| [Background]Skin aging is the most common human aging. Skin aging is not only seriously affect the beauty, but also associated with many skin diseases. With the development of social economy, how to delay the onset of skin aging, especially how to "beauty" become the focus of attention all over the world, therefore prevention and delay skin aging become one of the modern medical research hot spot. The oxidation injury of skin is one of the main reasons causing skin aging, therefore, to prevent skin oxidative damage to protect normal structure and function of skin tissue is of great significance of postpone skin aging.Reactive oxygen species were kinds of containing unpaired electrons of the atoms or molecules, including hydroxyl radicals (HO·), superoxide anion radical (O2·-), singlet oxygen (102) and hydrogen peroxide (H2O2)etc, studies showed that H2O2 as the main members of ROS, can directly or indirectly harm the cells, induce apoptosis and necrosis, is widely used to establish oxidative stress model in vitro. Researches showed that when intracellular redox steady destruction induced the balance between production and clearance of ROS was broken, it caused excessive ROS accumulation resulting in oxidative stress, and induced a series of oxidative damage. Especially, ROS can induce the matrix metalloproteinases (MMPs) matrix metalloproteinase activation or up-expression, and promote the degradation of collagen and elastin in the extracellular matrix (ECM), thus make skin loses elasticity and toughness, eventually lead to the occurrence of skin aging. A lot of researches have shown that NADPH oxidase (Nox) family was the key enzyme of induction of ROS, It is composed of multiple membrane-associated and cytosolic components, including gp91phox, p22phox, p40phox, p47phox, p67phox, and RacOver the last years, six homologs of the cytochrome subunit of the phagocyte NADPH oxidase were found:Nox1, Nox3, Nox4, Nox5, Duox1, and Duox2. Together with the phagocyte NADPH oxidase itself (Nox2/gp91phox), the homologs are now referred to as the Nox family of NADPH oxidases., Nox is no active in physiological state, its activation need cytoplasm submits combination and bit into the cell membrane submits. When the expression of Nox family were abnormal, ROS level has increased dramatically. Nox source of ROS was involved in a variety of diseases'development, but its research in skin aging was less. Some scholars found that Connexin43(Cx43) and Nox joint influencein the oxidative stress damage of kidney cells, and suggested that Cx43 can be used as a new indicator of podocyte oxidative stress and as a novel therapeutic target to reduce the podocyte damage. Cx43 as a major structural protein of gap junction channels play a key role in cell proliferation, differentiation and stable internal environment, the body's metabolism, growth and development, and lots of reseach fouend that the abnormal expression of Cx43 can lead to many diseases. In recent years the resreaches about the role of Cx43 in oxidative stress injury in myocardial cells and renal tubular epithelial cells have also reported, these studies suggest that Cx43 play the key role to adjust ROS mediated cell damage, which prompt Cx43 also played an important role in oxidative stress injury, but its role in the oxidative damage skin has not yet been reported. This paper intend to discussed the role of Nox and Cx43 in oxidative damage of skin.Contains antioxidant active ingredients of skin care products can help skin against oxidative damage to some extent. Many studies have shown that reasonable adding anti-oxidant in the cosmetics, or in the skin disease treatment using products containing antioxidants can effectively prevent ultraviolet light mediated skin damage. With Marine resources development, people understanding of Marine resources is becoming increasingly clear that has rich resources found in the ocean, the Squid ink polysaccharide (Squid ink polysaccharides, SIP) is a kind of extracted from Squid Murray in ocean Squid to highly reactive towards functional active ingredients. Squid ink by secretory synthesis of the ink sac, the ink bag storage, the main chemical composition is melanin and protein polysaccharide complexes. Polysaccharide was extracted from squid ink squid ink effective non-toxic Marine bioactive substances, Takaya etc were obtained from squid ink three polysaccharide composition Illexins A, B and C, by the glucuronic acid (GlcA) and fucose (Fuc) and N-acetyl galactose (GalNAc) of different molecular weight of size, structural formula. Its chemical formula is [1-4-3 glca?(GalNAca 1-3) Fucal-]n. Reported in recent years showed that the SIP have an impact on antioxidation, antitumor, antibacterial and chemotherapy protection, and the antioxidant function drawed a lot of attention. Rsearches showed that the SIP in vitro can effectively remove DPPH and HO· ect reactive oxygen species composition, and can effectively relieve cyclophosphamide (CP)-induced multiple tissues and organs of oxidative damage, such as bone marrow, heart, liver or kidney, and inhibited the increased of lipid peroxide malondialdehyde (MDA) c induced by CP, and restore antioxidant enzymes Superoxyde dismutase(SOD) and catalase(CAT) activity. However, there is no research on the effect of SIP in skin oxidative damage. So this experiment for the first time expolre the effect of SIP on in vitro antioxidant.To examine the effect of SIP esistance skin ageing and its mechanism, fully tap the potential of SIP resist skin aging, this experiment will use SIP as antioxidants skin, oxidation damage model caused by H2O2 as the research object, observe the damage occurs when the change of NADPH oxidase and Cx43, further explore the pathogenesis of oxidative stress damage. Then using SIP on the oxidative damage model determines the pharmacodynamic material basis of SIP anti-oxidative stress damage in skin, deepen the fundamental research of SIP in the fight against skin aging.