Bazi Bushen alleviates skin senescence by orchestrating skin homeostasis in SAMP6 mice

Abstract Bazi Bushen, a Chinese‐patented drug with the function of relieving fatigue and delaying ageing, has been proven effective for extenuating skin senescence. To investigate the potential mechanism, senescence‐accelerated mouse prone 6 (SAMP6) was intragastrically administered with Bazi Bushen for 9 weeks to induce skin homeostasis. Skin homeostasis is important in mitigating skin senescence, and it is related to many factors such as oxidative stress, SASP, apoptosis, autophagy and stem cell. In our study, skin damage in SAMP6 mice was observed using HE, Masson and SA‐β‐gal staining. The content of hydroxyproline and the activities of SOD, MDA, GSH‐PX and T‐AOC in the skin were measured using commercial assay kits. The level of SASP factors (IL‐6, IL‐1β, TNF‐α, MMP2 and MMP9) in skin were measured using ELISA kits. The protein expressions of p16, p21, p53, Bax, Bcl‐2, Cleaved caspase‐3, LC3, p62, Beclin1, OCT4, SOX2 and NANOG were measured by western blotting. The expression of ITGA6 and COL17A1 was measured by immunofluorescence staining and western blotting. Our findings demonstrated that Bazi Bushen alleviated skin senescence by orchestrating skin homeostasis, reducing the level of oxidative stress and the expression of SASP, regulating the balance of apoptosis and autophagy and enhancing the protein expressions of ITGA6 and COL17A1 to improve skin structure in SAMP6 mice. This study indicated that Bazi Bushen could serve as a potential therapy for alleviating skin senescence.

Therefore, research on how to delay skin ageing or how to maintain skin homeostasis is of great significance.
Skin homeostasis is related to many factors such as oxidative stress, senescence-associated secretory phenotype (SASP), apoptosis, autophagy and stem cell. Several studies have demonstrated that the level of oxidative stress and the balance of apoptosis and autophagy are crucial for maintaining skin homeostasis. 3,8,9 This oxidative stress results in reduced collagen production, synthesis and activation of matrix metalloproteinases (MMPs) responsible for degrading connective tissue and secretion of SASP which ultimately promotes ageing of the skin. 3,8 Meanwhile, the balance of apoptosis and autophagy is perturbed by oxidative stress and inflammatory factors. In addition, stem cells underlie tissue homeostasis. Adult stem cells are essential to replace cells in tissues, but their capacity declines with age. Stem cell exhaustion or the accumulation of senescent cells has previously been reported to be implicated in ageing. [10][11][12] Skin homeostasis is maintained through the presence of stem cells in epithelial tissues, which can replace those that are constantly lost during tissue turnover or following injury. 13,14 Recently, emphasis has been paid to the role of the hemidesmosome component collagen XVII (COL17A1) in stem cell competition and skin homeostasis. 15 Yan Qing E Wan, which have unique advantages in alleviating skin senescence. 18

| Animals and treatments
Twenty male SAMP6 mice (25-35 g) aged 9-10 weeks were pur- reports. [22][23][24][25] The mice in the BZBS group were orally administrated with 2.8 g/kg/day of Bazi Bushen for 9 weeks. Body weights of mice were recorded, and the skin appearance was observed during the experiments.

| Histological analysis of skin tissue
Skin tissue was fixed in 4% paraformaldehyde and embedded in paraffin, and 5 μm sections were stained with haematoxylin and eosin (H&E) and Masson's trichrome as described previously. 26 The skin sample was examined using Nikon ELWD 0.3T1-SNCP Microscope (Nikon, Japan). The dermal thickness was measured with Image J analyzer software.

| Measurement of hydroxyproline (Hyp) content
According to the manufacturer's instruction, 27 Hyp content in skin tissue was measured using commercial assay kits (Nanjing JianCheng Bioengineering Institute, Nanjing, China).

| Measurement of oxidative damage sign
According to the manufacturer's instructions, 28 SOD, MDA, GSH-PX and T-AOC in skin tissues were measured using commercial assay kits (Nanjing JianCheng Bioengineering Institute).

| Enzyme-linked immunosorbent assay (ELISA)
According to the manufacturer's instructions, 26 IL-6, IL-1β, TNFα, MMP2, MMP9 and VEGF in skin tissues were measured using commercial ELISA kits. The microplate reader (Thermo, USA) was used to determine the optical density (OD) value at 450 nm.

| Ageing-related β -galactosidase (SA-βgal) staining
Skin tissue was fixed in 4% paraformaldehyde and embedded in paraffin, and 5 μm sections were incubated for 24 h, as reported previously. 29 According to the manufacturer's instructions, the SAβgal staining kit (No. RG0039, Beyotime, China) was used for staining.

