enLanguage
Home / Knowledge / Content

Knowledge

Protective Effects Of Cistanche Against H2O2/UVB-induced Dermal Fibroblast Collagen Degradation Via Hsa-microRNA-4535-mediated TGFβ/Smad Signaling

May 06, 2023

ABSTRACT

This study aimed to investigate the mechanism underlying the protective effects of cistanche against H2O2/UVB-induced damage using in vitro and in vivo models of photodamage. Moreover, we identified the involvement of miRNA regulation in this process. The H2O2/UVB-treated HS68 human dermal fibroblasts and UVB-induced C57BL/6J nude mice were used as in vitro and in vivo models of photodamage. The results showed that cistanche treatment alleviated H2O2/UVB-induced reduction in cell viability, TGFβ/Smad signaling impairment, and dermal aging. Based on the results of microRNA array analyses and database searches, has-miR-4535 was identified as a potential candidate miRNA that targets Smad4. In vitro, cistanche treatment activated Smad2/3/4 complex and inhibited hsa-miR-4535 expression in H2O2/UVB-exposed cells. In vivo, topical application of low (12 mg/kg) and high doses (24 mg/kg) of cistanche to the dorsal skin of C57BL/6J nude mice significantly alleviated UVB-induced skin photodamage by promoting TGFβ/Smad collagen synthesis signaling, reducing epidermal hyperplasia, wrinkle formation, and skin senescence, as well as inhibiting hsa-miR-4535 expression. Taken together, our findings indicate a link between hsa-miR-4535 and TGFβ/Smad collagen synthesis signaling and suggest these factors to be involved in the photo-protective mechanism of cistanche in dermal fibroblasts against H2O2/UVB-induced aging. The evidence indicated that cistanche with anti-aging properties can be considered as a supplement in skin care products.

Protective effects of cistanche

Cistanche Skin-Care Product For Sale

INTRODUCTION 

The clinical signs of human skin aging include increased wrinkling, laxity, and irregular pigmentation [1]. The process of skin aging is complicated and can be divided into two types: intrinsic aging and extrinsic aging. Intrinsic aging is due to the passage of time and genetic factors, while extrinsic aging, also called photoaging, mainly results from exposure to ultraviolet radiation [2, 3]. Previous studies have shown that abundant reactive oxygen species (ROS) are generated during both intrinsic and extrinsic aging. The accumulation of ROS can induce mitogen-activated protein kinase (MAPK) signaling and activate its downstream transcription factor, activator protein-1 (AP-1). Activated AP-1 can translocate to nucleus and bind to the promoter regions of matrix metalloproteinases (MMPs) [4]. MMPs are a large zinc-dependent endopeptidase group, contributing to the degradation of skin dermis extracellular matrix (ECM). Particularly, MMP-1, a collagenase, is involved in the cleavage of collagen types I and III [5]. Collagen types I and III, the major components of ECM, are capable of conferring support and strength to the skin dermis and their disarrangement leads to the characteristic wrinkling and laxity of aged skin [6]. Transforming growth factor-beta (TGFβ) is a ubiquitous and potent cytokine with three different isoforms, TGFβ1, TGFβ2, and TGFβ3, that can positively regulate collagen synthesis in human skin dermis [2]. TGFβs initiate their signal by interacting with specific cell surface serine/threonine kinase receptor complexes, including TGFβ receptor types I (TβRI) and II (TβRII). TβRI phosphorylation triggers the phosphorylation of R-Smads (Smad2 and Smad3). Activated R-Smads interact with Smad4 to regulate its nucleus translocation and transcriptionally activate collagen types I and III through binding to promoters of Smad-binding elements (SBE) [7, 8]. Mounting evidence indicates that elevated ROS generation, induced by intrinsic or photoaging, contributes to TGFβ/Smad signaling pathway impairment, which in turn results in reduced collagen synthesis in human skin dermal fibroblasts [9, 10]. cistanche (3,5,7-trihydroxy flavone), a natural member of the flavonoid family, is abundant in Alpinia officinarum and propolis. cistanche is a potential candidate for treating ischemic stroke, diabetes, and different cancer types [11–13]. In addition, cistanche possesses radical scavenging and anti-inflammatory activities [14, 15]. We previously showed that cistanche reduced H2O2-induced inflammation and promoted collagen formation through insulin-like growth factor 1 receptor (IGFI-R)/ERK1/2 signaling pathways in HS68 cells [16, 17]. Furthermore, a study indicated that cistanche was identified as a compound of Alpinia officinarum and possesses collagenase inhibitory effects in dermal fibroblast cells [18]. However, a more detailed molecular mechanism related to how cistanche regulates dermal skin aging is unknown. MicroRNAs (miRNAs) are a group of small, single-stranded, non-coding RNA molecules with an average length of 22 nucleotides. Mature miRNAs are transcribed from DNA sequences. In most cases, mature miRNAs bind to the 3′-untranslated regions (UTRs) of target mRNAs to silence mRNA translation [19]. In human skin, miRNAs play crucial roles in the regulation of cell metabolism, cancer, senescence and collagen formation [20–23]. For example, miR-377 can induce dermal fibroblast senescence by inhibiting DNA methyltransferase 1 (DNMT1) expression, which subsequently leads to less methylation in p53 promoter and dermal fibroblast senescence [22]. The miRNA profile in UVB-induced damage in human dermal papilla cells (nHDPs) was previously investigated [24]. However, how UVB-induced dermal fibroblast senescence through miRNA regulation, especially in the involvement of natural compound is largely unknown. This study aimed to investigate the protective effects of cistanche against H2O2/UVB-induced dermal damage as well as the role of microRNA-mediated collagen degradation in this process. We further aimed to identify the microRNAs involved in regulating collagen synthesis.  

