Mechanism And Application Of IL-6 And Its Inhibitors In Urosepsis By Regulating JAK/STAT3 Pathway

BackgroundUrosepsis is a common and serious complication after percutaneous nephrolithotomy(PCNL).It onsets insidiously with rapid progression,poor prognosis and high mortality.Due to the characteristics of urolithiasis itself and the particularity of lithotripsy,it's hard to detect until it appears and burst out.Once it occurs,it progresses rapidly,and can progress to septic shock in a few hours or even endanger the patients' life.Studies have shown that the mortality of patients will increase by 7.6%for every one hour of delay in the diagnosis and intervention of the disease.According to statistics,the incidence of urosepsis after percutaneous nephrolithotomy is 0.3-4.7%,with a mortality rate of up to 30-40%,and the more severe the urosepsis is,the higher the mortality rate is.Although the current clinical front-line doctors have paid enough attention to the disease,urosepsis still severely restricts the development of percutaneous nephrolithotomy,and threatens doctors and patients.In view of this,early diagnosis and effective intervention are the keys to reduce the severity of urosepsis,improve the prognosis,and reduce postoperative mortality.This requires us to predict and diagnose the occurrence of urosepsis as early as possible,and actively explore precisely targeted and powerful intervention measures to reduce inflammatory reactions,control disease progression,improve prognosis and reduce mortality rate.Research and clinical experience tell us that urosepsis often occurs within 6 hours after percutaneous nephrolithotomy,and this requires us to explore and discover inflammatory markers with significantly early changes after surgery to get early warning and help the diagnosis of urosepsis.Once urosepsis occurs,a series of cascading systemic inflammatory reactions affect the body,destroying tissue and cell,affecting the body's metabolism,causing organ failure and even death.The treatments of the disease currently focus on infection control,adequate fluid resuscitation,vasoactive drugs and protect vital organs and so on,we can see that the above treatments focus on the result and damage of urosepsis,and comparing with the rapid progress of urosepsis there is a large lag in the treatment of the disease.Therefore,it is necessary to look for early warning and diagnosis of inflammatory markers in urosepsis.On the basis of sepsis,focusing on the upstream of the entire inflammatory response mediator network,we need to explore and find targeted and powerful interventions to inhibit the excessive inflammatory response and reduce its damage to the body,which may become a new direction and therapeutic method for intervention and treatment of urosepsis.Therefore,on the basis of searching for inflammatory markers with early predictive and diagnostic ability of urosepsis,focusing on the upstream of the entire inflammatory response mediator network,exploring and searching for targeted and powerful interventions to suppress excessive inflammatory reaction to reduce its damage to the body is a problem that we need to think and study,and it may also become a new direction for intervention and treatment of urosepsis.Interleukin-6(IL-6)is a pleiotropic chemokine secreted by T lymphocytes,B lymphocytes,fibroblasts,monocyte macrophages and endothelial cells.It widely participates in a variety of physiological reactions.When pathogenic microorganisms invade body,pathogenic microorganisms and their endotoxin activate the human immune system and trigger a series of cascade reactions.IL-6 is located upstream of the whole cascade reaction and in the center of the whole "inflammatory storm".Under normal circumstances,the content in human body is very low,about 1?5pg/ml.Once infection occurs,IL-6 responses abnormally rapid and the level rises rapidly,There will be a very significant increase within 1 hour after infection,and the peak value can be reached within 2 hours after infection,especially in patients with sepsis.The half-life in plasma is only 6h.Based on the physiological and metabolic characteristics of IL-6,we regarded 2 hours after percutaneous nephrolithotomy as the test time node of IL-6.Theoretically,it can meet the needs of very early predicting and diagnosis of urosepsis after percutaneous nephrolithotomy.After infection,IL-6 binds to interleukin-6 receptor(IL-6R)and then recruits glycoprotein 130(gp130)to form IL-6/IL-6R/gp130 hexameric complex,which activates tyrosine kinase(JAK)/signal transducer and activator of transcription(STAT)signal pathway.JAK is phosphorylated and activated by forming p-JAK,and then catalyzes the phosphorylation of STAT3 to form p-STAT3.The activated p-STAT3 enters