Protective effects of fermented pomegranate peel against UVB-induced photoaging in HaCaT cell and hairless mice
- 주제(키워드) skin photoaging , UVB , fermented pomegranate peel , wrinkles , moisture , skin barrier function , MAPK
- 발행기관 고려대학교 대학원
- 지도교수 서형주
- 발행년도 2023
- 학위수여년월 2023. 8
- 학위구분 석사
- 학과 대학원 의생명융합과학과
- 세부전공 바이오식의과학 전공
- 원문페이지 111 p
- UCI I804:11009-000000277549
- DOI 10.23186/korea.000000277549.11009.0000122
- 본문언어 영어
초록/요약
In this study, HaCaT cells, human-derived keratinocytes, and SKH-1 hairless mice were used to evaluate the inhibitory activity of ultraviolet B (UVB)-induced skin photoaging of fermented pomegranate peels (FP) with increased ellagic acid content through fermentation. To induce photoaging through UVB, HaCaT cells were irradiated with an intensity of 25 mJ/cm2 and treated with FP at concentrations of 30 and 40 μg/mL. SKH-1 hairless mice were irradiated with UVB at an intensity of 75-300 mJ/cm2 for 8 weeks and the FP were orally administered at concentrations of 200 and 400 mg/kg. FP significantly reduced matrix metalloproteinases (MMPs) and increased tissue inhibitor of metalloproteinases (TIMPs), type 1 collagen, filaggrin, and hyaluronic acid synthase, which play important roles in skin elasticity and moisturizing, and decreased hyaluronic acid degradative enzymes in HaCaT cells. According to the antioxidant activity of FP, antioxidant enzymes such as glutathione peroxidase (GPX) and superoxide dismutase 1 (SOD 1) increased significantly, and inflammatory cytokine interleukine 1 beta (IL-1β) and cyclooxygenase 2 (COX 2) generated by photoaging decreased. Oral administration of FP restored skin water-holding capacity, trans epidermal water loss, erythema value, and skin thickness to normal in SKH-1 hairless mice induced by photoaging. Oral administration of FP decreased the area, length, and depth of wrinkles, and the number of wrinkles increased as it suppressed conversion to deep wrinkles. In addition, FP administration significantly reduced UVB-induced inflammatory mediators, reactive oxygen species, and lipid peroxides through high expression levels of antioxidant enzymes. FP increased the expression of hyaluronic acid synthesis genes and decreased the expression of MMPs, thereby suppressing the decrease of collagen and increasing the expression of involucrin and filaggrin involved in the formation of keratinocyte membrane. In conclusion, FP inhibited UVB-induced photoaging-induced wrinkle formation in HaCaT cells and hairless mice and showed the effect of improving skin barrier function and the expression of related factors. FP is considered to have sufficient potential as a functional material to improve skin photoaging.
more초록/요약
본 연구에서는 사람 유래 각질형성 세포인 HaCaT 세포와 SKH-1 무모 쥐를 이용하여 발효를 통해 엘라그산의 함량을 높인 석류 껍질 발효물(FP)이 UVB에 의해 유도된 피부 광노화 억제활성을 평가하였다. UVB를 통한 광노화를 유도하기 위해 HaCaT 세포는 25 mJ/cm2의 강도로 조사하여 FP을 30 및 40 μg/mL의 농도로 처리하였으며, SKH-1 무모 쥐는 75-300 mJ/cm2의 강도로 8주간 강도를 높여가며 조사하여 200, 400 mg/kg의 농도로 경구 투여하였다. FP는 HaCaT 세포에서 MMPs를 현저히 감소시켰으며, 이와 동시에 TIMPs를 증가시켰고 피부에서 탄력과 보습 등 중요한 역할을 하는 1형 콜라겐, 필라그린 그리고 히알루론산 합성 효소를 증가시켰고 분해 효소를 감소시켰다. FP가 가지는 높은 항산화 활성에 따라 GPX 및 SOD 1 등 항산화 효소가 확연히 증가하였고, 광노화에 따라 발생한 염증성 매개인자 IL-1β, COX 2가 감소하였다. 광노화가 유도된 SKH-1 무모 쥐에서 FP의 투여로 피부 수분 보유력, 경 표피 수분 손실량, 홍반 지수 및 피부 두께가 정상 수준으로 회복되었고, 주름 면적, 길이, 깊이 등이 감소하였으며, 깊은 주름으로의 전환을 억제함에 따라 주름 수가 증가하였다. 또한, FP 투여는 항산화 효소의 높은 발현량을 통해 UVB에 의해 발생한 염증 매개인자와 활성 산소종 그리고 지질 과산화물을 현저히 감소시켰다. FP는 히알루론산 합성 유전의 발현을 증가시키고 MMPs의 발현을 감소시킴으로써 콜라겐의 감소를 억제하는 동시에 각질세포막 형성에 관여하는 인볼루크린과 필라그린의 발현을 증가시켰다. 결론적으로, FP는 HaCaT cell과 무모 쥐에서 UVB로 유발한 광노화에 의한 주름 생성을 억제하였고 피부 보호막 기능과 이와 관련 인자들의 발현량 개선하는 효과를 보였다. 따라서,FP는 피부 광노화를 개선하는 기능성 소재로서 충분한 가능성을 가지고 있을 것으로 판단된다.
