Titanium Dioxide Inhibits Inflammation And Endoplasmic Reticulum Stress To Promote Zebrafish Skin Wound Healing

The skin is the human body’s largest organ and its main function is to protect the body against external factors.Wounds that fail to heal in a timely manner may lead to serious tissue necrosis.Skin wound healing is a dynamic and highly coordinated biological process,which mainly includes three overlapping but completely distinct stages:inflammation,proliferation and remodeling.Inflammation is the body’s defense response against injury,which aims to resist the pathogen or remove necrotic tissue.However,loss of control of the inflammatory response will lead to overexpression of pro-inflammatory cytokines,resulting in persistent wound damage.The activation of NLRP3 inflammasome resists the infection of pathogens,but the uncontrolled activation of NLRP3 inflammasome will lead to the amplification of inflammatory effects and impaired wound healing.Activation of NF-κB regulates the expression of NLRP3.SIRT3 reduces the level of pro-inflammatory cytokines by inhibiting the expression of NF-κB.AMPK plays an anti-inflammatory role in rat liver fibrosis by upregulating SIRT3 expression.AMPK and SIRT3inhibit the release of pro-inflammatory cytokines in wounds,but the upstream and downstream regulatory relationship between AMPK and SIRT3 has not been reported in the inflammatory response of wound healing.Some stress conditions（e.g.,oxidative stress,hypoxia and nutrient deprivation）in the wound microenvironment cause endoplasmic reticulum stress,which affects re-epithelialization and delays wound healing by inhibiting keratinocyte migration.SIRT1alleviates endoplasmic reticulum stress by deacetylating XBP1s in HEK（human embryonic kidney）-293 cells.Reconstruction of skin function after injury remains a challenge due to the complexity of the wound healing process.TiO₂ nanoparticles exhibit excellent antibacterial and anti-inflammatory properties,and accelerate the skin wound healing process of mice and rats by promoting granulation tissue formation,angiogenesis and collagen deposition.However,the mechanisms of how TiO₂ promote wound healing need to be further explored.Zebrafish,which have similar physiological and anatomical features to higher animals,share more than 80%genomic homology with the human genome and exhibit the same three stages of wound healing as mammals,and have been identified as model organism for studies of wound healing.In the present study,a full thickness wound was created on the flank of zebrafish using a cylindrical metal rod（2 mm diameter）,heated over the open flame for 5 s and pressed to the target area surface for 1 s.Fifty zebrafish were randomly divided into five groups:control,250,500,750and 1000μg/L TiO₂ groups to detect effect of different concentrations of TiO₂ on skin wound healing.Fish in the control group were kept in clear water,while fish in the treatment group were immersed in different concentrations of TiO₂.The wound size was recorded starting at 2 days post wounding（dpw）and the wound healing rate was calculated based on the wound size at 2 dpw.The effects of TiO₂ on wound healing were determined by measuring the wound size（at 2,7,14 and21 dpw）and calculating the wound healing rate（at 5,7,10 and 14 dpw）.Immersion application with different concentrations of TiO₂ all decreased the wound area at 7,14 and 21 dpw and increased the wound healing rate at 5,7,10 and 14 dpw.Compared with other concentrations,500μg/L TiO₂ showed the most significant effect on accelerating wound healing.Therefore,500μg/L TiO₂ was used to treat zebrafish in subsequent experiments.A total of 163 zebrafish were used to explore the effects of TiO₂ on tissue structural changes,inflammation and endoplasmic reticulum stress during skin wound healing and its possible mechanisms.The results were as follows:1.The