the nucleus and binds with corresponding receptors to stimulate the transcription and expression of targeted response genes,and plays a role in inflammation,cell proliferation and differentiation,angiogenesis,immune regulation and tumor metastasis.We are committed to finding an IL-6 inhibitor to block JAK/STAT3 signal pathway,inhibit the activation of JAK and STAT3,and then inhibit the transcription and expression of corresponding targeted response genes caused by p-STAT3,which is expected to achieve the purpose of inhibiting excessive inflammatory response,reducing damage to body and improving prognosis.In this study,the expression of IL-6 and its activation of JAK/STAT3 signal pathway,as well as the inhibitory effect of IL-6 inhibitors on JAK/STAT3 signal pathway were studied through the cell experiment of E.coli stimulating monocytes and the animal experiment of establishing rat urosepsis model,then we explored the early predicting and diagnostic value of IL-6 on urosepsis and the inhibitory effect of IL-6 inhibitors on excessive inflammatory response.Combined with clinical practice,we verified the role and clinical application value of IL-6 on early predicting and diagnosis of urosepsis,and guided the early predicting,diagnosis and treatment of urosepsis.In addition,a new invention of colloidal gold test strip for rapid detection of IL-6,has been developed in this study,and the production of the strip,performance test and preliminary clinical verification of the production have been completed.Through this study,we strived to provide valuable theoretical basis,experimental basis and data support for predictive and diagnostic ability of IL-6 for postoperative urosepsis and its receptor inhibitors in the treatment of urosepsis.Objective1.To study the expression of IL-6 and its activation of JAK/STAT3 signal pathway,and the inhibitory effect of IL-6 inhibitors on the JAK/STAT3 signal pathway by the cell experiment of E.coli stimulating monocytes.2.To establish an animal model of urosepsis by injecting Escherichia coli into the renal pelvis of rats,and explore the early diagnostic value of IL-6 for urosepsis and the inhibitory effect of IL-6 inhibitors on excessive inflammatory response in urosepsis.3.To verify and confirm the value of IL-6 in the early predicting and diagnosis of urosepsis after percutaneous nephrolithotomy through clinical practice.4.To explore the clinical value of IL-6 as an early inflammatory marker for early predicting and diagnosis through clinical practice in the treatment of urosepsis after percutaneous nephrolithotomy.5.To provide a more convenient and rapid method for the diagnosis and treatment of urosepsis by the application of the colloidal gold test strip for rapid detection of IL-6 which was based on colloidal gold immunochromatography technology.Methods1.Expression of IL-6 in monocytes stimulated by Escherichia coli and the effect of its inhibitor on JAK/STAT3 signaling pathway1.1 Isolation and culture of rat peripheral blood mononuclear cellsFresh anticoagulant whole blood of rats was diluted with equal volume PBS,treated with rat peripheral blood monocyte isolation solution kit and centrifuged,absorbed white monocyte layer for treatment,monocyte culture medium for adherent culture,and subcultured in a cell culture box with 37? and 5%CO2 saturation humidity.1.2 Detection of IL-6,p-JAK,STAT3 and p-STAT315 EP tubes were and each 3 EP tubes were assigned as a group,added with 1ml of mononuclear ceIl suspension with a concentration of 1×106/mL.In the experimental group D,E.coli was added at the ratio of 1:10 of E.coli to monocytes.In the experimental group B,E.coli was added to E.coli at the ratio of 1:10 of E.coli to monocytes,and 0.5?L IL-6 receptor monoclonal antibody tocilizumab was added.In the experimental group C,E.coli was added at the ratio of 1:10 of E.coli to monocytes monocytes,and 80uM JAK antagonist AG490 was added.In the experimental group E,E.coli was added at the ratio of 1:10 of E.coli to monocytes,and 0.5?L IL-6 receptor monoclonal antibody tocilizumab,80uM JAK antagonist AG490 and an equal volume of DMEM containing 10%fetal bovine serum were added.Control group A was added with an equal volume of DMEM containing 10%fetal bovine serum.It was centrifuged after 6 hours for reaction,and the supernatant and cell components were collected.(1)Detection of IL-6The cell supernatant of each group was collected,and IL-6 was detected by ELISA.