Effect And Mechanism Of Qiang-Ji-Jian-Li Recipe On Regulating Macrophage Polarization And Myocyte Autophagy Mediated By ROCK1/AKT Signaling Pathway In Sepsis-Induced Myopathy

Background:Sepsis is one of the diseases affecting human health all over the world,and its incidence has gradually increased in recent years.With the continuous innovation of medical technology,the death threat of sepsis is gradually decreasing.However,the incidence of Sepsis-induced Myopathy(SIM)increases,with symptoms of persistent ICU acquired myasthenia in respiratory and limb muscles,which can lead to prolonged mechanical ventilation time and unplanned readmission due to long-term bed rest and expectoration weakness,which has become one of the main causes of mortality in the later stage of sepsis survivors.Due to its complex pathogenesis,modern medicine still lacks accurate treatment methods,and can only take symptomatic treatment such as nutritional support and early exercise.Therefore,studying the exact pathogenesis of muscle atrophy in patients with sepsis and finding more effective treatment methods has become a hotspot and difficult field of critical care medicine.Traditional Chinese medicine pays attention to the overall state of the body and takes syndrome differentiation and treatment as the basic program,which can adjust the deviation of Qi,blood,and Yin/Yang of the body as a whole.Moreover,traditional Chinese medicine has a variety of active substances.Various active components act on multiple targets and work together through a variety of signal pathways.They have good curative effects in targeting various acute and chronic diseases,and have been gradually recognized by the international community.Although the pathological mechanism of Sepsis-induced Myopathy is complex,SIM could be considered to the category of"flaccid syndrome"in Traditional Chinese Medicine according to the overall clinical manifestations.It is considered that the general pathogenesis starts from“Lung Heat Leaf Coke”and quickly turns into“Acute Deficiency”syndrome-spleen deficiency syndrome.The Inner Canon of the Yellow Emperor puts forward that"treating impotence only takes Yang-Ming",which mainly treats impotence syndrome by Tonifying the spleen and stomach.Therefore,the selection of prescriptions and drugs should pay attention to tonifying the spleen and stomach.Professor Deng Tie-Tao,a master of Chinese medicine,also believes that the pathogenesis of flaccidity syndrome is the deficiency of spleen and stomach qi,and the treatment should pay particular attention to the word deficiency.Qiang-Ji-Jian-Li(QJJL)prescription is a traditional Chinese medicine prescription created by Deng Tie-Tao,based on the concept of"treating impotence and taking Yang-Ming alone"and the characteristics of spleen and stomach deficiency in patients with myasthenia gravis.It has achieved a good curative effect in the clinical treatment of acquired myasthenia in ICU.This research mainly studies the molecular mechanism of the Qiang-Ji-Jian-Li formula(QJJLF)in the treatment of sepsis muscular atrophy,to provide a scientific theoretical basis for the treatment of this disease with traditional Chinese medicine and lay a foundation for the development of new drugs in the future.Objective:To investigate the molecular mechanism of the Qiang-Ji-Jian-Li formula in the treatment of Sepsis-induced Myopathy.Methods:Clinical studyStudy subjects:from October 2017 to May 2019,61 septic patients who met the study conditions were selected as the study subjects.All eligible patients in this study were followed up to the 28th day after ICU admission.Inclusion criteria:the patient's age should be?18 years old,and the diagnostic criteria are consistent with the diagnostic criteria of sepsis 3.0.The patient is the first time to enter the ICU for treatment.Exclusion criteria:neuromuscular diseases were new at admission;Less than 7 days after admission to ICU or the patient dies during admission;No ultrasonic test results;On the28th day,the survival status and FIM motor function score were missing;The patient had a malignant tumor.TCM Syndrome Classification:within 24 hours after ICU admission,the TCM syndrome analysis of the septic patient was evaluated through the syndrome elements recorded in the electronic medical record