Jianzheng Zhang

Mitochondrial DNA induces inflammation and increases TLR9/NF-κB expression in lung tissue

Mitochondrial DNA (mtDNA) contains unmethylated CpG motifs that exhibit immune stimulatory capacities. The aim of this study was to investigate whether mtDNA activates the Toll-like receptor 9 (TLR9)/nuclear factor-κB (NF-κB) pathway, thereby contributing to post-traumatic systemic inflammatory response syndrome (SIRS) and lung injury in rats. The effects of mtDNA on macrophage culture were examined in order to elucidate the putative cellular mechanisms. Rats and macrophage cultures were treated with phosphate-buffered saline, nuclear DNA, or mtDNA for 2, 4, 8 and 24 h. Histological analysis of lung tissue was undertaken following hematoxylin and eosin staining, and cytokine levels were assessed by ELISA. NF-κB and IκB-α phosphorylation levels, as well as TLR9 protein expression were determined by western blot analysis; NF-κB, IκB-α and TLR9 mRNA levels were analyzed by RT-PCR. A greater degree of inflammation and lung injury was observed in response to mtDNA. In addition, mtDNA increased serum tumor necrosis factor-α, interleukin (IL)-6 and IL-10 levels in vivo and increased their secretion by cultured macrophages (p<0.05). In lung tissue, mtDNA increased NF-κB, IκB-α and TLR9 mRNA levels (p<0.05); it also increased phosphorylated NF-κB p65 and TLR9 protein levels in the macrophage cultures. Thus, mtDNA may be part of the danger-associated molecular patterns, contributing to the initiation of sterile SIRS through the activation of the TLR9/NF-κB pathway and the induction of pro-inflammatory cytokine production.

Plasma concentrations of pro- and anti-inflammatory cytokines and outcome prediction in elderly hip fracture patients

BACKGROUND Hip fractures, particularly intertrochanteric fractures, frequently occur in the elderly, and they are associated with a high incidence of complications and mortality. The development of markers is essential to allow for adjustments to treatment strategies in patients, as it remains unclear why some patients endure organ failure and others do not under seemingly similar clinical conditions. OBJECTIVE Our objective was to determine the kinetics of tumour necrosis factor (TNF)-a, interleukin (IL)-6 and IL-10 during the hospitalisation of patients and to examine the relationship of these parameters to outcome (mortality and complications) 6 months and 12 months postoperatively. METHODS AND SUBJECTS A total of 127 elderly patients, who underwent hip fracture surgery, were prospectively followed up for 12 months, and 60 healthy elderly volunteers were enrolled in the control group to examine the effects of trauma and surgery on the inflammatory response. The epidemiological characteristics, chronic medical conditions and type of operation and anaesthetic were recorded. Cognition was evaluated using the Mini-Mental State Examination, and TNF-a, IL-6 and IL-10 levels were assessed during admission and preoperatively (post-anaesthesia) as well as 1 h, 1 day, 3 days and 5 days postoperatively. During the follow-up period, serious complications and mortality within 1 year were evaluated. RESULTS Overall, 96 patients survived, and 31 died within the 6-month postoperative period; 43 patients died, and 84 survived when examining the 12-month postoperative period. There were significant within-subject effects of time on TNF-a, IL-6 and IL-10 (P<0.001, P<0.001 and P<0.001). The above three cytokines were all significantly increased in the hip fracture patients compared with the control group. There were also differences in the kinetic patterns of all three parameters when the patients who died were compared with those who survived during the 6-month and 12-month postoperative periods. Multiple logistic regression analysis showed that TNF-a at 1 day (odds ratio (OR)=1.020, P=0.045) and 3 days (OR=1.034, P=0.037) postoperatively and IL-6 at 1 day (OR=1.048, P=0.000) postoperatively were independent predictors of mortality at 6 months; IL-6 (OR=1.019, P=0.025) and IL-10 (OR=1.018, P=0.042) at 1 day postoperatively were independent predictors of mortality at 1 year. The analysis of the receiver operating characteristics curve (ROC) showed that only IL-6 or IL-10 had the highest values for the area under the curve for mortality at 6 months and 12 months. Of the 84 patients who survived, 23 patients had 32 complications. The most common complication was pneumonia infection (11/84, 13%). TNF-a, IL-6 and IL-10 kinetics were found to differ in patients with complications compared to those without complications and in patients with infections compared with patients without complications. Multiple logistic regression analysis showed that IL-6 (OR=1.081, P=0.000) at 1 day postoperatively was an independent outcome predictor. CONCLUSION In elderly hip fracture patients, cytokine concentrations (TNF-a, IL-6 and IL-10) represented independent outcome predictors for adverse postoperative outcomes (mortality and complications). The inflammatory response played an important role in postoperative organ dysfunction in elderly hip fracture patients, and further study is needed to define whether decreasing the inflammatory response through cytokine antibodies or damage control strategies would decrease mortality and complication following hip fracture.