(2)Detection of STAT3 and p-STAT3Cell samples were collected,treated with trypsin solution and cell lysate,centrifuged to collect the supernatant,and the STAT3 and p-STAT3 were detected by Western Blotting.2.Research of IL-6 on the early predicing and diagnosis of urosepsis and antiinflammatory effects of its inhibitors on urosepsis2.1 Establishment of animal modelsIn this study,animal models of urinary infection in rats was established by injecting E.coli into the renal pelvis.20 SD rats were randomly divided into four groups.The rats of group A were injected with normal saline into the renal pelvis,and the rats of group B were injected into the large intestine.Bacillus(concentration 2.0×108cfu/ml),E.coli(concentration 12.0×108cfu/ml)was injected into the renal pelvis of rats in group C,E.coli(concentration 12.0×108cfu/ml)was injected into the renal pelvis of rats in group D and IL was injected into the abdominal cavity-6 receptor monoclonal antibody tocilizumab.2.2 24-hour survival rate of rats To observe and record the 24-hour survival rate of each of the four groups of rats.2.3 Monitoring vital signs(1)Arterial pressure:To puncture the left femoral artery and connect the arterial pressure transducer to monitor the arterial pressure in real time.(2)Body temperature:The body temperature of the rats was monitored through the anus before the operation,2h after the operation,4h after the operation,and 6h after the operation.2.4 Detection of blood inflammatory markers(1)Detection of white blood cell countBlood was collected from the punctured femoral artery before the operation,2 hours after the operation,and 6 hours after the operation.After the blood collection,the same volume of normal saline was refilled.The white blood cell count was detected by the automatic blood cell counter detects.(2)Detection of IL-6 and PCTBlood was collected from the punctured femoral artery before operation,2 hours after operation,and 6 hours after operation.After blood sampling,the same volume of normal saline was refilled,and IL-6 and PCT were detected by ELISA.2.5 Detection of the expression of IL-6,STAT3 and p-STAT3 in liverAfter the rats were killed 24h after the operation,the liver tissues were taken and rinsed with PBS solution 3 times.The liver tissues were ground with the assistance of liquid nitrogen.The RIPA lysate was lysed and centrifuged.The supernatant was collected and the level of IL-6 expression in the liver tissues was detected by ELISA.,Western Blotting was used to detect the expression of STAT3 and p-STAT3 in liver tissue.2.6 Assessment of the section of rat lung tissueAfter the rats were killed 24 hours after the operation,the middle lobe of lung were taken to prepare paraffin sections for HE staining to assess the degree of lung inflammation.3.Effects of IL-6 for early predicting and diagnosis of urosepsis after percutaneous nephrolithotomyThe clinical data of 90 patients in our center from April 2019 to September 2019 were enrolled.Among them,16 patients with postoperative urosepsis were included in experimental group 1(EXP1 group,16),74 patients without postoperative urosepsis were included in the control group(CON group,74).All patients performed a standardized surgical route(percutaneous nephrolithotomy under ultrasound guidance)and standardized anti-infective treatment.Collect and sort out the data characteristics of the enrolled patients,such as gender,age,diabetes,hypertension,hydronephrosis,stone burden,urine culture,pyuria,operation time and postoperative hospital stay,,2h after operation,and after operation.The leukocytes,neutrophil percentages,PCT and IL-6 of the patients were detected before operation and at 2 hours,1 day and 3 days after the operation,and statistical analysis was performed.4.Research on the clinical value of application of IL-6 in urosepsis after percutaneous nephrolithotomyAccording to the inclusion and exclusion criteria,the clinical data of a total of 16 patients with urosepsis after percutaneous nephrolithotomy from April 2019 to September 2019 were collected and included in experimental group 1(EXP1 group,16),Routine laboratory tests for IL-6 were taken in all patients during the perioperative period;clinical data of 25 patients with urosepsis after percutaneous nephrolithotomy from March 2018 to March 2019 were collected and included in the experimental group 2(EXP2 group,25),all patients did not detect IL-6 during the perioperative period.5.Research and development of the colloidal gold test strip for rapid detection of IL-6Colloidal gold immunochromatographic technology was used to develop the colloidal gold test strips for rapid detection of IL-6.We used the trisodium citrate reduction method to prepare a 40nm colloidal gold solution,evaluated the quality of the colloidal gold particles by observing and calculating the diameter of the colloidal gold particles by the UV-visible light absorption spectrum,used the salting-out method to determine the best labeling pH and the best labeled protein amount of the self-made colloidal gold particles,prepared