| Background:Wound healing is one of the most sophisticated processes in the human body.When the skin is deeply injured,multiple cell types within the epidermis,dermis and subcutaneous fat need to be coordinated in precise stages to achieve healing.Hemostasis,inflammation,angiogenesis,growth,re-epithelialization and remodeling of the wound occur in a chronological but overlapping fashion.Macrophages and fibroblasts play a pivotal role throughout trauma healing.Deep partial thickness burn wounds are highly susceptible to scarring during the healing process due to persistent inflammation and abnormal fibrosis.This has a profound impact on both the function and appearance of the patient.There is a paucity of research and a lack of relevant medications that can be applied early to reduce scar growth without affecting the healing of deep partial thickness burn.Metformin(Metformin)has been used as the drug of choice in the treatment of diabetes mellitus.As a classical activator of adenylate-activated protein kinase(AMPK),more and more scholars have found in recent years that metformin not only can lower blood glucose,but also has certain efficacy in anti-tumor,antiinflammatory,delaying aging and anti-fibrosis.Since metformin has been proven to have anti-inflammatory and fibrosis-inhibiting effects,there are no similar studies on whether its application to deep partial thickness burn wounds can regulate the level of inflammation and fibrosis in the wound microenvironment,thus improving the quality of wound healing and reducing scar formation.The aim of this study is to investigate the effect of metformin intervention on deep partial thickness burn wound healing,and to preliminarily elucidate the molecular mechanism of metformin regulation of macrophages and fibroblasts in inflammation and fibrosis formation,which is of great significance for the rapid translation to clinical application.Therefore,we firstly observed the effects of metformin intervention on the expression of the main components of the extracellular matrix(ECM): Collagen I,Collagen III,Fibronectin and inflammatory factors and chemokines in deep partial thickness burns in rats in vivo,and then observed the effects of metformin on the biological behavior of macrophages and fibroblasts through in vitro experiments.Then,the effects of metformin on the biological behaviors of macrophages and fibroblasts were observed through in vitro experiments,and the molecular mechanisms of NF-κ B and AMPK/m TOR,TGF-β 1/Smad3 signaling pathways were further explored.Through this study,the regulatory mechanism of metformin in improving the healing quality of deep partial thickness burns was preliminarily investigated at animal and cellular levels,and a new way and idea was provided for its reduction of scar proliferation in deep partial thickness burn wounds.Part I.Effect of metformin on wound healing of deep partial thickness burn in ratsObjective:To investigate the effects of metformin on type I collagen deposition,inflammatory factors and chemokines in deep partial thickness burn wounds in rats.Method:1.6 SD rats with deep partial thickness burns were constructed.The rats were injected subcutaneously with metformin at different concentrations(1m M,10 m M,100 m M)into the burn wounds immediately after the burns.five groups(negative control group(normal skin),positive control group(Met0m M),low dose group(Met1m M),medium dose group(Met10m M),high dose group(Met100m M))were used for the experiment.Immediately after successful modeling,metformin was injected every other day for one week,and the intervention was not continued for the following week.burn wound tissues were extracted after 14 d.HE staining was performed to measure the dermal thickness of post-burn wounds,and Masson staining was performed to observe the deposition of Collagen I.Western blot was performed to detect the expression of Collagen I.Sirius red staining andimmunohistochemistry were used to detect Collagen III expression.Immunohistochemical staining was performed to detect the expression of Fibronectin.2.According to the results of method 1,6 SD rats were taken,and the experiment was divided into three groups(negative control group(normal skin)normal skin group,burn positive control group(PBS10m M),and experimental group(Met100m M)).After successful modeling,metformin was injected immediately,and RNA was extracted from 0.5CM skin tissue at the perimeter of the burn wound after injection on alternate days until day 4.q RT-PCR was performed to detect the m RNA expression levels of associated inflammatory factor(IL1β)and chemokine(CCL2).Result:All rats survived after burn modeling without necrosis,ulceration,infection,or delayed healing of burn wounds.HE results showed that the dermal thickness in the Met100 m M group was significantly reduced compared with the Met0 m M group,and the difference was statistically significant(p < 0.05),while the dermal thickness in the Met1 m M and Met10 m M groups was not statistically different compared with the Met0 m M group.Masson staining results showed that