Experimental Study On Anti-aging Effect Of Black Ginseng Extract On Mice Skin By Regulating Inflammatory Factors

Skin aging,the most common phenomenon of aging,is characterized by sagging,dryness,wrinkling and degenerative changes in collagen and elastic fibres.The skin condition is closely related to the function of the intestine.With the onset of aging,the intestinal mucosal epithelium is vulnerable to damage,which allows large amounts of lipopolysaccharide(LPS)to enter the bloodstream.LPS induces the development of local and systemic inflammatory response syndromes through a receptor-dependent skin-gut axis.Black Ginseng,a processed ginseng nine times,has been reported to have stronger anti-inflammatory,antioxidant and anti-aging properties.At present,the research on anti-aging skin mainly focuses on topical skin care products.This study aims to explore whether black ginseng extract can play a protective role on the skin aging induced by D-galactose(D-gal)in mice,and whether the underlying mechanism is related to the Gut-Skin Axis,so as to provide a theoretical basis for the development of anti-aging medicine as the base material of functional foods.Objiective:To explore the role of Black Ginseng Extract in the occurrence and development of skin aging and the relevant mechanism of the protective effect through by regulating inflammatory factors.Method:1.Establishment of skin aging mouse model: 28 Kunming mice were randomly divided into 4 groups: normal control group(NC),D-galactose group(D-gal),Black Einseng high-concentration treatment group(HB)and low-concentration treatment group(LB).The skin aging model was established by subcutaneous injection of1000mg/kg D-gal in the neck.The LB group and the HB group were daily treated by intragastric administration of 300mg/kg or 1000mg/kg black ginseng extract,.and the D-gal group was given the same volume of normal saline by gavage for 60 days.The skin,blood and intestinal tissues of the mice were obtained.2.HE staining was used to observe the morphological changes of skin and colon Masson staining and Gomori aldehyde fuchsin staining were used to analyse the morphology and quantity of collagen fibers and elastic fibers in each group.4.The dermal cell apoptosis of mice in each group was detected by TUNEL method.5.Reactive Oxygen species(ROS)kit was used to detect the level of reactive oxygen species in the skin of mice in each group.6.Total DNA of fecal bacteria in mice was extracted,and 16 S r RNA genes of fecal bacteria were amplified by PCR.Changes in the species and richness of fecal bacteria in mice were detected by deform gradient gel electrophoresis(DGGE).7.The content of LPS in colon tissue and serum of mice was detected by lipopolysaccharide(LPS)kit.8.The expression of claudin-2,occludin,ZO-1 in colon and matrix metalloproteinase9(MMP9)in skin tissue were detected by immunohistochemical staining.9.Real-time PCR was used to detect the levels of inflammatory factors tumor necrosis factor α(TNF-α),interleukin 6(IL-6),interleukin 1β(IL-1β)genes in skin and colon of mice in each group.10.The expressions of nitric oxide synthase(iNOS),TNF-a,IL-6,IL-1β,TLR4 and NF-κB pathway-related proteins were detected by Western Bloting.11.TLR4 and CD68 were double-stained by immunofluorescence staining to detect TLR4 expression in macrophages in skin.12.After TLR4 of macrophage Raw264.7 cells activated by LPS,the cells were divided into four groups: Control group(NC),60 ng LPS induction group(LPS),30μM ginsenoside Rg3 administration group(Rg3),30μM Rg3+80n M TLR4inhibitor(Resatorviod)administration group(Rg3+Re).13.The expression of TLR4 in each group was detected by immunofluorescence staining.14.Real-time PCR was used to detect the m RNA levels of inflammatory cytokines TNF-α,IL-6 and IL-1β in each group.15.The expressions of iNOS,TNF-α,IL-6,IL-1β,and NF-κB pathway-related proteins were detected by Western Bloting.Result:1.Effects of extract of black ginseng on morphological changes by D-gal-induced aging skin