the colloidal gold marker,coated the nitrocellulose membrane,prepared and processed the sample pad,Tris-HCl buffer solution treated the glass fiber membrane,assembled test strips,prepared IL-6 calibrator and prepared semi-quantitative colorimetric cards,sequentially detected and verified the specificity,sensitivity,linear range,stability and repeatability of the test strips,and made preliminary clinical verification through clinical sample testing.6.Statistical analysisThe normality of the quantitative data was tested by the Kolmogorov-Smirnov test.Student's t test,the ?2 test,and receiver operating characteristic(ROC)curve were used to analyze these data.Two-tailed p values<0.05 were considered statistically significant.Results1.1 Escherichia coli stimulated monocytes to express IL-6 highly,highly expressed IL-6 activated JAK/STAT3 signaling pathway and then the expression of p-JAK and p-STAT3 increasedThe group D(monocytes+E.coli)expressed more IL-6 than group A(monocytes+DMEM).Highly expressed IL-6 activated the JAK/STAT3 signaling pathway and catalyzed the phosphorylation of JAK and STAT3,and p-JAK and p-STAT3 promotes the transcriptional expression of targeted response genes and aggravates the inflammatory response.1.2 Anti-IL-6R monoclonal antibody inhibited the phosphorylation of JAK and STAT3 by blocking JAK/STAT3 signaling pathway and the expression of IL-6 by monocytesComparing Group B,Group C,Group E with Group D,there were not significant difference in STAT3 levels,the levels of p-JAK and p-STAT3 were significantly lower,and the levels of IL-6 were significantly lower.Tocilizumab and AG490 blocked JAK/STAT3 signaling pathway,inhibited the phosphorylation of JAK and STAT3,reduced the expression of p-JAK and p-STAT3,and Tocilizumab and AG490 inhibited the expression of IL-6 by monocytes.2.1 Highly expressed IL-6 activated JAK/STAT3 signaling pathway and aggravation of inflammatory response(1)24-hour survival rate of ratsAfter the animal models were successfully established,the survival rate of rats were observed among groups A,B,and C within 24 hours after modeling.It was seen that all 5 rats in group A and B survived(5/5),and Only 2 rats survived in group C(2/5).(2)Monitoring of vital signs? Arterial pressureArterial pressure of rats was monitored in groups A,B and C before operation,2h after operation,4h after operation,and 6h after operation.Among them,there was no significant difference in mean arterial pressure of rats before operation and 2h after operation,it was significantly lower in mean arterial pressure of rats in group C comparing with group A and group B at 4h and 6h after operation.? Body temperature:Body temperature of rats was monitored in groups A,B and C before operation,2h after operation,4h after operation,and 6h after operation.Among them,there was no significant difference in body temperature before operation,it was significantly higher in body temperature of rats in group C comparing with group A and B at 2h after operation,it was significantly higher in body temperature of rats in group C and group B comparing with group A at 4h and 6h after operation,and the body temperature of rats in group C was significantly higher than that of group B at 4h and 6h after operation.(3)Detection of blood inflammatory indexes?White blood cell countWhite blood cell counts of rats in groups A,B,and C were detected by automatic blood cell counter before operation,2h after operation and 6h after operation.Among them,there was no significant difference in white blood cell count between groups before operation and 2h after operation;After 6h,the white blood cell count of group C rats was significantly higher than that of group A and group B rats.? ProcalcitoninThe three groups A,B and C were detected by ELISA before operation,2h after operation and 6h after operation.Among them,there was no significant difference in the level of procalcitonin among the groups before operation,the levels of procalcitonin in groups B and C were higher than that in group A and there was no significant difference between group B and group C at 2h after operation,the levels of procalcitonin in groups B and C were significantly higher than those in group A and the level of procalcitonin in group C was higher than that in group B at 6 hours after the operation.? Interleukin-6 ELISA method was used to detect the levels of IL-6 in groups A,B,and C before operation,at 2h after operation and 6h after operation.There was no significant difference in the level of IL-6 among them before operation,the level of IL-6 in group C was significantly higher than that of group A and B at 2h after operation,the level of IL-6 in group C was significantly higher than that in group A and group B at 6h after operation.