Cistanche Rb1 Attenuates Age-associated Vascular Impairment By Modulating The Gas6 Pathway

Results Rb1 alleviated senescence in the aortas of aging mice

To identify the antisenescence effect of Rb1, we first investigated cellular senescence, which is characterized by the expression of proteins involved in cell cycle inhibition and irreversible growth arrest (Yang et al. 2019). As the immunohistochemistry results are shown in Figure 1(A,B), compared with those in the Young group, Old þ Vehicle group mice showed that aging accelerated the upregulation of p21Cip1 and p16INK4a in thoracic aorta cells. However, Rb1 treatment alleviated cellular senescence in blood vessels, especially in the Old þ Rb1-20 group. In addition, the Western blot results were in accordance with the immunohistochemistry results. As shown in Figure 1(C), the protein expression of p21Cip1 and p16INK4a was significantly increased by approximately 2-fold in the thoracic aortas of aged mice compared with young mice. After treatment with 20 mg/kg Rb1, the protein levels of p21Cip1 and p16INK4a were just increased by 41.92% and 37.52% compared with those of the Young group, which were significantly lower than those of the Old + Vehicle group. In addition, there was no significant difference in the expression of p21Cip1 or p16INK4a between young mice with and without Rb1 treatment.

Figure 1. Effect of Rb1 on thoracic aorta senescence. Images of the immunohistochemical staining (400  ) of p21Cip1 (A) and p16INK4a (B) in mouse thoracic aorta cross sections. (C) Western blot analysis of mouse thoracic aortic p21Cip1 and p16INK4a expression. The data are expressed as the mean ± SD.

p < 0.01 vs. the Young group; # p < 0.05 vs. the Oldþ Vehicle group.

Rb1 ameliorated vascular reactivity in aged mice

A vascular ring experiment was used to evaluate endothelium-dependent vasodilatation. As shown in Figure 2, in all mouse thoracic aortic rings, the administration of Ach resulted in vasorelaxation. At maximal concentrations (10–5 mol/L), Ach induced approximately 88.84% ± 1.20% and 58.35% ± 2.50% vasorelaxation in the Young group and the Old þ Vehicle group, respectively, demonstrating that aging is associated with vascular reactivity impairment. However, 10 and 20 mg/kg Rb1 treatment resulted in approximately 70.48% ± 2.20% and 80.90% ± 3.24% relaxation, respectively, in aging mouse thoracic aortic rings in response to 10 5 mol/L Ach. 

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Rb1 reduced cellular adhesion molecule expression in the thoracic aortas of aging mice

We examined the protein expression of certain cell adhesion molecules to determine the inflammatory and anti-inflammatory effects of Rb1 on aged mice. The results showed that the protein expression of ICAM-1, VCAM-1, and PAI-1 was significantly increased by approximately 2-4 fold in the thoracic aortas of aged mice compared with those in the Young group (Figure 3). In contrast, Rb1 treatment significantly inhibited these changes in aged mouse thoracic aortas but had no effect on the Young mouse groups. The quantitative analysis indicated that 20 mg/kg Rb1 treatment resulted in approximately 36.58%, 46.66%, and 49.34% downregulation of ICAM-1, VCAM-1 and PAI-1 protein expression, respectively, in aging thoracic aortic tissue, compared with those of Old + Vehicle group mice.

Rb1 inhibited age-related vascular calcification and fibrosis

We performed HE staining and alizarin red S staining to assess aortic tissue structure and vascular medial calcification in aging mice respectively. HE staining showed that aging-induced thickened walls of the thoracic aorta and disorganization of the aortic extracellular matrix, could be relieved by Rb1 treatment (Figure 4(A)). As shown in Figure 4(B), aging mice developed more severe medial calcification (red-stained area) than young mice, while Rb1 intervention reduced this pathological change. Then, we examined thoracic aorta cross-sections by Masson’s trichrome staining to assess vascular medial fibrosis in aging mice. The results showed that there was a noticeable increase in collagen deposition (blue-stained area) in the Old þ Vehicle group compared with the Young group (Figure 4(C)), and this effect was significantly inhibited by Rb1 intervention. Moreover, Western blotting showed that the expression of collagen I and collagen III significantly increased by 4.34- and 2.62-fold in Old þ Vehicle group mice compared with young mice; however, Rb1 treatment downregulated the expression of collagen I and collagen III in aged mice (Figure 4(D)). Moreover, treatment with Rb1 had no influence on the degrees of thoracic aorta calcification and fibrosis in young mice (Figure 4(A–D)). 

Rb1 regulated the Gas6 signalling pathway

We further investigated whether Rb1 functioned through the Gas6/Axl signaling pathway to ameliorate age-associated thoracic aorta impairment in mice. The downregulation of Gas6 protein expression by 41.72% and mRNA expression by 52.73% in aged mice compared with young mice was abrogated by Rb1 treatment (Figure 5(A,B)). We also determined the protein expression of Axl by Western blotting. Interestingly, there was no significant difference in Axl protein expression among the five experimental groups (Figure 5(A)). We also did not detect a significant difference in the mRNA levels of Axl among the groups (Figure 5(B)).