The type I collagen volume fraction(CVF)in the Met100 m M group was significantly reduced compared to the Met0 m M group,and the difference was statistically significant(p < 0.05),and there was no statistical difference between the type I collagen volume fraction(CVF)in the Met1 m M and Met10 m M groups compared to the Met0 m M group.Western blot results showed that the expression of type I collagen in the Met100 m M group was significantly lower than that in the Met0 m M group,and the difference was statistically significant(p<0.001).Sirius red staining results suggested that the area percentage of type III collagen in the Met100 m M group,Met10 m M group,and Met1 m M group were not statistically different compared with the Met0 m M group(p>0.05).Immunohistochemical results showed that the relative absorbance of type III collagen and fibronectin in the Met100 m M,Met10 m M,and Met1 m M groups were not statistically different compared with the Met0 m M group(p > 0.05).q RT-PCR results showed that the expression levels of IL1β and CCL2 m RNA were significantly lower in the Met100 m M group compared with the PBS10 m M group(p<0.05).Conclusion:In this experiment,an animal model of deep partial thickness burn in rats was successfully constructed,and the expression of type I collagen deposition,inflammatory cytokines(IL1β),and chemokines(CCL2)in deep partial thickness wounds in rats could be inhibited by subcutaneous injection of 100 m M metformin every other day.Part II.Effect of metformin on the biological behavior of RAW 264.7 cellsObjective:To investigate the effect of metformin on the proliferation of murine macrophage leukemia cells(RAW 264.7);to investigate whether metformin inhibits the expression of inflammatory factors and chemokines;and to investigate the effect of metformin on LPS-induced apoptosis in RAW 264.7 cells.Method:1.In vitro culture of RAW 264.7 cells and verification of the effect of metformin on the proliferation of RAW 264.7 cells using CCK8 cell proliferation assay;2.RAW 264.7 cells were intervened with different concentrations(2.5m M,5m M,10 m M)of metformin for 24 h,and 1μg/L LPS was added at the 18 th hour.The protein expression levels of p NF-κB p65 and NF-κB p65 were detected by Western blot,m RNA expression levels of IL1 β and CCL2 were detected by q RT-PCR,and ELISA was used to detect The expression levels of CCL2 in cell culture supernatant were detected by ELISA;further,10 n M QNZ(NF-κB inhibitor)and different concentrations(2.5m M,5m M,10 m M)of metformin were used to simultaneously intervene in RAW 264.7 cells for 24 h,1μg/L LPS was added at the18 th hour,and the protein expression levels of p NF-κ B p65,NF-κB p65 were detected by Western blot expression levels,and q RT-PCR for m RNA expression levels of IL1β and CCL2;3.RAW 264.7 cells were intervened with different concentrations(2.5m M,5m M,10 m M)of metformin for 24 h,and the proportion of RAW 264.7apoptotic cells was detected by flow cytometry;RAW 264.7 cells were intervened with different concentrations(2.5m M,5m M,10 m M)of metformin for 24 h,and stimulated by adding 10μg/L LPS at the 16 th hour,the percentage of apoptotic RAW 264.7 cells was detected by flow cytometry.Result:The results of CCK8 assay showed that:the proliferation ability of RAW 264.7cells intervened with different concentrations(0.5m M,1m M,5m M,10 m M,20 m M)of metformin for 24 h was significantly decreased and showed a dose-dependent effect,and the difference was statistically significant(p<0.05);Western blot results showed that,compared with the control group,the p NF-κB p65 protein expression levels in the Met2.5m M,Met5 m M and Met10 m M groups were significantly downregulated(p < 0.001)and showed a dose-dependent effect,and there was no significant difference in NF-κB protein expression levels,and q RT-PCR results showed that IL1β and CCL2 m RNA expression levels in the Met2.5m M,Met5 m M and Met10 m M groups also showed a significant decrease compared with the control group,and the difference was statistically significant(p < 0.05);ELISA results suggested that the CCL2 protein expression in the cell culture supernatant of the Met2.5m M,Met5 m M and Met10 m M groups compared with the control group;After adding NF-κB inhibitor(10n M QNZ)and metformin to RAW 264.7 cells,Western blot showed that the p NF-κB protein expression level was significantly lower in each group after QNZ intervention compared with the negative control group(p < 0.001)and the p NF-κB protein expression level was significantly lower in each group after QNZ intervention compared with the positive control group(p < 0.001).There were no significant differences in the expression levels of p NF-κB and NF-κB protein in the control group and no significant differences in the expression levels of NF-κB protein in each group,and q RT-PCR results showed that the expression levels of IL1βand CCL2 m RNA in each group after QNZ intervention was not significantly different compared with the positive control group(p > 0.05);The results of flow cytometry showed that there was no significant