(4)Escherichia coli stimulated the high expression of IL-6 in liver tissue and catalyzed the increased phosphorylation of JAK and STAT3Rats were killed at 24 hours after the operation and IL-6 of liver were tested.The expression of IL-6 in the liver tissues increased which was stimulated by E.coli,the IL-6 levels were in the order of group C>group B>Group A,the more E.coli,the higher the expression of IL-6,IL-6 catalyzes the phosphorylation of JAK and STAT3 to p-JAK and p-STAT3 through JAK/STAT3 signaling pathway.The expression levels of p-JAK and p-STAT3 were in the order of group C>group B>group A,that is,the higher the expression of IL-6,the higher the expression of p-JAK and p-STAT3.(5)The more severe the infection caused by E.coli,the more severe the pulmonary inflammatory reactionInfection of Escherichia coli induced pulmonary inflammatory reaction.The pulmonary inflammatory reaction were in the order of group C>group B>group A.The more E.coli,the more severe the pulmonary inflammatory reaction.2.2 Anti-IL-6R monoclonal antibody blocked JAK/STAT3 signaling pathway and inhibited excessive inflammatory response(1)24-hour survival rate of rats After the animal model was successfully established,the survival rate of rats was observed in group C and D within 24 hours after modeling.It was seen that all the 5 rats in group D survived(5/5),and only 2 of the 5 rats in group C survived.(2/5).(2)Monitoring of vital signs? Arterial pressureThere was no significant difference in the mean arterial pressure of rats between group C and group D before operation and at 2h after operation,the mean arterial pressure of rats in group D was significantly higher than that of rats in group C at 4h and 6h after operation.?Body temperatureThere was no significant difference in the average body temperature of the rats between group C and group D before operation and at 2h after the operation,the average body temperature of the rats in the D group was significantly lower than that of the rats in the C group at 4h and 6h after operation.(3)Detection of blood inflammatory markers?White blood cell countWhite blood cell count of rats in group C and D were detected by automatic blood cell counter before operation,at 2h and 6h after operation.Between them,there was no significant difference before operation and at 2h after operation,and the white blood cell count of rats in group C was significantly higher than that in group D at 6h after operation.?ProcalcitoninELISA method was used to detect the level of procalcitonin before operation,at 2h and 6h after operation in rats of group C and D.Between them,there was no significant difference in the levels of procalcitonin before operation and at 2h after the operation,the level of procalcitonin in group D was significantly lower than that in group C at 6h after the operation,.? Interleukin-6ELISA method was used to detect the IL-6 levels of rats in groups C and D before operation,at 2h and 6h after operation.Between them,there was no significant difference in the levels of IL-6 before operation and at 2h after the operation,the level of IL-6 in group C was significantly higher than that in group D at 6h after the operation,.(4)Anti-IL-6R monoclonal antibody blocked JAK/STAT3 pathway,inhibited the phosphorylation of JAK and STAT3 and inhibited the expression of IL-6The rats were killed 24h after the operation,and the liver tissues were taken to detect the expression of IL-6,STAT3 and p-STAT3.The level of IL-6 in group D was significantly lower than that in group C.There was no significant difference in the expression of STAT3 between group C and D.The expression of p-STAT3 in group D was significantly lower than that in group C.Tocilizumab blocked JAK/STAT3 signaling pathway by binding to IL-6R and inhibited the phosphorylation of STAT3,the expression of p-STAT3 reduced.The anti-IL-6R monoclonal antibody tocilizumab inhibited IL-6 expression.(5)The anti-IL-6R monoclonal antibody blocked the JAK/STAT3 pathway and alleviated the inflammatory response of the lungGroup C and D received the same E.coli infection,and the inflammatory response of the lung of rats in group D was significantly milder than that of group C under the action of the anti-IL-6R monoclonal antibody.3.The early predictive and diagnostic ability of IL-6 for postoperative urosepsis after percutaneous nephrolithotomy3.1 General clinical dataThere was no statistical significance in the differences of gender,age,diabetes,hypertension,hydronephrosis,stone burden,urine culture,pyuria,and operation time between EXP1 group and CON group.The average postoperative hospital stay of the EXP1 group was greater than that of the CON group and there was statistical significance.3.2 Inflammatory markers(1)White blood cell countThere was no significant difference between the two groups in EXP1 group and CON group at 2h after operation,the average white blood cell count of EXP1 group was significantly higher than that of CON group patients on 1 day after operation,the white blood cell count of EXP 1 group was significantly higher than that CON group.