The Role And Mechanism Of Mitochondrial DNA In Hip Fracture-induced Inflammatory Reaction

IntroductionHip fracture is the proximal femur fractures within the area of 5 cm from the femoral head to femur lesser trochanter that include femoral neck fracture, intertrochanteric fractures and subtrochanteric fractures. In which, femoral neck fracture and intertrochanteric fractures are the most common and have occupied more than 90% of hip fracture in clinically. The incidence of hip fracture gradually rise with age, the elderly have been the most widely affected groups because of its special physical and related basic diseases.Current clinical research data showed that 87%-96% of hip fracture patients aged 60 or older, elderly hip fracture have very high morbidity (27%-36%) and mortality (2.7%-5.0%). How to obtain an ideal clinical outcome for the elderly patients with hip fracture, it has been the focus of clinical workers.As the modern inflammation doctrine show:trauma can trigger inflammation, but not all of the trauma can lead to uncontrolled systemic inflammatory response. Because when the trauma trigger the release of inflammatory mediators, the wound tissue and the body system will start and secretion of anti-inflammatory substances that control the inflammation induced by trauma. Only when the body suffer trauma exceeds its tolerance, the balance of pro-inflammatory/anti-inflammation is broken, it could induce systemic inflammatory response and multiple organ failure. Elderly patients easily induce traumatic inflammatory response as the body’s physiological function reserves decline. Even a mild trauma may induce severe self-destructive inflammatory mediators released, and anti-inflammatory medium can not restrain the development of inflammation. Recent researches showed that elderly hip fracture patients with high morbidity and mortality is associated with post-traumatic stress response. Therefore, it is of great significance for reducing the incidence of complications and mortality of elderly hip fracture to explore the molecular mechanism of inflammatory response induced by elderly hip fracture and look for new possible intervention targets.Endosymbiont content insist:eukaryotic progenitor cells swallow oxygen bacterial symbiosis that symbiosis and evolving into eukaryotic cell. After bacteria is engulfed in eukaryotes, they mutually adapt and gradually evolve into mitochondria in the process of long-term coexistence. Bacterial evolution confirmed mitochondria has great similarity with the bacteria in morphology, staining reaction, chemical composition, physical properties, active state and genetic system. Therefore, mitochondrial components that releasing from the cells of traumatic tissue burst activate innate immune response through the mitochondrial damage-associated molecular patterns (mitochondrial DAMPs, MTDs), the inflammation promoted by MTDs is similar to that of sepsis. MTDs including formyl peptide and mitochondrial DNA. Mitochondrial DNA can activate TLR9 (Toll-Like Receptor) in cells, trigger multiple inflammatory signaling pathways, cause systemic inflammatory reaction and the remote organ damage (lung injury is the most common).A large number of clinical studies have shown that pulmonary infection is one of the most common complications of elderly hip fracture, and multiple organ failure induced by lung infection is the most common cause of death. The trauma of hip fracture and surgery can cause the mitochondrial DNA released from the damaged tissue, promote inflammation, result in the abnormal immune function and the remote organs damage(lung injury is the most common). it closely relate to the occurrence of complications (such as lung infection, sepsis and multiple organ failure) after fracture. The immune compensatory ability of elderly is abate due to aging. Under hip fracture trauma and surgical strike, the elderly is more likely to suffer systemic inflammatory response and the remote organ damage induced by immune dysfunction. The outcome are often more serious, more prone to infectious complications and death. Presently, the study about the release model of mitochondrial DNA after hip fracture and mechanism of its related inflammatory reaction is less, there are also lack of effective interventions. At the same time, the related research of inflammation induced elderly hip fracture is still relatively rare, it does not accord with global population aging characteristics. Therefore, it is of great significance to explore the release patterns of mitochondrial DNA after hip fracture and molecular mechanism of its related inflammatory response for looking for new possible intervention targets to reduce the incidence of complications and mortality after hip fracture.The first chapter:The experimental research of mtDNA released and TLR9/NF-kappa B pathway activation after hip fractureObjective(1) To observe the release patterns of mtDNA after hip fracture and compare differences between the elderly hip fracture model and the younger in mtDNA release pattern.