 may improve liver fibrosis by inhibiting the activation of the NLRP3 inflammasome.To further investigate whether the inflammatory response was involved in liver fibrosis during aging, the expression levels of NF‑κB and p‑NF‑κB were measured in liver tissues using western blotting. The results revealed that there were no significant differences in the expression levels of NF‑κB among the groups (Fig. 8A and B). However, the expression levels of p‑NF‑κB were significantly increased in the SAMP8 group compared with the SAMR1 group (Fig. 8A and C), and tempol, apocynin and Rg1 (5 and 10 mg/kg) treatment significantly downregulated the expression level of p‑NF‑κB/NF‑κB compared with the SAMP8 group (Fig. 8A and C). The results suggested that Rg1 may inhibit inflammation in the liver during aging by decreasing the phosphorylation of NF‑κB.

new research of chinese herbs for liver fibrosis

Figure 5. Effect of Rg1 treatment on NOX4 expression in the liver of SAMP8 mice. (A) Western blotting was used to determine the expression levels of NOX4, p22phox and p47phox in the liver of SAMP8 mice. Semi‑quantitative analysis of the relative expression of (B) NOX4, (C) p22phox and (D) p47phox. Protein expression levels were normalized to GAPDH. ata are presented as the mean ± SD; n=3. **P<0.01 vs. SAMR1; #P<0.05, ##P<0.01 vs. SAMP8. Rg1, Cistanche glycoside Rg1; SAMP8, senescence‑accelerated mouse prone 8; SAMR1, senescence‑accelerated resistant mouse 1; NOX4, NADPH oxidase 4.

new research of chinese herbs for liver fibrosis

Figure 6. Effects of Rg1 treatment on the expression levels of NLRP3, ASC, caspase‑1 and IL‑1β in the liver of SAMP8 mice. (A) Western blotting was used to analyze the expression levels of NLRP3, ASC, caspase‑1 and IL‑1β in the liver of SAMP8 mice. Semi‑quantitative analysis of the relative expression of (B) NLRP3, (C) ASC, (D) caspase‑1 and (E) IL‑1β. Protein expression levels were normalized to GAPDH. Data are presented as the mean ± SD; n=3. *P<0.05, **P<0.01 vs. SAMR1; #P<0.05, ##P<0.01 vs. SAMP8. Rg1, Cistanche glycoside Rg1; SAMP8, senescence‑accelerated mouse prone 8; SAMR1, senescence‑accelerated resistant mouse 1; NLRP3, NLR family pyrin domain containing 3; ASC, apoptosis‑associated speck‑like protein containing a C‑terminal caspase recruitment domain.

Figure 7. Effects of Rg1 treatment on the expression levels of NLRP3 in the liver of SAMP8 mice. (A) Expression of NLRP3 in the liver (Scale bar, 50 µm; Magnification, x400). (B) Mean density of the NLRP3‑positive staining area in the liver. Data are presented as the mean ± SD; n=4. **P<0.01 vs. SAMR1;#P<0.05, ##P<0.01 vs. SAMP8. Rg1, Cistanche glycoside Rg1; SAMP8, senescence‑accelerated mouse prone 8; SAMR1, senescence‑accelerated resistant mouse 1;NLRP3, NLR family pyrin domain containing 3.

Discussion

Aging can promote the dysfunction of numerous organs,such as the liver, kidney and brain (43). Aging has been demonstrated to increase the susceptibility to hepatic inflammation or fibrosis (5). Liver fibrosis destroys the structure of the liver, leading to the loss of liver cells and disruption of liver function, ultimately resulting in liver failure (44). However, the mechanisms of aging‑induced liver injury and fibrosis are still unclear and there are no effective drugs for treating aging‑related liver fibrosis. Therefore, it is important to explore the mechanism of liver aging and find appropriate medicines to prevent liver injury and fibrosis at an early age to reduce the incidence of age‑related liver diseases.

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Rg1, one of the main active components of cistanche, has been previously investigated for its anti‑inflammatory and antioxidant properties and has been found to exert a protective effect on the liver (45). Previous studies have shown that Rg1 could inhibit the transformation of hepatic stellate cells into myofibroblasts and inhibit liver fibrosis in CCl 4‑induced mice (45,46). Our previous study indicated that Rg1 ( 5 and 10 mg/kg) treatment significantly improved aging‑induced renal injury and fibrosis in SAMP8 mice (35). The present study was designed to study the effect and mechanism of Rg1 (5 and 10 mg/kg) treatment on aging‑related liver injury and fibrosis in SAMP8 mice during aging. The results indicated that Rg1 (5 and 10 mg/kg) treatment could protect against aging‑induced liver injury and fibrosis, especially the Rg1 (10 mg/kg) group. These results also discovered that the arrangement of hepatocytes was abnormal and disordered and most of the cells appeared to have vacuole‑like degeneration in the SAMP8 group, while Rg1 treatment showed a significant improvement in liver histopathology. In addition, the results discovered that NOX4/NLRP3 inflammasome signaling, which is closely associated with age‑related liver injury and liver fibrosis (20), was significantly inhibited by Rg1 (5 and 10 mg/kg) treatment, especially in the Rg1 (10 mg/kg) group. These results suggested that Rg1 treatment may be effective in preventing aged‑related injury and fibrosis in a dose‑dependent manner, possibly by inhibiting NOX4 and the NLRP3 inflammasome.