Honokiol downregulates Kruppel-like factor 4 expression, attenuates inflammation, and reduces histopathology after spinal cord injury in rats.

Study Design. Randomized experimental study. Objective. To investigate the neuroprotective effect of honokiol (HNK) on rats subjected to traumatic spinal cord injury (SCI) and the molecular mechanisms. Summary of Background Data. Inflammation contributes to the secondary injury to the spinal cord. Honokiol has been used as a neuroprotective agent because of its strong antioxidant and anti-inflammatory properties. Kruppel-like factor 4 (Klf4) is a newly identified critical target for the anti-inflammatory effect of HNK. Whether HNK can inhibit inflammatory response in rat model of SCI through mediating the expression of Klf4 has yet to be elucidated. Methods. Eighty-four adult female Sprague-Dawley rats were randomly divided into 4 groups as sham, SCI, SCI + Vehicle (0.1% propylene glycol in saline, intraperitoneally), and SCI + HNK (20 mg/kg, intraperitoneally) groups. The influences of HNK on the proinflammatory cytokines, microglial activation, neutrophil infiltration, histological changes, and improvement in motor function were assessed. Results. In the SCI group, proinflammatory cytokines, microglial activation, neutrophil infiltration, and Klf4 expression levels were increased compared with the sham group (P < 0.001). HNK intervention downregulated the expression of Klf4, reduced the production of proinflammatory cytokines, inhibited microglial activation, and neutrophil infiltration (P < 0.05). Furthermore, HNK also reduced histopathology and improved functional outcome after traumatic SCI. Conclusion. HNK reduces secondary tissue damage and improves locomotor function recovery after SCI through suppressing inflammatory response, and can be used as a potential therapeutic agent for SCI. Level of Evidence: NA

Systemic Inflammatory Responses and Lung Injury following Hip Fracture Surgery Increases Susceptibility to Infection in Aged Rats

Pulmonary infections frequently occur following hip fracture surgery in aged patients. However, the underlying reasons are not fully understood. The present study investigates the systemic inflammatory response and pulmonary conditions following hip fracture surgery as a means of identifying risk factors for lung infections using an aged rodent model. Aged, male Sprague-Dawley rats (8 animals per group) underwent a sham procedure or hip fracture plus femoral intramedullary pinning. Animals were sacrificed 1, 3, and 7 days after the injury. Markers of systemic inflammation and pulmonary injury were analyzed. Both sham-operated and injured/surgical group animals underwent intratracheal inoculation with Pseudomonas aeruginosa 1, 3, and 7 days after surgery. P. aeruginosa counts in blood and bronchoalveolar lavage (BAL) fluid and survival rates were recorded. Serum TNF-α, IL-6, IL-1β, and IL-10 levels and markers of pulmonary injury were significantly increased at 1 and 3 days following hip fracture and surgery. Animals challenged with P. aeruginosa at 1 and 3 days after injury had a significantly decreased survival rate and more P. aeruginosa recovered from blood and BAL fluid. This study shows that hip fracture and surgery in aged rats induced a systemic inflammatory response and lung injury associated with increased susceptibility to infection during the acute phase after injury and surgery.

Significance of Serum mtDNA Concentration in Lung Injury Induced by Hip Fracture.

ABSTRACT Acute lung injury is the most serious and fatal complication of the elderly patients with hip fracture, but the mechanisms are unknown. Recent studies demonstrated the mitochondrial DNA (mtDNA) release was associated with lung injury after trauma. This study aimed to examine the differential release of mtDNA between younger and elderly rats suffering from hip fracture and to investigate the possible mechanism of mtDNA in the lung injury induced by hip fracture. In the first part of the study, we investigated the effects of hip fracture on the rats. The elderly and younger rats, respectively, received hip fracture operations. The degree of lung injury was evaluated, toll-like receptor 9 (TLR9) and nuclear factor kappa B (NF-&kgr;B) were determined using Western blot, and mtDNA were analyzed by fluorescent quantitative polymerase chain reaction. In the second part of the study, we investigated the effects of mtDNA on the rats. The elderly and younger rats directly received intravenous injections with mtDNA. After 24 h, the specimens were collected and detected as the first part. Hip fracture resulted in significant mtDNA release, TLR9 and NF-&kgr;B p65 expression, and lung injury in the rats. Meanwhile, the mtDNA injection could indirectly induce lung injury. Compared to the younger ones, the elderly rats suffered more serious lung injury after hip fracture and mtDNA injection. These results suggest that the lung injury induced by hip fracture may be involved with the mtDNA release and its TLR9/NF-&kgr;B pathway.