| Immunofluorescence staining
Immunofluorescence was performed as described previously. 16 Skin tissue was fixed in 4% paraformaldehyde and embedded in paraffin, and 5 μm sections were treated with xylene for deparaffinization.
The sections were treated with 3% hydrogen peroxide to prevent endogenous peroxidase activity. After blocking with 10% goat serum for 1 h, the slides were incubated with ITGA6 and COL17A1 anti-

| Western blot analysis
Western blot analysis of the skin samples was conducted as described previously. 16 The skin samples were put into an ice-cold RIPA buffer solution for homogenate preparation. After centrifugation, we used a BCA kit to measure the protein concentration of the lysates. After denaturation, protein samples were separated GAPDH was used as a normalization control. Each experiment was repeated at least three times.

| Statistical analysis
PRISM8 software (Graph Pad Software) was used to assess statistical significance. To determine the significance between the two groups, comparisons were performed using an unpaired two-tailed Student t-test or Mann-Whitney U-test for parametric and nonparametric data, respectively. p < 0.05 was considered statistically significant. **p < 0.01, *p < 0.05.

| Bazi Bushen improved body weight and skin structure in SAMP6 mice
SAMP6 mice used to be a model of senile osteoporosis and exhibited obesity and loss of skin glossiness. [30][31][32] During the experiments, body weight of mice was recorded and the skin appearance was observed.
As shown in Figure 1A, the body weight decreased in the BZBS group compared with the SAMP6 group. We assumed that treatment with Bazi Bushen may improve the obesity of SAMP6 mice. Interestingly, compared with the BZBS group, the mice in the SAMP6 group exhibited hair loss and loose skin. To further explore the changes in the skin, we observed the skin structure by HE and Masson staining. The results revealed that dermis thickness of skin reduced, collagen fibres disordered and the amount of collagen decreased in the SAMP6 group compared with the BZBS group ( Figure 1B-D). Furthermore, the skin Hyp content in the SAMP6 group was lower than that in the BZBS group ( Figure 1E). To investigate the effects of Bazi Bushen on the hemidesmosome component, we measured the expression of ITGA6 by immunofluorescence staining and western blotting. The results revealed that the protein expression of ITGA6 increased in the BZBS group compared with the SAMP6 group ( Figure 1F-H). These findings suggested that BZBS could improve body weight and skin structure in SAMP6 mice, and could rescue it from skin senescence.

| Bazi Bushen delayed skin senescence in SAMP6 mice
To further explore skin senescence visually, we observed the skin structure by SAβ-gal staining. We observed a decrease in SAβ-gal activity after Bazi Bushen treatment (Figure 2A). At the same time, we measured the expression of senescence markers p16, p21 and p53 using western blotting. In the BZBS group compared with the SAMP6 group, we observed a decrease in the levels of senescence markers p16, p21 and p53 ( Figure 2B-E). These findings suggested that Bazi Bushen may delay skin senescence by preventing the senescence of internal cells and improving cell proliferation.

| Bazi Bushen alleviated skin oxidative stress in SAMP6 mice
Oxidative stress regulates skin homeostasis and mediates the occurrence of skin senescence. 8 Therefore, we examined the effect of Bazi Bushen on redox status in skin tissue of SAMP6 mice. As shown in Figure 3, compared with the SAMP6 group, Bazi Bushen treatment enhanced the activities of SOD, GSH-PX and T-AOC in skin tissue. Simultaneously, the skin tissue levels of MDA were significantly lower than those in the SAMP6 group. These findings indicated that Bazi Bushen could increase antioxidant capacity, which alleviated skin senescence.

| Bazi Bushen decreased the levels of inflammatory cytokines in skin tissue of SAMP6 mice
In addition, oxidative stress drives inflammageing. 3

| Bazi Bushen improved the levels of MMPs and VEGF in skin tissue of SAMP6 mice
The release of inflammatory factors increases the expression of MMPs, degrades collagen, leads to the loss of collagen and aggravates skin senescence. 8 In ageing skin tissue, the expression of MMPs and the degradation of collagen degradation increased. At

| Bazi Bushen regulated the balance of apoptosis and autophagy in skin tissue of SAMP6 mice
Under normal conditions, a balance exists between apoptosis and autophagy that maintains skin homeostasis. 9,33 This balance is perturbed by oxidative stress and inflammatory factors such as skin senescence. Inflammation often causes apoptosis. In ageing skin tissue, pro-apoptotic proteins increase and anti-apoptotic proteins decrease. As expected, the expressions of pro-apoptotic proteins Bax and Cleaved caspase-3 were significantly decreased and the expression of anti-apoptotic protein Bcl-2 was markedly increased in skin tissue of the BZBS group compared with the SAMP6 group ( Figure 5A-E). Autophagy is a cellular mechanism for eliminating damaged organelles. 34 To confirm Bazi Bushen's effect on cell autophagy, we measured the conversion of LC3 I to LC3 II and the level of the autophagy receptor SQSTM1 and the autophagy protein Beclin 1 in skin tissue by western blotting. After treatment with Bazi Bushen, the conversion of LC3 I to LC3 II and the expression of the autophagy protein Beclin1 increased and the expression of the autophagy receptor SQSTM1 decreased in the BZBS group compared with the SAMP6 group, which indicated that the level of autophagy increased ( Figure 5F-I). These findings indicate that Bazi Bushen may inhibit cell apoptosis and promote cell autophagy to alleviate skin senescence in SAMP6 mice.