system and the intensive care auxiliary diagnosis system.The evaluation process strictly follows the Chinese guidelines for the treatment of severe sepsis/septic shock(2014).According to the guideline classification standard,this case is divided into acute deficiency syndrome(34 cases),toxic heat syndrome(15 cases),blood stasis syndrome(12 cases),visceral Qi obstruction syndrome(0 cases).Finally,the change rate of rectus femoris cross-sectional area(R F _{C S A}),length of stay in intensive care unit(LOS),the relationship between motor function scores in 28-day functional independence scale(FIM),and the difference between 28-day survival rates were compared.Animal studiesThe model was established by intraperitoneal injection of LPS(dose:10mg/kg).The experiment was divided into two parts.The first part:10-week-old C57/BL6 mice(each weighing 18-25g)were randomly divided into three groups,with 20 mice in each group.They were divided into sham group,LPS group and LPS+inhibitor group(LPS+ROCK1inhibitor group)LPS group and LPS+inhibitor group were injected intraperitoneally with LPS,while sham group mice were injected intraperitoneally with an equal volume of normal saline.After injection,mice in each group were treated with ROCK1 inhibitor 72 hours before LPS injection and treated continuously until 48 hours after LPS injection.Part ?:12010-week-old C57/BL6 mice were randomly divided into four groups:sham group and sham group(30 rats),LPS group(30 rats),Qiang-Ji-Jian-Li formula group(QJJLF)(30 rats),dexamethasone group(DXMS)(30 rats).Each mouse in the LPS group,QJJL formula group and DXMS group was intraperitoneally injected with 10 mg/kg LPS,and the sham group was given the same amount of 0.9%normal saline.After the injection,the mice in each group were given the following treatment:QJJL formula group and DXMS group were given Qiang-Ji-Jian-Li formula and DXMS injection 72 hours before LPS injection,all once a day.QJJL formula group was given 0.1ml Qiang-Ji-Jian-Li formula solution,DXMS group0.3mg/kg per day.After intraperitoneal injection of LPS,the survival of mice was recorded;the morphological changes of mouse muscle tissue were observed by HE staining;the serum(eyeball blood)was collected 48 hours after the intervention,and the peripheral serum IL-1?,IL-6,TNF-?(Proinflammatory factors),IL-10,TGF-?(the expression levels of anti-inflammatory factors),C-reactive protein,as well as the expression levels of CK and myoglobin(two muscle injury markers)were detected by ELISA;the m RNA expression levels of Caspase-3 and Caspase-8 in caspase were detected by qPCR,and trim63(MuRF1)and fbx032(MAFbx/Atrogin-1)in UPS system were measured by western blot.The two tests jointly identified the changes of proteolytic system in mice with different treatments;the levels of macrophage related markers were detected by immunofluorescence,q PCR and Western blot to clarify the polarization of macrophages in mice with different treatments;the expression levels of LC3 and Beclin1 proteins were evaluated by Western blot to clarify the changes of autophagy level of myofibroblasts in mice with different treatments;The changes of signal protein AKT(down-stream of ROCK1)were detected by Western blot.Results:Part?:Retrospective cohort study on TCM syndrome types,R F _{C S A} and 28-day survival rate in patients with sepsisThe confounding factors such as age,length of invasive ventilation,gender,infection site,BMI and simplified acute physiological score(SAPS)were adjusted.Among eligible sepsis patients,the change rate of R F _{C S A}in blood stasis syndrome group was-14%(95%CI:-27%-1%,P<0.05).After adjusting the confounding factors of age,invasive ventilation duration,gender,infection site,BMI,acute physiology and chronic health score system ?(APACHE ?),the change rate of R F _{C S A}was:-14%(95%CI:-26%?