difference in the proportion ofapoptotic cells in each group after 24 h intervention of RAW 264.7 cells with different concentrations(2.5m M,5m M,10 m M)of metformin;Different concentrations(2.5m M,5m M,10 m M)of metformin intervened in RAW 264.7 cells for 24 h,and the proportion of apoptotic cells in the Met2.5m M group,Met5 m M group and Met10 m M group decreased significantly compared with the control group when 10μg/L LPS was added for stimulation at the 16 th hour,and the difference was statistically significant(p<0.001).Conclusion:Metformin inhibited the proliferation of RAW 264.7 cells,suppressed the expression of LPS-induced inflammatory factors(IL1 β)and chemokines(CCL2)probably through the NF-κB pathway,and inhibited apoptosis of RAW 264.7 cells induced by high concentrations of LPS.Part III.The effect of metformin on the biological behavior of NIH 3T3 and its mechanismObjective:To observe the effects of metformin on the proliferation,migration and Collagen I expression of NIH 3T3 through activation of AMPK,and to investigate the mechanism.Method:1.NIH 3T3 cells were cultured in vitro and the effect of metformin on the proliferation of NIH 3T3 cells was verified by CCK8 cell proliferation assay;the interference efficiency of AMPK in NIH 3T3 cells was verified by Western blot after the transfer of AMPK-si RNA in NIH 3T3 cell line;the effect of AMPK down-regulation on the proliferation of NIH 3T3 cells was investigated by Ed U cell proliferation assay;2.NIH 3T3 cells were intervened with different concentrations(2.5m M,5m M,10 m M)of metformin for 24 h,Transwell migration assay was performed to observe cell migration,Western blot to detect the expression of pm TOR,m TOR,p AKT,AKT,MMP2,MMP9;further in NIH 3T3 cell line was transfected with After AMPK-si RNA was transfected into NIH 3T3 cell line,Transwell migration assay was performed to observe cell migration and Western blot to detect the expression of pm TOR,m TOR,p AKT,AKT,MMP2,MMP9;lentivirus carrying AMPK interference sequence was constructed,and AMPK down-regulated stably transfected cell line was screened by 1/2 small volume infection method transfection and puromycin The expression of pm TOR,m TOR,p AKT,AKT,MMP2,MMP9 was detected by Western blot;3.NIH 3T3 cells were intervened with different concentrations(2.5m M,5m M,10 m M)of metformin for 24 h,and the expression of TGF-β1,p Smad3,Smad3 and Collagen I was detected by Western blot;in NIH 3T3 cell lines,after transferring AMPK-si RNA,the expression of TGF-β1,p Smad3,Smad3 and Collagen I was detected by Western blot.Lentiviral constructs of NIH 3T3 cell lines stably down-regulating AMPK expression,Western blot for TGF-β1,p Smad3,Smad3 and Collagen I expression.Result:The results of CCK8 experiment showed that the proliferation ability of NIH3T3 cells was significantly decreased by different concentrations(0.1m M,0.5m M,1m M,5m M,10 m M,20 m M)of metformin intervention for 24 h,and the difference was statistically significant(p<0.05);The results of Ed U assay showed that after the transfer of AMPK-si RNA into NIH 3T3 cell line,the proportion of Ed U-positive cells in si AMPK group was significantly higher than that in si NC group,and the difference was statistically significant(p < 0.05);The number of migrating cells decreased significantly and showed a dose-dependent difference with statistical significance(p<0.001)after 24 h intervention of metformin at different concentrations(2.5m M,5m M,10 m M)in NIH 3T3 cells compared with the control group;After 24 h intervention of metformin at different concentrations(2.5m M,5m M,10 m M)in NIH3T3 cells Western blot results showed that compared with the control group,the expression levels of pm TOR,p AKT,MMP2 and MMP9 proteins were significantly down-regulated in the Met2.5m M group,Met5 m M group and Met10 m M group(p <0.001)and showed a dose-dependent effect,while the expression levels of m TOR and AKT proteins were not significantly different;After the transfer of AMPK-si RNA in NIH 3T3 cell line,the results of Transwell migration assay showed that the number of migrating cells in NIH 3T3 cells was significantly increased after AMPK down-regulation compared with the si NC group,and the difference was statistically significant(p < 0.001);After the transfer of AMPK-si RNA in NIH 3T3 cell line,Western blot results showed that the expression levels of pm TOR,p AKT,MMP2 and MMP9 were significantly up-regulated compared with the si NC group(p<0.05),while the expression levels of m TOR and AKT were not significantly different;The lentiviral constructs stably down-regulated the expression of AMPK in NIH 3T3 cell line,and the western blot results showed that the expression levels of pm TOR,p AKT,MMP2 and MMP9 were significantly up-regulated compared with the sh NC group(p<0.05),while the expression levels of m TOR and AKT were not significantly different;After the intervention of metformin at different concentrations(2.5m M,5m M,10 m M)in NIH 3T3 cells for 24 h,the expression levels of TGF-β1,p Smad3,Collagen I protein in the Met2.5m M group,Met5 