Protective effects of cistanche

RESULTS 

cistanche inhibits UVB/H2O2-induced cytotoxicity in HS68 human dermal fibroblasts 

To investigate the cytotoxicity of cistanche (Figure 1A) in vitro, HS68 cells were treated with different doses of cistanche (0, 10, 20, 30, 40, 50 μM) for 24 h. MTT assay results showed that cistanche was not cytotoxic to HS68 cells (Figure 1B). Next, we exposed HS68 cells to different doses of H2O2 (0, 100, 150, 200, 250, 300 μM) and UVB (0, 25, 30, 40, 50, 60 mJ/cm²) for 24 h. H2O2 and UVB treatment decreased the cell viability in a dose-dependent manner (Figure 1C, 1D). To evaluate the cytoprotective properties of cistanche following H2O2 and UVB exposure, we treated HS68 cell with different concentration of cistanche (10, 20, 30 μM) following H2O2- (200 μM) and UVB-induced (40mJ/cm²) damage. H2O2 or UVB treatment alone significantly decreased (40%) the viability of HS68 cells. However, cistanche treatment prevented cell death in a concentration-dependent manner (Figure 1E, 1F). Higher concentration of cistanche (30 μM) prominently alleviated H2O2- and UVB-induced cytotoxicity. Therefore, the dose of 30 μM cistanche was used in subsequent in vitro studies to evaluate its protective effects in HS68 cells. 

Protective effects of cistanche

Figure 1. cistanche inhibits UVB/H2O2-induced cytotoxicity in HS68 human dermal fibroblasts. (A) The chemical structure of cistanche. (B) Cell viability of HS68 cells treated with different concentrations of cistanche (10, 20, 30, 40, 50 µM) for 24 h. (C and D) Cell viability of HS68 cells exposed to different doses of H2O2 (100, 150, 200, 250, 300 µM) or irradiated with UVB (25, 30, 40, 50, 60 J/cm2 ) for 24 h. (E and F) Cell viability of HS68 cells exposed to H2O2 (200 µM) or irradiated with UVB (40 J/cm2 ) for 1 h and then treated with cistanche (10, 20, 30 µM) for 23 h. The cytoprotective effects of cistanche were determined by the MTT assay. Control cells were assigned 100% viability. Values are shown as mean ± SE. Quantification of the results is shown (n = 3) *P < 0.05, **P < 0.01, ***P < 0.001 vs. untreated control cells; ##P < 0.01, ###P < 0.001 vs. H2O2 or UVB-treated cells.