(2) To explore the correlation between mtDNA released and inflammation after hip fracture, and preliminary study on the correlation between mtDNA released and TLR9/NF-kappa B pathway activation after hip fracture.Materials and Methods(1) Forty elderly rats and forty younger rats were randomly divided into two groups respectively (sham group and fracture group). Two sham groups only received anesthesia, cannulation and observation. Two fracture groups received hip fracture operations. The rats were respectively sacrificed at 8,24,48, and 72 h after hip fracture or anesthesia. Blood samples, bronchoalveolar lavage fluid (BALF) and lung tissues were collected for further examination.(2) The degree of lung injury was evaluated by lung tissue pathological histological grading, bronchoalveolar lavage fluid (BALF) protein content determination, and examination of myeloperoxtidase(MPO) activity and neutrophil elastase (NE) concentration. The concentrations of IL-6 and IL-10 in serum and BALF were determined by enzyme-linked immunosorbent assay (ELISA). Toll-like receptor 9 (TLR9) and nuclear factor kappa B (NF-kB) were determined using Western blot, and mtDNA were analyzed by real time fluorescent quantitative polymerase chain reaction (Rt-PCR).(3) SPSS 20.0 analysis statistical software was used for date analysis. Data was expressed as mea±SD (x± s), One-way analysis of variance (one-way ANOVA) was used to compare the overall mean when the variance was Homogeneity, and Bonferroni method was used for Multiple comparisons among the groups; When the variance was not homogeneity, Welch method was used to compare the overall mean, Dunnett’s T3 was used for multiple comparisons among the groups. Quantitative data were compared between two groups using the Independent-Samples T test. Significance was accepted when P<0.05.Results(1) The serum concentration of mtDNA, IL-6 and IL-10 within two fracture groups were significantly higher than the respectively sham group at 8,24,48 and 72 h after hip fracture (t= 9.016, P= 0.001 and t= 36.366, P= 0.000). These variables of the elderly fracture group were significantly higher than the younger fracture group at these time points (except P of IL-6 at 8h(0.242)and 48h(0.055), all P<0.05).(2) The protein content as well as the concentration of IL-6 and IL-10 in the BALF of two fracture groups were significantly higher than the respectively sham group at 8,24,48 and 72 h after hip fracture (PI:elderly fracture group vs elderly sham group; P2:younger fracture group vs younger sham group,except P2=0.340 of IL-10 at 72h, all P< 0.05).These variables of the elderly fracture group were significantly higher than the younger fracture group at these time points (all P<0.05).(3) Lung tissue pathological histological grading, MPO activity, NE concentration, and protein content of TLR9 and NF-kB of two fracture groups were significantly higher than the respectively sham group at 8,24,48 and 72 h after hip fracture(all P<0.05). These variables of the elderly fracture group were significantly higher than the younger fracture group at these time points (except P=0.254 of MPO at8h, all P< 0.05).(4) Compared with serum IL-6 (B=-0.003, P=0.178) and IL-10 (B=0.025, P=0.000), mtDNA (B=0.965, P=0.000) has higher correlation with lung tissue pathological histological grading.Conclusion(1) Hip fracture can cause the release of mtDNA, systemic inflammation, and lung injury.(2) Elderly hip fracture rats suffered more severe systemic inflammation and lung injury than the younger under the same hip fracture strike.(3) The release of mtDNA and the TLR9/NF-kB pathway activation after hip fracture may be the cause of post-traumatic systemic inflammation and lung injury.The second chapter:experimental research of mtDNA trigger systemic inflammation and acute lung injury via TLR9/NF-kB pathway activationObjective(1) To investigate the correlation between mtDNA and systemic inflammation as well as acute lung injury.