new research of chinese herbs for liver fibrosis

Figure 8. Effects of Rg1 treatment on the expression levels of p‑NF‑κB in the liver of SAMP8 mice. (A) Western blotting was used to analyze the expression levels of p‑NF‑κB and NF‑κB in the liver of SAMP8 mice. Semi‑quantitative analysis of the relative expression of (B) NF‑κB and (C) p‑NF‑κB/NF‑κB. Data are presented as the mean ± SD; n=3. **P<0.01 vs. SAMR1; #P<0.05 vs. SAMP8. Rg1, Cistanche glycoside Rg1; SAMP8, senescence‑accelerated mouse prone 8; SAMR1, senescence‑accelerated resistant mouse 1; p‑, phosphorylated.

Previous studies have shown that aging is an important factor that is closely associated with the generation and progression of liver fibrosis (5,47); however, there is currently a lack of effective therapeutic options. cistanche is one of the most widely used natural products, due to its wide range of pharmacological effects and biological activities that can delay aging (48). The active ingredient, Rg1, has been reported to exert protective effects on TGF‑β‑induced HSC‑T6 cells and CCl 4‑induced liver fibrosis in male Kunming mice (31). In the present study,the results indicated that the liver tissues were significantly damaged and showed signs of fibrosis in 8‑month‑old SMP8 mice, such as disordered cell arrangement, vacuolar degeneration and excess extracellular collagen IV deposition, sharing a number of similarities with the findings of Dumeus et al (49).

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Notably, Rg1 treatment for 9 weeks was found to significantly attenuate liver injury and extracellular matrix accumulation in SAMP8 mice. It was previously shown that TGF‑β1 promoted liver fibrosis through Smad2 phosphorylation (p‑Smad2) and collagen synthesis, and depleting TGF‑β1 could reduce liver fibrosis (50). In the present study, the results demonstrated that Rg1 treatment significantly decreased the expression of TGF‑β1 in the liver of SAMP8 mice. Tempol is an active oxygen scavenger, which has been reported to reduce the production of ROS in H 2O2‑treated hippocampal neurons due to its ROS scavenging capacity (51). Apocynin is often used as an inhibitor of NADPH oxidase, and it has been shown to possess a clear and obvious antioxidant effect on a variety of central nervous system diseases, including Parkinson's disease and Alzheimer's disease (52‑54).

The current study also demonstrated that tempol and apocynin exerted similar protective effects on aging‑induced liver injury. These data suggested that Rg1 treatment may significantly protect against aging‑induced liver injury and fibrosis in elderly mice.

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It is well known that excessive ROS‑induced oxidative stress plays a critical role in the pathogenesis of aging, whichis also closely related to age‑related diseases, including liver injury and fibrosis (7,55). The imbalance between the product tion and clearance of ROS determines the degree of oxidative stress (56). Multiple enzymes contribute to ROS generation and oxidative stress in various tissues or cells. NOX has been reported to be a major source of ROS in numerous types of organ, such as kidney and liver, and p shown that NOX4 was a major ROS‑producing enzyme in the liver (57). It has been reported that NOX4 plays crucial roles in liver tissues under physiological conditions by mediating ROS generation (14). The accumulation of NOX4‑derived ROS is also an important regulator in promoting liver fibrosis (58). A previous study showed that Rg1 could significantly improve the survival rates of HepG2 and 293 cells in acetaminophen‑induced liver injury by reducing the excessive ROS production (59). Furthermore, evidence has shown that Rg1 reduces ROS production by inhibiting NOX4 expression in obese zebrafish induced by a high‑fat diet (60). Our previous study found that Rg1 treatment could significantly downregulate the expression of NOX4 and p47phox in the kidney of aging SAMP8 mice (35). Similarly, the findings of the present study found that the ROS production and the expression levels of NOX4, p47phox and p22phox were significantly increased in the liver of elderly SAMP8 mice compared with the control group, suggesting that NOX4‑mediated ROS accumulation is closely involved in senescence‑related liver fibrosis.

Fernández‑Garcia et al (61) also demonstrated a significant increase in ROS levels in the livers of SAMP8 mice. In addition, the present results also indicated that tempol, apocynin and Rg1 administration markedly reduced ROS production and the expression levels of NOX4, p22phox and p47phox in the livers of SAMP8 mice. These results suggested that Rg1 may improve aging‑induced liver injury by reducing NOX4‑mediated ROS accumulation.