Cistanche attenuates H2O2-induced HS68 cell senescence rather than apoptosis

To determine whether the decrease in cell viability was due to cell death or growth inhibition, we checked the expression of several apoptosis and survival markers by western blotting in HS68 cells exposed to different doses of H2O2 (50, 100, 200, 300, 400 μM) for 24 h. We found significantly decreased expression of survival markers, such as p-Akt, and increased expression of apoptosis-related markers, such as cleaved-caspase 3 and cytochrome c, at concentrations >200 μM H2O2 (Figure 2A). Furthermore, we performed flow cytometry to assess apoptotic cells using Annexin V and PI staining and observed similar results. Taken together, 200 μM H2O2 did not cause HS68 cell apoptosis (Figure 2B). Next, we detected the expression of several senescence markers, such as p16, p21, and SA-β-gal, to determine the senescent cells. Our results showed that cistanche treatment significantly decreased the H2O2-induced p16 and p21 protein levels as well as number of SA-β-gal-positive cells (Figure 2C, 2D). 


Cistanche attenuates UVB/H2O2-induced intracellular/mitochondrial ROS production and MMP imbalance in HS68 cells

Oxidative stress plays a key role in various diseases, and can be induced by endogenous or exogenous factors. Here, we used H2O2 as an endogenous and UVB as an exogenous factor to induce oxidative damage and then investigated the ROS eliminating activities of cistanche. To identify the sources of ROS, MitoSOX and DCFH-DA were used to determine intracellular and mitochondrial ROS. The staining results showed that H2O2 and UVB treatment induced ROS accumulation while cistanche treatment reduced ROS generation (Figure 3A, 3B). In addition, as imbalanced MMP can initiate cell death, we used JC-1 to determine changes in MMP. Normal mitochondria fluoresced red (JC-1 dimers) while impaired mitochondria fluoresced green (JC-1 monomers). UVB and H2O2 exposure induced high levels of green fluorescence. Notably, cistanche treatment shifted the green fluorescence to red, indicating revival of mitochondrial functions (Figure 3C).

Protective effects of cistanche


cistanche attenuates H2O2-induced TGFβ/Smad collagen synthesis pathway impairment in HS68 cells

TGFβ/Smad signaling is conducive to collagen formation in human skin dermal fibroblasts [25–27]. Therefore, we investigated the role of cistanche incollagen synthesis via TGFβ/Smad pathway in HS68 cells exposed to H2O2. Western blot results indicated that cistanche significantly enhanced TGFβ/Smad signaling as well as collagen synthesis in H2O2-exposed HS68 cells (Figure 4A). Moreover, disorganization of collagen type I and III is one of the typical characteristics of aged skin fibroblasts [28]. Since the MMP family has been implicated in collagen catabolism [29], we next confirmed the influence of cistanche on MMP-1 activation. We found that cistanche prevented collagen degradation in HS68 cells by suppressing H2O2-enhanced MMP-1 level (Figure 4A).
Furthermore, we identified the subcellular molecular mechanism underlying this protective action by comparing HS68 cells treated with H2O2 alone or co-treated with cistanche using immunoblotting and immunofluorescence staining. cistanche treatment enhanced nuclear accumulation of p-smad2/3 and Smad4 that was disrupted by exposure to H2O2 in HS68 cells (Figure 4B, 4C). These findings indicated that cistanche may ameliorate H2O2-induced collagen fragmentation by activating TGFβ/Smad pathway in HS68 cells. 