m M group and Met10 m M group decreased significantly(p<0.05)and showed a dose-dependent effect compared with the control group,while there was no significant difference in Smad3 protein expression levels;After the transfer of AMPK-si RNA into NIH 3T3 cell line,Western blot showed that the expression levels of TGF-β1,p Smad3 and Collagen I were significantly up-regulated compared with the si NC group(p<0.01),while the expression levels of Smad3 were not significantly different;The lentiviral constructs stably down-regulated the expression of AMPK in NIH 3T3 cell line,Western blot showed that the expression levels of TGF-β1,p Smad3 and Collagen I were significantly up-regulated compared with sh NC group(p<0.01),while the expression levels of Smad3 were not significantly different.conclusion:Metformin may inhibit the proliferation,migration and Collagen I synthesis of NIH 3T3 cells by activating AMPK,where the inhibition of NIH 3T3 cell migration and Collagen I synthesis may be related to the activation of AMPK by metformin andthus downregulation of m TOR and TGF-β1/Smad3 pathways,respectively.Part IV.Effect of metformin on TGF-β1 secretion from NIH 3T3 and RAW264.7 in the inflammatory microenvironmentObjective:To construct an in vitro cellular inflammatory microenvironment and investigate the effect of metformin on TGF-β1 secretion in RAW 264.7 and NIH 3T3 cells in a RAW 264.7/NIH 3T3 cell co-culture system.Method:ELISA was performed to detect the expression of TGF-β1 in RAW 264.7 cell culture supernatant under different concentrations(2.5m M,5m M,10 m M)of metformin intervention;NIH 3T3 cells were inoculated in the lower chamber of Transwell and RAW 264.7 cells were inoculated in the upper chamber of Transwell as the experimental group,and the upper and lower chambers were simultaneously added or not with 1μg/ ml LPS and Met(10 m M)were added simultaneously to the upper and lower chambers,and the two types of cells were co-cultured for 48 h(NIH3T3 and NIH 3T3 co-cultures were used as the control group).Each group was divided into 3 subgroups(LPS-Met-,LPS+Met-,LPS+Met+)according to the interventions of LPS(1 μg/ml)and Met(10 m M),for a total of 6 groups.Western blot was performed to detect the expression level of Collagen I in NIH 3T3 cells in the lower chamber of each group,and ELISA was performed to detect the expression level of TGF-β1 in the cell culture supernatant in the Transwell chamber of each group.Result:After pre-intervention of NIH 3T3 cells with different concentrations(2.5m M,5m M,10 m M)of metformin for 18 h,the ELISA results showed that the expression of TGF-β1 was significantly higher in the Met0 m M group compared with the control group(p < 0.05);Compared with the Met0 m M group,the TGF-β1 expression levels in the Met2.5m M,Met5 m M,and Met10 m M groups were significantlydown-regulated(p<0.001)and showed a dose-dependent effect,and the differences were statistically significant;Cell culture supernatants from each group in the co-culture system were extracted and the TGF-β1 levels were measured by ELISA,the results showed that the TGF-β1 expression in cell culture supernatants of the"LPS+Met-" group was significantly reduced compared with that of the "LPS-Met-"group(p < 0.001),while TGF-β1 expression in cell culture supernatants of the"LPS+Met+" group with the addition of metformin was significantly reduced compared with that of the "LPS+Met-" group(p < 0.001).LPS+Met-" group also showed a significant decrease compared to the " LPS+Met-" group(p<0.05);In the RAW 264.7/NIH 3T3 group,the expression of TGF-β1 in the supernatant of cell culture in the "LPS+Met-" group was significantly increased compared with the"LPS-Met-" group(p<0.001),while the expression of TGF-β1 in the supernatant of cell culture in the "LPS+Met+" group was significantly decreased compared with the"LPS+Met-" group by adding metformin(p<0.001);In the NIH 3T3/NIH 3T3 group,after 1 μg/m L LPS stimulation for 48 h,the expression of Collagen I in NIH 3T3 cells in the lower chamber of the "LPS+Met-" group was significantly reduced compared with that in the "LPS-Met-" group(p < 0.001),while the expression of Collagen I in NIH 3T3 cells in the lower chamber of the "LPS+Met+" group with the addition of metformin was also significantly reduced compared to the "LPS+Met-" group(p<0.05);In the RAW 264.7/NIH 3T3 group,after 1 μg/m L LPS stimulation for 48 h,the expression of Collagen I in NIH 3T3 cells in the lower chamber of the"LPS+Met-" group was significantly increased compared with that in the "LPS-Met-"group(p<0.001),while the expression of Collagen I in NIH 3T3 cells in the lower chamber of the "LPS+Met+" group was significantly decreased compared with that of the "LPS+Met-" group(p<0.001)and the difference was statistically significant.Conclusion:Metformin in the in vitro cellular inflammatory microenvironment may inhibit TGF-β 1 secretion from RAW 264.7 cells in the upper Transwell,which in turn inhibits Collagen I expression in NIH 3T3 cells in the lower Transwell. |
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