(2) Preliminary investigate the role of mtDNA release and TLR9/NF-kB pathway in the pathological process of systemic inflammation and acute lung injury.Materials and Methods(1) 20 elderly rats and 20 younger rats were randomly divided into two groups respectively (sham group and mtDNA group). Two sham groups only received anesthesia, cannulation and observation. Two mtDNA groups received mtDNA injection. The rats were sacrificed at 24h after mtDNA injection or anesthesia. Blood samples, bronchoalveolar lavage fluid (BALF) and lung tissues were collected for further examination.(2) The degree of lung injury was evaluated by lung tissue pathological histological grading, bronchoalveolar lavage fluid (BALF) protein content determination, and examination of myeloperoxtidase (MPO) activity and neutrophil elastase (NE) concentration. The concentrations of IL-6 and IL-10 in serum and BALF were determined by enzyme-linked immunosorbent assay (ELISA). Toll-like receptor 9 (TLR9) and nuclear factor kappa B (NF-kB) were determined using Western blot, and mtDNA were analyzed by real time fluorescent quantitative polymerase chain reaction (Rt-PCR).(3) SPSS20.0 analysis statistical software was used for date analysis. Data was expressed as rnean±SD (x±s), One-way analysis of variance (one-way ANOVA) was used to compare the overall mean when the variance was Homogeneity, and Bonferroni method was used for Multiple comparisons among the groups; When the variance was not homogeneity, Welch method was used to compare the overall mean, Dunnett’s T3 was used for multiple comparisons among the groups.Quantitative data were compared between two groups using the T test. Significance was accepted when P<0.05.Results(1) The serum concentration of IL-6 and IL-10 within two mtDNA groups were significantly higher than the respectively sham group at 24 h after mtDNA injection (all P<0.05).(2) The protein content as well as the concentration of IL-6 and IL-10 in the BALF of two fracture groups were significantly higher than the respectively sham group at 24h after mtDNA injection (all P<0.05).(3) Lung tissue pathological histological grading, MPO activity, NE concentration, and protein content of TLR9 and NF-kB of two fracture groups were significantly higher than the respectively sham group at 24h mtDNA injection(all P< 0.05).ConclusionMtDNA can cause the systemic inflammation and lung injury, it may be related to the TLR9/NF-κB pathway activation.The third chapter:The effect of mtDNA in bronchial epithelial cells physiological functionObjectiveTo study the effect of mtDNA in bronchial epithelial cells physiological functionMaterials and MethodsThe bronchial epithelial cell of SD rat was cultured. The cell was divided into sham group and mtDNA group after the cell culture built. The cell of mtDNA group was cultured in medium with 10μg/ml mtDNA for 24h. Trans Epithellal Electric Resistance (TEER) were used to detect the change of cell permeability, the concentrations of IL-6 and IL-10 were determined by enzyme-linked immunosorbent assay (ELISA), the expression level of TLR-9, NF-κB P65, bronchial epithelial cell adhesion connection protein (E-cadherin, beta-catenin) and tight junction protein (Occludin, ZO-1) were detected by Western Blot, the distribution of bronchial epithelial cells connected protein (E-cadherin, beta-catenin, Occludin and ZO-1) were detected by immunity fluorescence. SPSS20.0 statistical software is used to analyze the results.ResultsAfter the bronchial epithelial cells were cultured in medium with mtDNA for 24h, the TEER significantly reduce(t= 9.016, P= 9.016), the FITC-dextran permeability (Pa/Pc) increased significantly (t= 4.022, P= 4.022), the expresssion of IL-6, IL-10, NF-κB P65 and TLR-9 increased, the expression level of adhesion junction protein(E-cadherin) and tight junction protein (ZO-1) reduced, the immune fluorescent displayed mtDNA could destroy the continuity and integrity of junction protein (E-cadherin, beta-catenin, Occludin and ZO-1) in the cell membrane.ConclusionMtDNA can destroy the bronchial epithelial barrier function by changing the distribution and expression of junction protein. It is related to the TLR9/NF-kB pathway activation and the releasing of IL-6 and IL-10.