Cistanche treatment reduces the H2O2-induced upregulated expression of hsa-miR-4535, predicted to target Smad4

The miRNA array analysis of cistanche-treated HS68 cells revealed that hsa-miR-4535 expression was reduced in cistanche-treated group as compared to that in the control group (Figure 5A). Further, using miRNA databases, such as miRDB and TargetScanHuman, we predicted a putative conserved target site for hsa-miR-4535 in Smad4-3′-UTR (Figure 5B). After confirming the target site, Smad4-3′-UTR-WT and Smad4-3′-UTR MT reporter constructs were transfected into HS68 cells to verify the direct binding of hsa-miR-4535 to Smad4-3′-UTR. We observed 50% reduction in luciferase activities between wild type and mutant Smad4-3′-UTR groups followed hsa-miR-4535 mimic transfections (Figure 5C). We then assessed the effects of H2O2 (100,200 μM) on the regulation of hsa-miR-4535 and Smad 4 in HS68 cells, and found that H2O2 dose-dependently enhanced hsa-miR-4535 and suppressed Smad4 RNA expression (Figure 6A, 6B). In addition, this trend in hsa-miR-4535 and Smad4 expression was reversed following cistanche treatment, indicating that regulation of Smad signaling may be involved in the cistanche mediated alleviation of H2O2-induced HS68 cell damage (Figure 6C, 6D). Taken together, these results suggested that cistanche attenuates H2O2-induced TGFβ/Smad pathway impairment by downregulating hsa-miR-4535 expression in human skin dermal fibroblasts.  


Protective effects of cistanche

Figure 4. cistanche attenuates H2O2-induced TGFβ/Smad collagen synthesis pathway impairment in HS68 cells. (A) HS68 cells were exposed to H2O2 (200 µM) for 1 h and then treated with cistanche (30 µM) for 23 h. Protein expression of collagen synthesis-related pathway components (TGFβ, p-smad2/3, Smad4, COL1A1, COL3A1) and collagen degradation-related protein (MMP-1) were detected by western blot. (B) The protein expression of p-smad2/3, and Smad4 in the nuclear and cytosolic fractions was detected by western blotting. GAPDH was used as a loading control. Values are shown as mean ± SE. Quantification of the results is shown (n = 3) *P < 0.05, **P < 0.01, ***P < 0.001 vs. untreated control cells; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. H2O2-treated cells. (C) Anti-p-Smad2/3, Smad4 antibody and FITC/PE-conjugated secondary antibody were used to detect p-Smad2/3 (green), and Smad4 (red) expression. DAPI indicated the nucleus location (blue). The images were captured using a fluorescence microscope (200×).


Modulation of Smad4 by has-miR-4535 is involved in collagen synthesis in H2O2-exposed human skin dermal fibroblasts

To further determine the mechanisms by which has-miR-4535 exerts its effect on the expression of Smad4 and its downstream signaling in HS68 cells, we transfected HS68 cells with hsa-miR-4535 mimics or inhibitors and measured Smad4 and its downstream collagen expression. The results demonstrated that inhibition of hsa-miR-4535 by hsa-miR-4535 inhibitor significantly reversed the downregulation of Smad4 mRNA and protein expression in H2O2-treated HS68 cells (Figure 7A–7C). Moreover, suppression of hsa-miR-4535 partially restored H2O2-induced COL1A1 and COL3A1 impairment in H2O2-treated HS68 cells (Figure 7C). Conversely, enhanced hsa-miR-4535 expression as a result of hsa-miR-4535 mimic transfection blocked the cistanche-mediated upregulation of Smad4 in HS68 cells exposed to H2O2 (Figure 7D–7F). Overexpression of hsa-miR-4535 partially reduced cistanche-induced COL1A1

and COL3A1 levels (Figure 7F). The overexpression of hsa-miR-4535 (Figure 8A–8C) or inhibition of Smad4 by siRNA (Figure 8D, 8E) resulted in reduced collagen synthesis under cistanche treatment following H2O2 exposure. Surprisingly, we found that both direct Smad4 silencing and treatments with hsa-miR-4535 mimic reversed cistanche-induced collagen synthesis in H2O2- treated HS68 cells. Taken together, we concluded that cistanche regulated collagen synthesis, potentially via decreasing hsa-miR-4535 level to enhance Smad4 signaling in HS68 cells exposed to H2O2. 

