| ObjectiveTrauma is a global public health problem.According to the data of the World Health Organization,there are about 3～9 million new trauma patients in developed countries every year.The number of patients caused by trauma in China is as high as 62 million per year,and among them,the number of fatalities has reached 700,000 to 800,000.Today,trauma is the leading cause of death for people under the age of 45.If infection occurs after trauma,it can lead to sepsis,multiple organ dysfunction syndromes,and other infection complications,which can cause high morbidity and even death of trauma patients.Over the past few decades,the emergence of drug-resistant bacterial strains has caused a more complex problem of post-traumatic bacterial infection.E.coli strains that are resistant to multiple antibiotics are an important problem for post-traumatic infectionsThe concept of oxidative stress has been introduced for research in redox biology and medicine in 1985.Oxidative stress is a phenomenon caused by an imbalance between production and accumulation of oxygen reactive species(ROS)in cells and tissues and the ability of a biological system to detoxify these reactive products.There are many ways to produce oxidative stress,including hydrogen peroxide,arsenite,glutamate,6-hydroxydopamine and so on.The toxic effects of severe oxidative stress on the human body include facilitating aging,diabetes,cardiovascular and neuropathy,etc.However,studies have shown that low-dose oxidative stress can protect the cell.For example,hydrogen peroxide preconditioning PC 12 cells against apoptosis induced by oxidative stressMacrophages is the part of the innate immune system and are present in almost all tissues.Macrophages play a vital role in maintaining body homeostasis and also contribute to inflammatory and repair responses during pathogenic infections and tissue damage.In terms of inflammation,mouse peritoneal macrophages are one of the most studied macrophage populations.Peritoneal macrophages play an important role in the control infection,the pathological inflammation and the maintenance of immune function.Due to the protective effect of low-dose H2O2 on the body,in order to explore whether there is a correlation between the low-dose H2O2 and peritoneal macrophages,we built a protective model of mice with lethal wound infection,and selected the peritoneal macrophages as the research object.First of all,we studied whether the early low-dose hydrogen peroxide induction affects the survival and protecte the organs of mice.Secondly,we studied the effect of low-dose hydrogen peroxide induction on the peritoneal macrophage function,including its phagocytosis and chemotaxis.Finally,we built a model of wound infection protection,and studied the internal relationship among them,and this model can resist a variety of bacterial invasion.MethodsPart Ⅰ:The effect of low-dose hydrogen peroxide induction on survival and organ protection in mice after traumatic infection1.We divided the mice into groups,including untreat group,post-trauma infection group,and low-dose hydrogen peroxide-induced infection after trauma group(hereinafter referred to as induced infection group).First,the mice were causer blood loss to establish the model in the post-trauma infection group and the induced infection group.Secondly,we used low-dose hydrogen peroxide to induce mice in the induced infection group for 3 days(once every 24 h)by intraperitoneal injection(60 mg/kg,100 μl/mouse),and the mice in the untreat group and post-trauma infection group were simultaneously induced by intraperitoneal injection(100 μl/mouse)for 3 days(once every 24 hours)with sterilized water at the same time.After 24 hours,all mice were intraperitoneally injected with the same dose of E.coli(3 × 108 CFU/ml)At the same time,we will simultaneously inject intraperitoneal hydrogen peroxide(60 mg/kg,100 μl/mouse)into the untreat and post-trauma infection group.Finally,we will draw a 72 h mouse survival curve.(See Figure 1 for details)2.The mouse model is the same as above.We will extract the organs(heart,liver,lung,kidney)and eyeballs in the blank infection group and the trauma infection group after 4 hours of E.coli injection,and take blood from the eyeball,and 4 hours and 48 hours after injection of E.coli After hours,the organs(heart,liver,lung,kidney)and eyeballs of the trauma-infected group were taken for blood collection.Finally save separately3.We centrifuge each extracted blood to retain serum,and then test AST,Crea,CK,CK-MB4.We will take 2 batches of heart,liver,lung,and kidney for HE stainingPart Ⅱ:The effect of low-dose hydrogen peroxide induction on the chemotaxis of peritoneal macrophages in miceThe untreat group,the post-trauma infection group and the induced infection group were used to establish the model of post-traumatic infection.After the induction,half of the mice in each group were injected with E.coli.Four hours later,six groups of mice were divided into two groups.Macrophages were counted by cell counterPart Ⅲ:The effect of low-dose hydrogen peroxide induction on phagocytosis of peritoneal macrophages in mice1.We divided the mice into groups,including untreated group,post-trauma infection group and induced infection group.After the induction,the three groups of mice were not injected intraperitoneally with Escherichia coli,and the mice of each group were killed,and we extracted the peritoneal macrophages of the mice at the same time.Then,centrifuge to remove the supernatant and plate the cells to construct an in vitro E.coli infection model.One hour after infection,the same count was taken for each group of cells and plated on LB solid agar plates.After incubating the bacteria incubator at 37℃ for 12 h,single colony counting was performed2.We will establish an in vitro model of low-dose hydrogen peroxide-induced protection.First,we took peritoneal macrophages from normal mice,and the cell numbers were plated in 6-well plates(blank 12 h group and induced 12 h group,blank 24 h group and induced 24 h group,blank 36 h group and induced 36 h group)Hydrogen peroxide induction(10 μM/time)was performed once every 12 hours.Finally,Trizol is used to collect the cells in each well.Finally,we will use qRT-PCR to detect the expression levels of phagocytosis-related proteins(Aif1,Marco,and Fgr)in cells.ResultsPart I:The effect of low-dose hydrogen peroxide induction on survival and organ protection in mice after traumatic infection1.The results of the lethal mouse model after traumatic infection showed that the survival rate of mice infected with traumatic infection induced by low-dose hydrogen peroxide was significantly improved.2.The biochemical test results showed that the AST,Crea,CK,and CK-MB indexes of mice induced by low-dose hydrogen peroxide decreased.3.Pathological section HE staining results show that low-dose hydrogen peroxide induces a certain protective effect on mouse organs after traumatic infectionPart II:The effect of low-dose hydrogen peroxide induction on the chemotaxis of peritoneal macrophages in miceThe peritoneal macrophages in mice infected with low-dose hydrogen peroxide-induced wounds increased compared with the number of macrophages in the wound-infected group.Part III:The effect of low-dose hydrogen peroxide induction on phagocytosis of peritoneal macrophages in mice1.The peritoneal macrophages of mice infected with low-dose hydrogen peroxide-induced wounds increased the number of phagocytic bacteria compared with the wound-infected group.2.The expression of Aifl、Marco and For in mice peritoneal macrophages induced by low-dose hydrogen peroxide was higher than that in mice infected with trauma.Conclusions:1.The induction of low-dose hydrogen peroxide can increase the survival rate of infected mice by trauma.2.Low-dose hydrogen peroxide has a certain protective effect on the organs of mice after traumatic infection.3.The peritoneal macrophages induced by low-dose hydrogen peroxide-induced wound infection in mice promoted the expression of Aifl than the wound infection group and had stronger phagocytosis and chemotaxis ability. |
PDF Full Text Request