-1%,P<0.05)in patients with blood stasis syndrome group and acute deficiency syndrome group.The survival curve was drawn.Kaplan-Meier method was used to analyze the survival rate of different components by log rank test,P=0.15.The results showed that there was no significant difference in the 28-day survival rate of sepsis patients with different TCM syndrome types.After adjusting for age,gender and other confounding factors,the results showed that there were significant differences in the length of stay in the intensive care unit and FIM motor function score among sepsis patients with different TCM syndrome types.Part?:The role of ROCK1/AKT mediated macrophage polarization and autophagy in septic muscular atrophy1.The expression level of ROCK1 protein in muscle tissue of LPS group was significantly up-regulated.2.Compared with the LPS group,the survival rate of the LPS+inhibitor group was significantly improved.3.Compared with LPS group mice,the muscle fibers of rectus femoris in LPS+inhibitor group were arranged relatively orderly,the muscle structure was relatively complete,the infiltration of inflammatory cells was relatively less,and the damage of muscle tissue structure was less.4.Changes of serum inflammation-related indexes in each group4.1.Compared with the LPS group mice,the proinflammatory cytokine IL-1?,IL-6,and TNF-?in serum of the LPS+inhibitor group mice were significantly lower.4.2.Anti-inflammatory cytokines IL-10 and TGF in peripheral serum of mice after LPS application.The expression level of IL-10 and TGF was significantly lower than that of the sham group.Inhibition of ROCK1 can significantly increase the in vivo anti-inflammatory cytokines IL-10 and TGF in septic mice level.4.3.Inhibition of ROCK1 can significantly reduce the level of C-reactive protein in mice after sepsis.5.The levels of MYO and CK-MB in peripheral blood of LPS group mice increased significantly,and the expression levels of MYO and CK-MB decreased significantly after inhibition of ROCK1 protein.6.Inhibition of ROCK1 can reduce the m RNA expression levels of Caspase-3 and Caspase-8 and the protein expression levels of trim63 and fbx032 in mouse muscle after LPS modeling.7.The results of immunofluorescence,Western blot and q PCR showed that inhibiting the expression of ROCK1 protein could inhibit the infiltration of macrophages and migration to pro-inflammatory M1 macrophages.8.To some extent,inhibiting the expression of ROCK1 protein can be achieved by inhibiting the expression levels of LC3 and Beclin1 protein.9.Inhibiting the expression of ROCK1 protein can inhibit the phosphorylation level of down-stream protein AKT.Part?:The role and mechanism of QJJLF in regulating ROCK1/AKT mediated macrophage polarization and autophagy in the prevention and treatment of septic muscular atrophy1.QJJLF can reduce the mortality of LPS model mice.2.QJJLF can increase the mass and thickness of rectus femoris muscle in mice after LPS modeling,protect the integrity of muscle structure and reduce the infiltration of inflammatory cells.3.QJJL recipe reduces the proinflammatory cytokine IL-1?,IL-6,TNF-?and the expression level of C-reactive protein in the peripheral blood of LPS model mice,increasing the anti-inflammatory cytokines IL-10 and TGF-?Its curative effect is equivalent to that of DXMS.4.QJJL formula reduces the levels of MYO and CK-MB in the serum of mice after modeling,and the curative effect is consistent with DXMS.5.QJJL formula can reduce the m RNA expression levels of Caspase-3 and Caspase-8,and the protein expression levels of trim63 and fbx032 in the muscles of mice after modeling.The curative effect is equivalent to that of DXMS.6.Both the QJJL formula and DXMS can reduce the M1 polarization of macrophages7.QJJL formula and DXMS can inhibit the expression levels of autophagy-related proteins LC3 and Beclin1.8.QJJL formula can reduce the phosphorylation level of AKT protein in mice after LPS modeling.Conclusion:1.Through the method of a retrospective cohort study,the muscle loss process of hospitalized sepsis patients in the intensive care unit was analyzed.The muscle loss process of sepsis patients with blood stasis syndrome in traditional Chinese medicine was faster than that of sepsis patients with acute deficiency syndrome;However,there was no significant difference in the length of stay in ICU,28-day survival rate and FIM motor function score between patients with different TCM syndrome types.2.Qiang-Ji-Jian-Li formula can reduce the inflammatory level in mouse muscle after LPS modeling,inhibit the migration of macrophages to M1 type and promote myofibroblast autophagy,so as to improve mouse muscle tissue injury and inhibit the process of SIM.Its effect might be realized by regulating ROCK1/AKT signal pathway.