and colon of mice: compared with NC group,the D-gal group showed irregular epidermis,dermis wrinkling and thinning.Basal membrane was not obvious;collagen fibers were broken,curled and uneven staining;they were less and loosely arranged;elastic fibers were also less,wrinkling and breaking.Compared with the D-gal group,the epidermis of LB group and HB group had uniform thickness,obvious boundary of basal membrane,thickened collagen fiber layer and dense arrangement,with a large number of fine filamentous elastic fiber bundles.Compared with the LB group,the epidermis in HB group was more regular and complete in structure,and the dermis was thickened.The morphology and quantity of collagen fibers and elastic fibers were similar to that of the normal control group.Compared with the NC group,a large number of inflammatory infiltrates appeared,and the number of colon glands and goblet cells decreased.Compared with D-gal group,the morphology of intestinal glands and goblet cells was almost normal.2.The effect of black ginseng extract on apoptosis of mouse epidermal cells:compared with NC group,the number of TUNEL positive cells was significantly increased in D-gal group(p<0.001),while the number of positive cells in administration group was significantly was decreased compared with D-gal group(p<0.001),and the number of positive cells in HB group was significantly decreased compared with LB group(p<0.05).3.Effects of extract of Black ginseng on antioxidant capacity of mice: Compared with NC group,serum ROS content in D-gal group was significantly increased(p<0.001),while ROS content in LB group and HB group was significantly decreased compared with D-gal group(p<0.01,p<0.001),and ROS content in HB group was significantly decreased compared with LB group(p<0.05).4.Effect of black ginseng extract on expression of MMP9 protein in skin tissue:Immunohistochemical staining results showed that compared with the NC group,the expression of MMP9 in the D-gal group was significantly increased(p<0.001),while the expression of MMP9 in LB group and HB group was significantly decreased compared with the D-gal group(both p<0.01),and the expression of MMP9 in HB group was significantly decreased compared with the LB group(p<0.01).5.Effects of black ginseng extract on colonic flora abundance and mechanical barrier of intestinal epithelium in mice: the results of DGGE showed no significant changes in flora richness and flora species between groups.Immunohistochemistry showed that the expression of intestinal Claudin-2,Occludin and ZO-1 in D-gal group was significantly lower than that in NC group(p<0.001),and the expression of these proteins in LB group was significantly higher than that in D-gal group(p<0.001,p<0.01,p<0.001,p<0.001),and in HB group was higher than that in LB group(p<0.01,p<0.01,p<0.001).6.Effects of Black ginseng on intestinal mucosa and blood of mice with LPS content:the LPS content of the D-gal group was significantly higher than that of the NC group(p<0.001),the LPS content of the LB group and HB group was significantly lower than that of the D-gal group(p<0.01,p<0.001),the HB group was significantly lower than that of the LB group(p<0.001).Serum LPS content in D-gal group was significantly higher than NC group(p<0.001),and LPS content in HB group was significantly lower than that in D-gal group(p<0.01,p<0.001).7.Effects of black ginseng extract on skin and colon inflammation in mice: the levels of TNF-α,IL-6 and IL-1β genes in the D-gal group were significantly higher than those in the NC group(p<0.001),and the levels of genes in the LB group were lower than those in the D-gal group(p<0.001,p<0.01,p<0.001).The levels of inflammatory cytokines in HB group were lower than those in LB group(p<0.01,p<0.05,p<0.001).In colon tissues,the levels of TNF-α,IL-6 and IL-1β genes in D-gal group were significantly higher than those in NC group(p<0.001),the levels of genes in LB group were significantly lower than those in D-gal group(p<0.001),and the expression levels of