(2)Proportion of neutrophils(%)The average proportion of neutrophils of EXP1 group patients was significantly higher than that of CON group patients at 2h after operation and on 1 day after operation.(3)PCTThere was no significant difference between EXP1 group and CON group at 2h after operation,the average value of procalcitonin in EXP1 group patients was significantly higher than that in CON group on 1 day after operation,the average value of procalcitonin in EXP1 group was significantly higher than that in CON group on 3 days after operation.(4)IL-6The average IL-6 value of EXP 1 group was significantly higher than that in CON group at 2h after operation,the average IL-6 of EXP 1 group was significantly higher than that in CON group on 1 day after operation.(5)Evaluation of diagnostic valueAt 2h after operation,the ROC curve showed that the AUC values of different inflammatory markers in descending order were IL-6(AUC=1.000),the proportion of neutrophils(AUC=0.788),PCT(AUC=0.719),white blood cells(AUC)=0.511).On 1 day after operation,the ROC curve showed that the AUC values of different inflammatory markers in descending order were IL-6(AUC=0.987),PCT(AUC=0.932),the proportion of neutrophils(AUC=0.896),white blood cells(AUC)=0.855).On 3 days after surgery,the ROC curve showed that the AUC values of different inflammatory markers in descending order were PCT(AUC=0.954),the proportion of neutrophils(AUC=0.763),white blood cells(AUC=0.726),IL-6(AUC)=0.656).4.The clinical value of the application of IL-6 in early predicting and diagnosis of urosepsis after percutaneous nephrolithotomy4.1 General clinical dataThere was no statistical significance in the differences of gender,age,diabetes,hypertension,hydronephrosis,stone load,urine culture,pyuria,and operation time between EXP1 group and EXP2 group.4.2 Clinical resultsThe postoperative hospital stay of EXP1 group was significantly shorter than that of EXP2 group.The postoperative intervention time of EXP1 group was significantly shorter than that of EXP2 group.2 patients in EXP1 group suffered severe urosepsis(2/16),and 12 patients suffered severe urosepsis in EXP2 group(12/25),the incidence of severe urosepsis in EXP1 group was significantly lower than that in EXP2 group.5.Research and development of the colloidal gold test strips for rapid detection of IL-6We successfully developed the colloidal gold test strips for rapid detection of IL-6 with the help of colloidal gold immunochromatography technology.A semi-quantitative colorimetric card was made.According to verification,the strip had high specificity,high sensitivity of 5pg/ml,a good linear relationship between 5 and 2000pg/ml,and had good stability and repeatability.The reference value of the paper strip was 7pg/ml,and the strip had a high consistency with the clinical verification results of inflammatory specimens.Conclusion1.Escherichia coli stimulated monocytes to highly express IL-6,activated JAK/STAT3 signaling pathway,catalyzed the phosphorylation of JAK and STAT3,and the expression of p-JAK and p-STAT3 increased.Tocilizumab blocked JAK/STAT3 signaling pathway by binding to IL-6R,inhibited the phosphorylation of JAK and STAT3,and the expression of p-JAK and p-STAT3 reduced.Tocilizumab inhibited the expression of IL-6 in monocytes.2.The injection of Escherichia coli with a concentration of 12.0×108cfu/ml into the renal pelvis of rats was used to establish animal models of urosepsis.IL-6 was used for early predicting and diagnosis of urosepsis.Anti-IL-6R monoclonal antibody effectively inhibited the excessive inflammatory response of the body.3.There were different diagnostic values of WBC,proportion of neutrophils,PCT and IL-6 for urosepsis at different time points.IL-6 was a valuable inflammatory marker for early predicting and diagnosis of urosepsis.4.With the help of IL-6 as an inflammatory marker,intervention was given in advance,the severity of urosepsis reduced,prognosis was improved and postoperative hospital stay was shortened after percutaneous nephrolithotomy.5.Development and clinical verification of the colloidal gold test strips for rapid detection of IL-6 was completed.The detection of the strips was convenient and rapid,and could be finished on site.Semi-quantitative detection could be completed in 10 minutes.It provided more time and better opportunities for the treatment of critically patients with urosepsis.