Figure 2. Rb1 treatment attenuated age-associated endothelium-dependent vasorelaxation impairment in thoracic aorta vascular rings. Figures showing the analysis results. The data are expressed as the mean ± SD.
p < 0.05,

p < 0.01 vs. the Young group; # p < 0.05, ##p < 0.01 vs. the Old þ Vehicle group.

Figure 3. Rb1 reduced cellular adhesion molecule expression in aged mouse thoracic aortas. Western blot analysis of ICAM-1, VCAM-1 and PAI-1 expression in thoracic aorta tissues. The data are expressed as the mean ± SD.

p < 0.01 vs. the Young group; # p < 0.05, ##p < 0.01 vs. the Oldþ Vehicle group.

Discussion

In the present study, we demonstrated that Rb1 ameliorated age-related vascular impairment by suppressing calcification and fibrosis through the regulation of Gas6 expression but not Axl expression, which provides a potential therapeutic strategy for preventing age-related vascular disease. It is well-accepted that vascular cell senescence, which is characterized by endothelial dysfunction and the phenotypic transition of smooth muscle cells, can result in increased vascular stiffness and increased thickness of vascular walls (Liu et al. 2019). Vascular cell senescence is a common feature of various complex, age-related diseases, particularly cardiovascular diseases (O’Rourke et al. 2010). With the development of vascular cell senescence, vascular function is impaired (Gong et al. 2014). Age-related vascular dysfunction is exemplified by pathophysiological alterations, including a progressive decline in endothelium-dependent vasodilatation (Herrera et al. 2010; Gong et al. 2014). In our study, we confirmed cellular senescence in the thoracic aortas of aged mice, which was indicated by increased expression of the age-related proteins p21Cip1 and p16INK4a, as well as a decline in endothelium-dependent vasodilatation. The consistency of our results with the above-mentioned studies suggests that there were some important molecular mechanisms associated with the changes in vascular cell pathophysiology during aging. Agents that act on these mechanisms would probably greatly reduce and delay vascular cell senescence.

Figure 4. Rb1 treatment ameliorated aging-induced vascular calcification and fibrosis. (A) Haematoxylin and eosin (HE) staining (400  ), (B) Alizarin red S staining (400  ), and (C) Masson’s trichrome staining (400  ) of representative thoracic aorta sections in each group. (D) Western blot analysis of collagen I and collagen III expressions in thoracic aorta tissues. The data are expressed as the mean ± SD.

p < 0.01 vs. the Young group; # p < 0.05, ##p < 0.01 vs. the Oldþ Vehicle Group

Figure 5. The influence of Rb1 on the Gas6/Axl signaling pathway. (A) Western blotting was performed to examine the protein expression of Gas6 and Axl. (B) qPCR was performed to examine the mRNA expression of Gas6 and Axl. The data are expressed as the mean ± SD.
p < 0.05,

p < 0.01 vs. the Young group; # p < 0.05 vs. the Old þ Vehicle group.

Accumulating evidence has revealed that Rb1 is considered a promising drug for preventing vascular damage (Zhou et al. 2017, 2019a, 2019b; Zheng et al. 2020). Previous studies have revealed that Rb1 prevents vascular cell senescence and inhibits vascular calcification (Nanao-Hamai et al. 2019; Zhou et al.  2019b; Zheng et al. 2020). However, it is still uncertain whether Rb1 can protect arterial functions against aging and the relevant molecular mechanism. Here, we found that Rb1 reduced and delayed arterial senescence in aged mice, as demonstrated by decreased expression of related proteins and improved vascular vasodilatation, indicating that Rb1 can efficiently block excessive senescence in aging mouse thoracic aortas and might be used as an effective agent to treat or prevent age-related arterial impairment.

Previous evidence has demonstrated the accumulation of chronic low-grade arterial inflammation with advancing age, which contributes to age-associated vascular structural and functional alterations (Wang et al. 2014). Additional studies have indicated that a microenvironment enriched in inflammatory profiles induces a phenotypic shift in VSMCs from the contractile to the synthetic type. This phenotypic shift is characterized by the attenuated expression of SMC-specific contractile proteins and the secretion of additional proinflammatory cytokines, chemokines, ECM proteins, and cell adhesion molecules, such as collagen, ICAM-1, VCAM-1, and PAI-1 (Wang et al. 2011; Lacolley et al. 2018). Moreover, adhesion molecules contribute to inflammation-induced remodeling of the arteries, particularly by reorganizing ECM-VSMC interactions and influencing the phenotypic modulation of VSMCs (Intengan et al. 1999). Furthermore, studies have shown that Rb1 has anti-inflammatory effects in vivo and in vitro (Miao et al. 2017; Zhou et al. 2017). In the present study, we found that Rb1 treatment could significantly decrease the expression levels of cell adhesion molecules related to inflammatory processes in aged mice, suggesting that Rb1 might act as an anti-inflammatory drug and protect the vasculature during aging.