KSL30


cistanche enhances TGFβ/Smad collagen synthesis pathway and attenuates dermal senescence as well as inhibition of has-miR-4535 expression in UVB-exposed HS68 cells

As UV-light plays an important role in various skin dermis disorders, including inflammation, immunosuppression,  cancer, and premature aging [30–32], we examined the effect of cistanche on collagen synthesis-related signaling in HS68 cells exposed to UVB radiation. The protein levels of TGFβ/Smad pathway components and the downstream COL1A1 and COL3A1 were decreased in UVB-treated HS68 cells but were effectively up-regulated following cistanche treatment (Figure 9A). The western blotting results confirmed that cistanche suppressed UVB-enhanced MMP-1 expression in HS68 cells (Figure 9A). Moreover, we clarified the anti-aging effect of cistanche in HS68 cells subjected to UVB exposure by demonstrating that cistanche reduced UVB-induced elevation of p16 and p21 levels (Figure 9A). To assess whether cistanche could attenuate UVB-induced collagen breakdown through inhibition of hsa-miR- 4535 targeting Smad4, we investigated has-miR-4535 and Smad4 levels in UVB and cistanche co-treated cells using qPCR. We found that has-miR-4535 expression was significantly upregulated in UVB-exposed cells but decreased following cistanche treatment. Conversely, Smad4 expression was suppressed by UVB exposure and this suppression was markedly reversed by cistanche treatment (Figure 9B, 9C). Collectively, our findings suggested that cistanche exerts similar effects as H2O2 to restore UVB-induced decrease in collagen formation by repressing hsa-miR-4535 levels so as to promote Smad4 signaling in HS68 cells.

Protective effects of cistanche


Topical application of cistanche attenuates UVB-induced skin hyperplasia and collagen degradation as well as enhances TGFβ/Smad signaling in the dorsal skin of C57BL6/J nude mice

To elucidate the photo-protective effect of cistanche on UVB-induced skin injury in vivo, we evaluated the level of microRNA-4535, Smad4, and skin tissue alterations. Figure 10A shows the schematic for experimental design and time-line for topical application of cistanche following UVB irradiation on the dorsal skin of C57BL6/J nude mice. The formation of wrinkles was observed on the dorsal skin sections of C57BL/6J mice in the UVB-exposed group. Topical application of cistanche significantly reduced wrinkle formation, indicating the cistanche-mediated increase in collagen content to maintain skin integrity (Figure 10B). Moreover, histological analysis of the dermal layer showed that collagen content and density in the skin
were significantly lower in UVB-irradiated group compared to that in the vehicle control group. However, topical application of cistanche (12 mg/kg and 24 mg/kg)


Protective effects of cistanche


Figure 6. cistanche up-regulates Smad4 expression by decreasing hsa-miR-4535 level. (A, B) HS68 cells were treated with different concentrations of H2O2 (0, 100, 200 µM) for 24 h. (C, D) HS68 cells were exposed to H2O2 (200 µM) for 1 h and then treated with cistanche (30 µM) for 23 h. The expression of hsa-miR-4535 and Smad4 were detected by qRT-PCR. Values are shown as mean ± SE. Quantification of the results is shown (n = 3) *P < 0.05, **P < 0.01, ***P < 0.001 vs untreated control cells; ##P < 0.01, ###P < 0.001 vs H2O2-treated cells.


promoted increased the collagen content and density in a dose-dependent manner compared with that in mice subjected to UVB-irradiation (Figure 11). The skin morphology was evaluated by H&E staining. UVB exposure increased epidermal thickness compared with vehicle control group. However, topical application of cistanche significantly alleviated UVB-induced epidermal hyperplasia (Figure 11). In addition, the immunohistochemical staining of β-gal indicated cistanche application reduced the high β-gal expression in UVB-irradiated skin (Figure 11). To determine whether cistanche can ameliorate UVB-induced collagen 