formation of granulation tissue at 2,4,6 and 8 dpw was analyzed by H&E staining in wounds of zebrafish.The results showed that the granulation tissue in the control group was first visible at 2 dpw,reached maximal size at 4 dpw,and gradually regressed at 6 and 8 dpw.The granulation tissue area increased at 2 and 4 dpw and decreased at 6 and 8 dpw after TiO₂ treatment.Cell proliferation in wounds at 2,4,6 and 8 dpw was detected with PCNA immunohistochemical staining.Comparing to the unwounded group,the number of PCNA positive cells in wounds of the control and TiO₂ groups increased at 2,4,6 and 8 dpw,and TiO₂ increased the number of PCNA positive cells in wounds at 2 and 4 dpw and reduced the number of PCNA positive cells at6 and 8 dpw compared with the control group.Masson’s trichrome staining was used to assess collagen deposition in wound healing.The results indicated that compared with the unwounded group,collagen deposition in wounds of zebrafish skin decreased at 2 dpw,increased at 4 and 6dpw,and had no obvious difference at 8 dpw in the control group.Compared with the control group,TiO₂ promoted collagen deposition in wounds at 2 and 4 dpw,decreased collagen deposition at 6 dpw,and had no significant difference at 8 dpw.2.The levels of pro-inflammatory cytokines in wounds were determined by qPCR,which revealed that the expression of pro-inflammatory cytokines IL-1β,TNF-αand IL-6 were higher in wounds at 1 and 3 dpw than those in the unwounded group.TiO₂ decreased the m RNA levels of IL-1β,TNF-αand IL-6 in wounds at 1 and 3 dpw.3.The m RNA levels of NF-κB and NLRP3 inflammasome components（NLRP3,ASC and caspase-1）in wounds were detected by qPCR.The results indicated that the m RNA levels of NF-κB and NLRP3 inflammasome components（NLRP3,ASC and caspase-1）were higher in wounds at 1 and 3 dpw than those in the unwounded group.TiO₂ down-regulated the m RNA levels of NF-κB,NLRP3,ASC and caspase-1 in wounds at 1 and 3 dpw.Western blot results showed that TiO₂inhibited the expression of NF-κB p65,NLRP3 and mature IL-1β,and up-regulated the p-AMPK/AMPK ratio and SIRT3 expression in wounds at 1 dpw.4.To explore the upstream and downstream regulatory relationship between AMPK and SIRT3 in wound healing,nine zebrafish were randomly divided into three groups:control group,500μg/L TiO₂ group and 500μg/L TiO₂+CC group.After treating zebrafish with AMPK inhibitor CC（15μg/m L）by immersion treatment for 2 h,TiO₂ was added to continue the treatment for 4 h.Western blot results showed that the ratio of p-AMPK/AMPK was decreased at the wound site and SIRT3 expression increase induced by TiO₂ was reversed after AMPK inhibitor CC added,indicating that AMPK was upstream of SIRT3.TiO₂ might regulate AMPK/SIRT3 pathway to inhibit NF-κB/NLRP3 inflammasome activation,thereby decreased the expression of pro-inflammatory cytokines.5.The expression of endoplasmic reticulum stress related genes in wounds were detected by qPCR.It was found that the m RNA levels of endoplasmic reticulum stress related genes IRE1α,XBP1s and e IF2αwere higher than those in the unwounded group,and TiO₂ decreased their m RNA levels in wounds at 1 and 3 dpw.Western blot results showed that TiO₂ up-regulated SIRT1expression and down-regulated the expression of XBP1s and GRP78 in wounds at 1 dpw,thus inhibiting endoplasmic reticulum stress.In conclusion,TiO₂ accelerated wound healing by increasing the number of PCNA positive cells,granulation tissue formation and collagen deposition.TiO₂ inhibited NF-κB/NLRP3inflammasome activation by upregulating AMPK/SIRT3 pathway to decrease the expression of pro-inflammatory cytokines IL-1β,TNF-αand IL-6,thus inhibiting the inflammatory response and accelerating wound healing.TiO₂ may up-regulate the expression of SIRT1 and suppress XBP1s and GRP78 expression,thereby alleviating endoplasmic reticulum stress and promoting wound healing.