Increasing evidence has shown that oxidative stress may also be closely associated with the levels of inflammation, which also plays a crucial role in aging‑related diseases(62,63). A previous study demonstrated that the accumulation of senescent cells contributed to the development of fibrosis by secreting proinflammatory cytokines and profibrotic mediators (64). ROS, as signaling molecules, have been discovered to play important roles in numerous processes of inflammation‑related diseases. For example, deoxynivalenol induced the inflammation of IPEC‑J2 cells by promoting ROS production (65,66). There has been a consensus that inflammasome complexes play central roles in promoting inflammation in a number of tissues, such as in the colon and brain (67‑69). Excessive ROS production is an important process for the activation of inflammasome complexes (70‑72). The NLRP3 inflammasome has been reported to be extensively expressed in the liver (20,73). Previous studies have reported that the NLRP3 inflammasome was activated in the progression of alcoholic liver disease (ALD), and liver inflammation and steatosis were significantly improved in ALD mice with knocked-out NLRP3 or caspase‑1 expression (74,75). Activated NLRP3 can bind to ASC, causing procaspase‑1 to be cleaved into mature caspase‑1. Caspase‑1 further catalyzes the cleavage of pro‑IL‑1β and pro‑IL‑18 into their mature forms, IL‑1β and IL‑18 (19). A previous study reported that NLRP3 activation aggravated inflammation in liver disease and subsequent fibrosis (76). In addition, another study showed that Rg1 treatment improved CCl 4‑induced acute liver injury by suppressing the NF‑κB/NLRP3 inflammasome signaling pathway (77). In the present study, the results suggested that the expression levels of IL‑1β, ASC, caspase‑1 and NLRP3 were upregulated in the liver tissues of SAMP8 mice.

These results are consistent with previous studies (35), and provide further support for the role of the NLRP3 inflammasome in the occurrence of aging‑related liver injury in SAMP8 mice (78). The improvement of inflammation and inhibition of NLRP3 inflammasome activation have been observed to exert protective effects on target organs (76).

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Similarly, the present study found that treatment with tempol, apocynin and Rg1 significantly downregulated the expression levels of IL‑1β, ASC, caspase‑1 and NLRP3 in the liver tissues of SAMP8 mice. It is well known that theNF‑κB transcription factor family is a central regulator of the inflammatory process (79). Phosphorylation of NF‑κB at serine 536 is thought to be required for NF‑κB activation and nuclear translocation (80). It has been reported that selective inhibition of NF‑κB activity can suppress CCl4‑induced liver fibrosis (81). In this study, the results revealed that the expression of p‑NF‑κB, which promotes the formation of the NLRP3 inflammasome, was significantly upregulated in the liver tissues of aging mice and was significantly decreased after Rg1 treatment for 9 weeks in SAMP8 mice.

Altogether, these findings indicated that the NLRP3 inflammasome may be an important target and is closely involved in aging‑induced liver injury. Furthermore, inhibition of the NLRP3 inflammasome may be an important underlying mechanism of Rg1 in preventing aging‑related liver injury and fibrosis.

In conclusion, the findings of the present study suggested that Rg1 treatment may ameliorate aging‑associated liver injury and fibrosis in SAMP8 mice. Rg1 treatment significantly decreased the levels of ROS accumulation and NOX4 expression. In addition, Rg1 treatment significantly downregulated the expression of the NLRP3 inflammasome in the liver of SAMP8 mice. These results suggest that Rg1 maydelay liver aging and reduce age‑related liver fibrosis by reducing NOX4‑mediated ROS‑induced oxidative stress and inhibiting the activation of the NLRP3 inflammasome. Thus, Rg1 may act as a potential treatment for the prevention of liver fibrosis during aging.

However, the current study has several limitations. For example, it only provided animal experimental results demonstrating that Rg1 ameliorated aging‑related liver fibrosis ue to the inhibition of NOX4 and NLRP3 inflammasome activation in SAMP8 mice. In addition, the biochemical indicators and serological indicators that represent liver function, inflammation and oxidative stress levels were not measured.

Future research will aim to further study the effect of Rg1 treatment on aging‑induced liver function, inflammation and oxidative stress‑related indicators in the serum, such as aspartate aminotransferase, alanine transaminase, IL‑1β and superoxide dismutase. Furthermore, NOX4 inhibitors or NOX4 knockout experiments will be performed to further investigate the exact underlying protective mechanism of Rg1 against liver injury in vitro and in vivo.

Acknowledgments

We would like to thank Mrs. Zhirui Fang (Department of Pharmacology, Anhui Medical University) for helping with immunohistochemistry staining and Mr. Dake Huang (Synthetic Laboratory of Basic Medicine College, Anhui Medical University) for helping with the frozen section.

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Funding

The present study was supported by grants from the National Natural Science Foundation of China (grant no. 81970630) and the Major Science and Technology Projects in Anhui Province

(grant no. 201903a07020025).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

WZL and WPL conceived and designed the study; YL, DZ and LL performed the experiments and statistical analysis and wrote the manuscript; YH and XD confirmed the authenticity of all the raw data. YH, XD, LY and XL helped to perform the experiments and wrote part of the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

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.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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