inflammatory cytokines in HB group were lower than those in LB group(p<0.01,p<0.001,p<0.001).Western Bloting showed that the expression levels of iNOS,TNF-α,IL-6 and IL-1β in skin tissue of D-gal group were significantly higher than those of NC group(p<0.001,p<0.01,p<0.001,p<0.01).The protein expression of LB group was lower than that of D-gal group(p<0.01),and the protein expression level of HB group was significantly lower than that of D-gal group(p<0.001,p<0.01,p<0.001,p<0.001).8.The expression of LPS receptor TLR4 in macrophages showed that: The expression of TLR4 receptor in skin macrophages of mice in D-gal group was significantly higher than that in NC group(p<0.001),and the expression of TLR4 receptor in macrophages of LB and HB groups was significantly decreased compared with that in D-gal group(p<0.01,p<0.001).TLR4 expression in HB group was significantly decreased compared with LB group(p<0.05).9.The effect of black ginseng extract on NF-κB signaling pathway in mice: the expression levels of TLR4,p-NF-KB,My D88 and P-IPKb-α in D-gal group were significantly higher than those in NC group(p<0.001,p<0.001,p<0.001,p<0.01);The protein expression of LB group was decreased compared with D-gal group(p<0.01,p<0.01,p<0.001,p<0.05),and the protein expression level of HB group was significantly lower than that of LB group(p<0.001,p<0.05,p<0.01,p<0.05).10.Effects of Rg3 on TLR4 receptor expression in macrophages: In vitro studies,TLR4 protein expression in Raw264.7 cells in LPS group was significantly higher than that in NC group(p<0.001),and TLR4 protein expression in Rg3 group and Rg3+Re group was significantly decreased compared with LPS group(p<0.01,p<0.001).Compared with Rg3 group,TLR4 expression in Rg3+Re group was significantly decreased(p<0.05).11.Effect of Rg3 on the level of macrophage inflammatory factor-related genes: The expression levels of TNF-α,IL-6 and IL-1β in Raw264.7 cells in LPS group were significantly higher than those in NC group(p<0.001),the expression levels in Rg3 group were significantly lower than those in LPS group(p<0.001,p<0.001,p<0.01),and the expression levels in Rg3+Re group were significantly lower than those in Rg3group(p<0.001,p<0.05,p<0.05).12.Effect of Rg3 on NF-κB signaling pathway in macrophages: the expression levels of iNOS,p-NF-κB,My D88,p-i Kb-α,TNF-α,IL-6 and IL-1β in LPS group were significantly higher than those in NC group(p<0.001,p<0.001,p<0.001,p<0.01,p<0.01,p<0.01).The histone expression in Rg3 group was significantly decreased compared with LPS group(p<0.05,p<0.01,p<0.01,p<0.01,p<0.05,p<0.05),and the protein expression in Rg3+Re group was significantly decreased compared with LPS group(p<0.01,p<0.001,p<0.001,p<0.01,p<0.01,p<0.01).Conclusion:1.Black ginseng aqueous extract can improve the morphological characteristics of D-gal induced skin aging in mice,and repaired the morphology and quantity of collagen fibers and elastic fibers in skin.2.Black ginseng extract can reduce the production of ROS induced by D-gal,and significantly inhibited the apoptosis in dermal cells to play an antioxidant role.3.D-gal caused the destruction of the intestinal barrier in mice,LPS accumulation in the intestine and "leak" into the blood circulation,leading to the occurrence of skin inflammatory diseases and exacerbating skin aging.Black cucumber extract can increase the expression of intestinal claudin-2,Occludin,ZO-1 to protect intestinal barrier and decrease the "leak",and inhibit the transcription and release of inflammatory factors through the Gut-Skin Axis,consequentially alleviate skin aging in mice.4.Black ginseng extract may exert its anti-inflammatory effect through one of its main components Rg3 which can inhibit the activation of NF-κB signaling pathway by inhibiting the over-activation of TLR4 induced by LPS accumulation in macrophages,reducing inflammatory response and the release of matrix metalloproteinase(MMP9),thus alleviating the hydrolysis of collagen fibers and the degradation of extracellular matrix.