more목차
Contents
ABSTRACT i
Abstract in Korean iⅴ
Contents vⅰ
List of Abbreviations x
List of Figures xⅲ
List of Tables xvⅱ
1. Introduction 1
2. Materials and methods 4
2.1 Sample preparation 4
2.2 Determination of hydrolysable tannins, polyphenol and flavonoid contents 4
2.3 Estimation of radical scavenging 5
2.4 Analysis of ellagic acid 5
2.5 HaCaT cell culture 6
2.5.1 HaCaT cells viability (WST) 6
2.5.2 Gene expression level analysis by qRT-PCR in HaCat cell 7
2.6 Experimental animal treatment 9
2.6.1 Measurement of body weight, food intake, water intake and organ weight 9
2.6.2 Measurement of serum biochemical parameters 10
2.6.3 Measurement of skin parameter and wrinkle formation 10
2.6.4 Histopathological Analysis 11
2.6.5 Enzyme linked immunosorbent assay (ELISA) of hyaluronic acid 12
2.6.6 Reactive oxygen species (ROS), and malondialdehyde (MDA) assay 12
2.6.7 Gene expression level analysis using quantitative real-time polymerase chain reaction (qRT-PCR) 13
2.6.8 Analysis of proteins expression levels involved in MAPK pathway by western blotting 16
2.7 Statistical analyses 17
3. Results 18
3.1 Hydrolysable tannins, phenolic compounds and dry weight of pomegranate peel (PP) and fermented pomegranate peel (FP) 18
3.2 Radical scavenging activity of pomegranate peel (PP) and fermented pomegranate peel (FP) 20
3.3 Ellagic acid content in pomegranate peel (PP) and fermented pomegranate peel (FP) 22
3.4 Effects of pomegranate peel (PP) and fermented pomegranate peel (FP) on cell viability in UVB-irradiated HaCaT cells 25
3.4.1 Effects of fermented pomegranate peel on mRNA expression of MMPs and TIMPs in UVB-irradiated HaCaT cells 28
3.4.2 Effects of fermented pomegranate peel on mRNA expression of HYAL 1, HAS 1, and COL 1a1 in UVB-irradiated HaCaT cells 31
3.4.3 Effects of fermented pomegranate peel on mRNA expression of FLG and IVL in UVB-irradiated HaCaT cells 33
3.4.4 Effects of fermented pomegranate peel on mRNA expression of CAT, GPX, and SOD 1 in UVB-irradiated HaCaT cells 35
3.4.5 Effects of fermented pomegranate peel on mRNA expression of IL-1β and COX 2 in UVB-irradiated HaCaT cells 38
3.4.6 Effects of ellagic acid on mRNA expression of MMP 2 and TIMP 1 in UVB-irradiated HaCaT cells 40
3.4.7 Effects of ellagic acid on mRNA expression of FLG and IVL in UVB-irradiated HaCaT cells 42
3.5 Changes of body, food intake, water intake and organ weight in UVB-irradiated hairless mice 44
3.5.1 Effects of fermented pomegranate peel on serum biochemical parameters in UVB-irradiated mice 47
3.5.2 Effects of fermented pomegranate peel on skin parameters in UVB-irradiated mice 49
3.5.3 Effects of fermented pomegranate peel on wrinkle formation in UVB-irradiated mice 52
3.5.4 Effects of fermented pomegranate peel on skin thickness in UVB-irradiated mice 57
3.5.5 Effects of fermented pomegranate peel on skin hyaluronic acid in UVB-irradiated mice 59
3.5.6 Effects of fermented pomegranate peel on skin ROS and MDA in UVB-irradiated mice 61
3.5.7 Effects of fermented pomegranate peel on mRNA expression of MMPs and TIMPs in UVB-irradiated mice 63
3.5.8 Effects of fermented pomegranate peel on mRNA expression of HYAL 1, HAS 1, and COL 1a1 in UVB-irradiated mice 67
3.5.9 Effects of fermented pomegranate peel on mRNA expression of FLG, IVL, and AQP 3 in UVB-irradiated mice 69
3.5.10 Effects of fermented pomegranate peel on mRNA expression of CAT, GPX, and SOD 1 in UVB-irradiated mice 71
3.5.11 Effects of fermented pomegranate peel on mRNA expression of IL-1β and TNF-α in UVB-irradiated mice 73
3.5.12 Effects of fermented pomegranate peel on the protein expressions of p-ERK/ERK, p-JNK/JNK, p-P38/P38 and COX 2 in UVB-irradiated mice 75
4. Discussion 79
References 84