[Objective]This study through research in H2O2 damage dermal fibroblasts the ROS levels, the expression of NADPH oxidase and of Cx43 in H2O2-induced dermal fibroblasts damage, analysis the role of Cx43 and NADPH oxidase in oxidative stress injury induced by H2O2, and on this basis, to explore the squid ink polysaccharide's protective effect and mechanism on H2O2-induce dermal fibroblasts damage. For effective clinical prevention and treatment of skin oxidative damage provide a new ideal.[Methods](1) Using MTT assay to detect the cell vitality of human dermal fibroblasts treated by concentrations of H2O2 at different times:the logarithmic phase HFbs were treated by different concentration of H2O2, for 100,200,400,600,700 and 800?mol/L respectively and for 2h,4h,8h,12h and 24 h, at each end time the cell vitality was determined by MTT. Then the appropriate concentration and time was select to establish HFbs oxidative damage model, and used in subsequent experiment.(2) Using microscope to examine HFbs morphological changes and MTT assay to detect cell vitality after treated with different ways:5 x 103/HFbs were seeded in 96-well plate, and classified into six groups:normal control group,700?mol/L H2O2 oxidation injury group, the 700?mol/L H2O2 add SIP 0.5 mg/mL oxidative damage protection group, 700?mol/L H2O2 add NADPH oxidase inhibitors (Diphenyleneiodonium, chloride, DPI) 1 ?mol/L oxidatie damage protection group, H2O2 add NADPH oxidase inhibitors Apocynin(APO) 100?mol/L oxidatie damage protection group, H2O2 add Connexin43 inhibitor (18-alpha-Glycyrrhetinicacid,18-a-GA) 5?mol/L oxidative damage protection group. Then use microscope to examine HFbs morphological changes and MTT assay to detect cell vitality after treated with different ways(3) DCFH-DA fluorescent probe to detect each group ROS changes:1 x 105/mL HFbs were seeded in 6-well plate. After the treatment with different ways as above collection the cells and adjust the cell number to agree, then exposed to DCFH-DA fluorescent probe for 30 min, and then to detect ROS levels of groups by Multiscan Spectrum.(4) Using Flow cytometry to detect the apoptosis rate of HFbs after treated with different ways:2.5 x 105/mL HFbs were seeded in the diameter of 60mm petri dish. After the treatment with different ways as above, the flow cytometry was used to measure the cell apoptosis rata with Annexin V-FITC/PI double staining assay.(5)The detection of malondialdehyde (MDA) content, superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activity:1 x 106/mL HFbs were seeded in the diameter of 100mm petri dish. After the treatment with different ways as above, the MDA content, SOD and GSH-PX activity were detected by kits.(6) Using western blot to detect the expression of Noxl, Nox2, Nox4, Nox5, Connexin43, MMP1 and MMP9 after different treatments:1 x 106/mLHFbs were seeded in the diameter of 100mm petri dish. The western blot was used to detect the expression of Noxl, Nox2, Nox4, Nox5, Connexin43, MMP1 and MMP9 after different treatments. The ratio of interest protein and GAPDH was used to reflect relative expression of protein.[Results](1) Human dermal primary fibroblasts presented with long spindle shaped, vortex shaped or parallel arrangement growth. As shown by immunofluorescence:the nucleus was blue, ovoid and the cytoplasm presented green fluorescence which high expression of relatively specific marker Vimentin.(2) HFbs were treated with concentrations of H2O2 at different times.100 to 800?mol/L of H2O2 significantly decreased the cell viability in a dose-dependent manner. At the same time point, the results of the time-response study in which the cell viability was reduced to approximately 40.8 after a 24h incubation with 700?mol/L H2O2. Then the cell morphological observations show that H2O2 results in significant injury to HFbs. Based on these results, we chose to use a 24h exposure of 700?mol/L H2O2 to establish HFbs oxidative stress damage model. HFbs exposed to 700?mol/L H2O2 without DPI and APO, 18-a-GA and SIP, the cell survival rate were significantly increased compared with the oxidative damage group, and the cell morphology compared to the normal group had no obvious change.(3) Different concentrations of H2O2 treatment HFbs after 2h increased the intracellular ROS leve in a concentration-dependent manner, which preprocessing with DPI and APO,18-a-GA and SIP can effectively reduce the H2O2-induced ROS (P< 0.05);(4) After HFbs exposed to H2O2700?mol/L 24h, the apoptosis rate was increased significantly (P< 0.05), and pretreatment with DPI, APO,18-a-GA and SIP could obviously reduce the apoptosis of H2O2 caused increased (P< 0.05).(5) After HFbs exposed to H2O2700?mol/L 24h, the MDA content was increased, and the activity of SOD and GSH-PX were decreased, and preprocessing HFbs with DPI, APO,18-a-GA and SIP could effectively relieve the H2O2 induction of MDA increased, activity of SOD and gsh-px decreased (P< 0.05);(6) Different concentrations of H2O2 treatment HFbs after 24 h, Western blot results show that the NADPH oxidase (Nox1, Nox2, Nox4 and Nox5), Connexin43, MMP1 and MMP9 expression increased in a concentration-dependent manner. and pretreatment HFbs with DPI, APO,18-a-GA and SIP could obviously reduce the H2O2 caused Noxl, Nox2, Nox4, Nox5, Connexin43, MMP1 and MMP9 expression increased;[Conclusions](1) H2O2 can make excessive intracellular ROS generation, increase the expression of NADPH oxidase and Cx43, thus induce HFbs oxidative damage.(2) NADPH oxidase and Connexin43 can effectively protect the fibroblast injury induced by H2O2, which show that NADPH oxidase and Connexin43 play an important role in the oxidative damage, and for the first time discover Nox and Cx43 can influence each other, mutual regulation of fibroblasts oxidative stress injury(3) For the first time find that Squid ink polysaccharide can obviously relieve HFbs oxidative stress injury caused by H2O2.(4) we firstly speculate that the squid ink polysaccharide could get though inhibition the expression of NADPH oxidase and Connexin43 to develop its antioxidant effect. |
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