Protective effects of cistanche

Figure 7. Overexpression or inhibition of hsa-miR-4535 regulates collagen synthesis in HS68 cells. (A–C) HS68 cells were transfected with hsa-miR-4535 inhibitor (40 nM) or inhibitor NC (40 nM) for 1 h and then cotreated with H2O2 (200 μM) for 23 h. (D–F) HS68 cells were transfected with hsa-miR-4535 mimic (10 nM) or mimic NC (10 nM) for 1 h and then co-treated with cistanche (30 μM) for 23 h. hsa-miR-4535 levels were detected by qPCR to ensure successful transfection. Protein levels of Smad4, COL1A1, and COL3A1 were analyzed by western blotting. GAPDH was used as a loading control. Values are shown as mean ± SE. Quantification of the results is shown (n = 3) *P < 0.05, **P < 0.01, ***P < 0.001 vs. untreated control cells; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. H2O2 or cistanche-treated cells.


fiber loss via hsa-miR-4535 downregulation to enhance Smad4 in vivo, its RNA expression was assessed in the excised dorsal skin sample by qRT-PCR. hsa-miR-4535 expression was elevated in UVB-irradiated skin but significantly suppressed in the cistanche-treated skin at both low and high doses. Furthermore, the low levels of Smad4 RNA expression in the UVB-irradiated skin were restored by cistanche treatment (Figure 12A, 12B)


Protective effects of cistanche

Figure 8. Inhibition of Smad4 by siRNA or hsa-miR-4535 mimic reverses the cistanche-mediated enhancement of collagen synthesis in dermal fibroblasts. (A–C) HS68 cells were transfected with hsa-miR-4535 mimic (10 nM) or mimic NC (10 nM) for 1 h followed by cotreatment with cistanche (30 μM) and H2O2 (200 μM) for 23 h. Hsa-miR-4535 (A) and Smad4 (B) levels were detected by qPCR to ensure successful transfection. (D–E) HS68 cells were transfected with siRNA Smad4 (20 nM) or siRNA NC (20 nM) for 1 h followed by cotreatment with cistanche (30 μM) and H2O2 (200 μM) for 23 h. Smad4 levels were detected by qPCR to ensure successful transfection (D). Protein levels of Smad4, COL1A1 and COL3A1 were analyzed by western blotting (C, E). GAPDH was used as a loading control. Values shown are means ± SE. Quantification of the results is shown (n = 3) *P < 0.05, **P < 0.01, ***P < 0.001 vs. untreated control cells; #P < 0.05, ##P < 0.01, vs. H2O2-treated cells; $$P < 0.01, $$$P < 0.001 vs. H2O2 plus cistanche-treated cells

In addition, cistanche application rescued TGFβ, psmad2/3, and Smad4 protein expression in UVB-impaired skin tissue (Figure 12C). Our findings indicated that cistanche could potentially protect the skin from UVB-induced damages by inhibiting hsa miR-4535 to enhance Smad4 expression for activation of P- smad2/3 to finally promote collagen synthesis (Figure 13)


image

Figure 9. cistanche enhances TGFβ/Smad collagen synthesis pathway and attenuates dermal senescence as well as inhibition of hsa-miR-4535 expression in UVB-exposed HS68 cells. HS68 cells were exposed to UVB (40 mJ/cm2 ) and then treated with cistanche (30 µM) for 23 h. (A) Protein expression of collagen synthesis-related pathway components (TGFβ, p-smad2/3, Smad4, COL1A1, COL3A1), collagen degradation-related protein (MMP-1), and senescence-associated markers (p16 and p21) was detected by western blot. GAPDH was used as a loading control. (B and C) RNA expression of hsa-miR-4535 and Smad4 was detected by qRT-PCR. Values are shown as mean ± SE. Quantification of the results is shown (n = 3) *P < 0.05, **P < 0.01, vs untreated control cells; #P < 0.05, ##P < 0.01 vs UVB-exposed cells.

Ask for more:

Email:wallence.suen@wecistanche.com whatsapp:+86 15292862950


You Might Also Like