In addition to inflammatory damage, remodeling of the arterial wall characterized by calcification and fibrosis is the other main pathophysiological change in age-related avascular modifications and causes vascular stiffness that contributes to a decline in vasodilatation (McEniery et al. 2005; Iyemere et al. 2006; Kovacic et al. 2011). Large-scale studies have demonstrated that coronary atherosclerotic calcification increases with age (Tesauro et al. 2017). Other research also demonstrated the presence of increased calcification with aging in the carotid artery region (Wendorff et al. 2015). Similarly, our results indicated that ageing mice developed more severe vascular medial calcification than young mice. Rb1 intervention substantially inhibited these changes. This result suggested that Rb1 administration alleviated the degree of age-related vascular calcification in vivo. Sufficient evidence has shown that vascular calcification involves a variety of pathobiological processes rather than simple calcium deposition, among which VSMC switching into osteoblast-like cells play a critical role (Zhou et al. 2019b). One limitation of our study is that, despite revealing that Rb1 intervention significantly inhibited vascular calcification in aging mice thoracic aortas, contractile VSMC markers (such as A-smooth muscle actin and calponin) and osteogenic VSMC markers (such as runt-related transcription factor 2) require further exploration.

Moreover, in the present study, we also observed decreased interstitial collagen levels and decreased related-protein expression in Rb1-treated aged mice compared with vehicle-treated aged mice, which suggested that Rb1 might have a beneficial effect on vascular function in aging thoracic aortas by ameliorating age-induced vascular fibrosis. Harvey et al. (2016) reported that arterial stiffening caused by excessive fibrosis and increased collagen deposition could result in increased vasomotor tone and altered tissue perfusion. Agents that attenuate fibrosis are considered to protect against age-related vascular diseases (Kim et al. 2018).

It can be inferred that age-related vascular impairment is a complex process that involves various abnormal changes, including inflammation, calcification, and fibrosis. More importantly, we found that Rb1 was able to inhibit these known abnormal changes in the thoracic aortas of aged mice. However, the underlying mechanism is still unknown. Gas6 was identified as the ligand for the TAM receptor family, especially Axl tyrosine kinase receptors (Rothlin et al. 2015). A previous study demonstrated the roles of Gas6/Axl in regulating multiple cellular functions (Chen et al. 2019). A recent study suggested that Gas6/Axl played a crucial role in vascular calcification (Son et al. 2010; Kaesler et al. 2016; Nanao-Hamai et al. 2019) and may also be involved in treatment-induced downregulation of collagen synthesis in VSMCs and aging heart tissue (Chen et al. 2016; 2019). Moreover, previous studies revealed that Rb1 activated Gas6 transcription at the specific ARE site in the promoter region (Son et al. 2010; Nanao-Hamai et al. 2019). Here, we demonstrated that Gas6 protein expression was downregulated in aging mice but was reversed by Rb1 treatment, indicating that Gas6 may participate in Rb1-mediated regulation of arterial impairment in aged mice. However, our results show that there was no change in Axl protein and mRNA expression, which was inconsistent with a previous study (Chen et al. 2019). The TAM receptor family includes Tyro3, Axl and MerTK. Although Gas6 shows the strongest affinity for Axl among the three members (Rothlin et al. 2015), Sun et al. (2019) found that Gas6 activation attenuated inflammatory injury and apoptosis in mice, but no changes in Axl activation occurred among the experimental groups. Other research suggested that Axl expression, which inhibits apoptosis and thus vascular calcification, was also unaltered in Gas6–/– mice (Kaesler et al. 2016). Our results clearly indicated that Rb1 specifically attenuated age-related Gas6 downregulation but had no influence on the expression of Axl, which was reported to be the primary event responsible for vascular calcification and fibrosis, to improve age-related vascular inflammation and endothelium-dependent vasodilatation. The stable expression of Axl may result from a sufficient quantity before Gas6 activation or indicate that Gas6 functions through other receptors. Further investigation exploring the effects of Rb1 on other TAMs that could also be activated by Gas6, such as Tyro3 and MerTK, is needed.

Conclusions

In the present study, we confirmed that Rb1 inhibited vascular calcification and fibrosis and decreased the expression of cellular adhesion molecules, all of which subsequently ameliorated age-related vascular endothelium-dependent vasodilatation at least in part by regulating Gas6 expression but not Axl expression. With uncontrollable aging, finding agents that can prevent or treat age-related vascular cell impairment is of great importance. Our study provides evidence validating the effects of Rb1 on amelio-rating vascular injury in aging mice. 

Disclosure statement

No potential conflicts of interest were reported by the author(s).

Funding This work was supported by grants from the National Natural Science Foundation of China [81370447], the Science and Technology Planning Project of Guangdong Province of China [2016A050502014], the Shenzhen Key Medical Discipline Construction Fund [SZXK002], and the Medical Scientific Research Foundation of Guangdong Province of China [A2019079].

Data availability statement 

Data and materials are available upon request to the corresponding author.

Author contributions 

SYK, DHL and XXQ contributed to the conception and design of this work. LW, GYS and MW contributed to data analyses. SYK, LW and JMZ wrote the manuscript. All authors read and approved the final version.

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