The Immediate Intramedullary Nailing Surgery Increased the Mitochondrial DNA Release That Aggravated Systemic Inflammatory Response and Lung Injury Induced by Elderly Hip Fracture

Conventional concept suggests that immediate surgery is the optimal choice for elderly hip fracture patients; however, few studies focus on the adverse effect of immediate surgery. This study aims to examine the adverse effect of immediate surgery, as well as to explore the meaning of mtDNA release after trauma. In the experiment, elderly rats, respectively, received hip fracture operations or hip fracture plus intramedullary nail surgery. After fracture operations, the serum mtDNA levels as well as the related indicators of systemic inflammatory response and lung injury significantly increased in the rats. After immediate surgery, the above variables were further increased. The serum mtDNA levels were significantly related with the serum cytokine (TNF-α and IL-10) levels and pulmonary histological score. In order to identify the meaning of mtDNA release following hip fracture, the elderly rats received injections with mtDNA. After treatment, the related indicators of systemic inflammatory response and lung injury significantly increased in the rats. These results demonstrated that the immediate surgery increased the mtDNA release that could aggravate systemic inflammatory response and lung injury induced by elderly hip fracture; serum mtDNA might serve as a potential biomarker of systemic inflammatory response and lung injury following elderly hip fracture.

Patterns of cytokine release and evolution of remote organs from proximal femur fracture in COPD rats

BACKGROUND Chronic obstructive pulmonary disease (COPD) is at increased risk for developing osteoporosis (OP) with subsequent proximal femur fracture. The presence of COPD is suggested to be a strong risk factor for proximal femur fracture or hip fracture. However, what happen behind it is not clearly understood. OBJECTIVE To investigate the pattern of cytokine (TNF-a, IL-6, and IL-10) releases in pulmonary and hepatic in rats with COPD suffering from proximal femur fracture, and its possible adverse effect on pulmonary and hepatic. METHODS AND SUBJECTIVE This paper has two parts. In the first part, we describe the procedure of COPD model in detail. In the second part, we study the influences of fracture on the COPD rats. 5 months WISTAR rats with 37 weeks cigarette smoking exposure (CS group) were dynamically determined for pulmonary function, inflammatory response in bronchoalveolar lavage fluid (BALF), histological changes in pulmonary in the first part. When the COPD model is proved to be successful, we begin the second part. COPD rats were euthanized at 2, 24, 48, 72, and 96h after proximal femur fracture (fracture group) or anaesthesia (control group). Cytokines (TNF-a, IL-6, and IL-10) and myeloperoxidase activity of pulmonary and hepatic (MPO) were measured with enzyme-liked immunosorbent assay technique. Permeability changes of the lung were assessed via bronchoalveolar lavage, and those of the liver via assessment of oedema formation. Tissues were further examined microscopically. RESULTS The current sidestream cigarette smoke induced rat COPD model has been proved an adequate animal model with several advantages as assessed by dynamically monitored lung mechanics and pathological changes for 37 weeks. In the second part, TNF-a, IL-6, and IL-10 levels of pulmonary tissue were significantly increased after proximal femur fracture compared to control rats. TNF-a, and IL-6 levels in pulmonary peaked at 2h, 24h in fracture group, whereas IL-10 level peaked at 24h and 96h. Pulmonary myeloperoxidase activity, permeability and histological score in fracture group were remarkably elevated, and peaked at 24h. In addition to TNF-a, all above parameters did not return to normal through our study. Hepatic in COPD rats showed notable increase of cytokines (TNF-a, IL-6, and IL-10), myeloperoxidase activity, histological score, and permeability in fracture group compared to control rats, and severity of these changes were much lower than in pulmonary. Apart from TNF-a, the peak of these parameters was at 24h after fracture. Changes of cytokines, MPO activity, permeability and histological score in pulmonary and hepatic in control rat were little changed. CONCLUSION COPD rats produced a remarkably increase of inflammatory response (TNF-a, IL-6, IL-10) in lung (liver) after proximal femur fracture, which lead to lung (liver) injury, as evidence by changes of MPO, permeability, and histological scores in local organs.

Less wound complications of a sinus tarsi approach compared to an extended lateral approach for the treatment of displaced intraarticular calcaneal fracture: A randomized clinical trial in 64 patients.