| Bazi Bushen regulated epidermal stem cells in SAMP6 mice
Stem cells underlie tissue homeostasis. 16 Adult stem cells are vital for replacing cells in tissues, but their capacity declines with age and stem cell exhaustion or the accumulation of senescent cells have previously been implicated in ageing. 10,11 To further explore the causes of structure and homeostasis changes in skin senescence, we measured the expression of stemness biomarkers NANOG, SOX2 and OCT4 in skin tissue by western blotting. In the BZBS group compared with the SAMP6 group, we observed an increase in the levels of stemness biomarkers ( Figure 6A intervening with epidermal stem cells, which may therefore be beneficial for the reduction in ageing.

| DISCUSS ION
In this study, we first demonstrated that Bazi Bushen alleviated skin senescence by orchestrating skin homeostasis in SAMP6 mice. As shown in Figure 7, Bazi Bushen delayed skin senescence by improving skin structure, reducing the level of oxidative stress and the expression of SASP, regulating the balance of apoptosis and autophagy and maintaining the protein expression of COL17A1 in SAMP6.
With the increasingly prominent problems of ageing population, environmental pollution and social pressure, the problems of skin ageing have attracted more attention. Skin ageing is associated with many factors, the most important of which is to orchestrate skin homeostasis. TCM exerts many health benefits, including ameliorative effects on skin ageing. By improving skin structure and orchestrating skin homeostasis, TCM can prevent or delay skin ageing. Bazi Bushen, a TCM formula, is used to relieve fatigue and delay ageing. As described previously, 14 compounds have been identified in Bazi Bushen, including neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isoquercitrin, hyperin, verbascoside, epimedin A, icariin, baohuoside I, imperatorin, osthole, catalpol, deoxyschizandrin and γ-Schizandrin. [22][23][24][25] These identified compounds may underlie the beneficial effects of Bazi Bushen to delay skin senescence.
The senescence-accelerated mouse (SAM), consisting of senescence-prone (SAMP) and senescence-resistant (SAMR), has been developed and utilized worldwide for the study of human ageing and age-associated diseases. 35,36 Compared with SAMR mice, SAMP mice showed signs of advanced senescence, such as loss of skin glossiness, hair loss and short life span. 30  In addition, hemidesmosomes, connecting basal keratinocytes to the basement membrane, are crucial for an integral part of the skin. 16 Our study demonstrated that Bazi Bushen improved skin structure, enhanced dermis thickness and increased the content of collagen and Hyp and the protein expression of ITGA6. Notably, the main compounds in BZBS, such as neochlorogenic acid, 37 chlorogenic acid, 38 cryptochlorogenic acid, 39 isoquercitrin, 40   Recently, an increasing number of studies have shown that stem cell competition orchestrates skin homeostasis and skin ageing. 16 COL17A1 is a good marker for epidermal stem cells and reflects individual cellular potential and quality for self-renewal. In the present study, we found that Bazi Bushen treatment enhanced the levels of stemness biomarkers NANOG, SOX2 and OCT4 in skin tissue. In addition, the expression of COL17A1 markedly increased.
Altogether, these findings suggest that Bazi Bushen may alleviate skin senescence through the enhancement of stem cells and COL17A1 by orchestrating skin homeostasis in SAMP6 mice.
However, there are some limitations of the present study. We did not evaluate the effect of Bazi Bushen on the natural ageing mice. However, SAMP6 mice showed signs of advanced senescence, such as loss of skin glossiness, hair loss and short life span. 30,31 Meanwhile, treatment with Bazi Bushen can improve skin structure and delay skin senescence. The multidose studies will be needed and the dose-response curve in the experimental setup should be further clarified and detailed. 50 And future investigations could evaluate the relationship between COL17A1 and the balance of apoptosis and autophagy.

| CON CLUS ION
In summary, based on our findings, Bazi Bushen can serve as a potential therapy for alleviating skin senescence. Its protective F I G U R E 7 Mechanism by which Bazi Bushen alleviated skin senescence by orchestrating skin homeostasis. function on skin structure and homeostasis has been demonstrated, followed by its attenuation of oxidative stress and SASP, and regulation of the balance of apoptosis and autophagy in skin.
The significance of COL17A1 in orchestrating skin homeostasis has also been revealed.

CO N FLI C T O F I NTE R E S T S TATE M E NT
All authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data can be accessed by emailing the corresponding authors.