Background:We conducted a prospective randomized clinical trial to compare the clinical and radiological outcomes of the sinus tarsi and extended lateral approaches for the surgical treatment of displaced intraarticular calcaneal fractures. Methods:Between January 2009 and January 2014, patients with displaced intraarticular calcaneal fracture were randomly assigned to receive surgical treatment by the sinus tarsi approach or the extended lateral approach using block randomization. We recorded and analyzed data on demographics, time to surgery, wound complications, Böhler angles pre- and postoperatively, and American Orthopedic Foot & Ankle Society score. Results:Sixty-four patients met the inclusion criteria and were randomly assigned to the 2 groups: 32 patients underwent sinus tarsi approach, and 32 patients the extended lateral approach. Baseline characteristics of both groups were similar. The time to surgery in the sinus tarsi approach group was significantly shorter than in the extended lateral approach group (P = 0.04). The wound complication rates were 6.3% and 31.2% in the sinus tarsi approach and extended lateral approach groups, respectively, which was significantly different (P = 0.01). Regarding the clinical outcomes, the groups did not differ significantly on walking visual analogue scale or American Orthopedic Foot & Ankle Society scores at 6 months and 1 year postoperatively. No significant differences existed between groups regarding the Böhler angle at different times and reduction quality of the articular surface and the medial wall. Conclusion:Compared with the extended lateral approach, the sinus tarsi approach decreased wound complications and preoperative waiting time, and achieved similar functional and radiological outcomes for displaced intraarticular calcaneal fractures.

The imbalance between regulatory and IL-17-secreting CD4+T cells in multiple-trauma rat

BACKGROUND It has been well recognised that a deficit of numbers and function of CD4(+)CD25(+)Foxp3(+)cells (Treg) is attributed to the development of auto-immune diseases, inflammatory diseases, tumour and rejection of transplanted tissue; however, there are controversial data regarding the suppressive effect of Treg cells on the T-cell response in auto-immune diseases. Additionally, interleukin-17 (IL-17)-producing cells (Th17) have a pro-inflammatory role. The balance between Th17 and Treg may be essential for maintaining immune homeostasis and has long been thought as one of the important factors in the development/prevention of auto-immune diseases, inflammatory diseases, tumour and rejection of transplanted tissue, but their role in multiple trauma remains unclear. OBJECTIVE This study aims to investigate whether an imbalance of Treg and Th17 effector cells is characteristic of rats suffering from multiple trauma. METHODS AND SUBJECTIVE Sixty Sprague-Dawley (SD) rats were randomly divided into three groups. The control group (n=20, group I) no received procedures (normal). The sham group (n=20, group II) only received anaesthesia, cannulation and observation. The bilateral femoral shaft fractures with haemorrhagic shock groups (n=20, group III). Rats in groups II and III were killed at the end of 4h after models were established. Peripheral blood samples were collected for assessment of Treg cells, Th17 cells and cytokines (IL-17, IL-6, IL-2, transforming growth factor beta (TGF-β)) and intestine tissue was collected for intestine histological analysis. RESULTS We observed decreased Treg/Th17 ratios in CD4(+)T cells in rats with multiple trauma and a strong inverse correlation with disease activity (intestinal histological scores). CONCLUSION We suggest a role for immune imbalance in the pathogenesis and development of multiple trauma. The alteration of the index of Treg/Th17 cells likely indicates the therapeutic response and progress in the clinic.

Impact of Cement Placement and Leakage in Osteoporotic Vertebral Compression Fractures Followed by Percutaneous Vertebroplasty.

Study Design:Retrospective study. Objectives:Assessment of the impact of cement placement and leakage in osteoporotic vertebral compression fractures (OVCFs) followed by percutaneous vertebroplasty (PVP) on patient pain relief and new vertebral fracture occurrence. Summary of Background Data:Previous studies have not specifically addressed cement placement in the context of pain outcomes and subsequent vertebral fracture. Methods:We included a total of 192 patients who underwent PVP for OVCFs. We assessed imaging data, and patients rated their pain over a 24-month period. The patients were divided into 3 groups based on image analysis: group 1 [31 cases: 5 thoracic, 15 thoracolumbar (TL) junction, 11 lumbar] included patients with no cement extension to the endplate(s), group 2 (121 cases: 19 thoracic, 64 TL junction, 38 lumbar) was comprised of patients with cement extension to the endplate(s) but no leakage into the disk space, and group 3 (40 cases: 8 thoracic, 21 TL junction, 11 lumbar) included patients with cement extension to the endplate(s) and leakage into the disk space(s). We assessed the correlation between cement location and pain ratings and changes in pain scores, as well as the proportions of new fracture. Results:Postprocedure pain numeric scores and changes in pain scores were similar among the 3 groups (P>0.05). Cement location did not significantly correlate with pain ratings or changes in pain scores for any follow-up points. There was no significant difference in new adjacent fracture rate among the groups (P>0.05). Conclusions:Neither extension of cement to the endplate nor cement leakage into the disk space had a significant impact on postprocedural pain. Furthermore, intradisk cement leakage was not a risk factor for new fracture after PVP in patients with OVCF. However, lower fill volumes should be used to lessen the risk of leakage.