Based On RNA-Seq To Explore The Immune Regulation Of Vagus Nerve Stimulation On Endotoxemia Rats

Part I: Determination of the safety zone of vagus nerve stimulationObjective: The vagus nerve(VN)is anatomically complex and has an impact on the heart,brain and other vital organs.By observing the effects of different parameters of VNS in different parts on the rat heart,the safety zone of vagus nerve stimulation(VNS)were determined.Methods: The left and right cervical VN were stimulated separately with automatic voltage(0 V,automatic increase of 0.1 V,2 ms,1 Hz),current(0 m A,automatic increase of 0.01 m A,2 ms,1 Hz)and frequency(5 V,2 ms,0 Hz,automatic increase of 0.05 Hz).The safety zone was defined through observing ECG changes and evaluating the impact of VNS on the heart.Results: With the increase of voltage,the R-R interval became longer,bradycardia and occasional arrhythmia were occurred over 10 V.With the increase of current,the R-R interval became longer and irregular.Over 1 m A,channel II recorded arrhythmia,and channel AVR and V1 showed obvious abnormal waveform,which is abnormal QRS complex.With the increase of frequency,the R-R interval gradually became longer,and occasional arrhythmia began to be observed at 3 Hz.At 5 Hz,channel AVR and V1 showed obvious bradycardia.With the further increase of frequency,the R-R intervals of channel II,AVR and V1 gradually extended,asystole was induced by 10 Hz.When the voltage was changed,the percentage of heart rate variability(HRV%)in the right cervical VNS was higher than that in the left with statistical significance,and the corrected p value(hereinafter referred to as p value)was 0.03.As current and frequency changed,The HRV% in the right cervical VNS was slightly higher than that in the left,but there was no statistical significance,and the p values were 0.144 and 0.967 respectively.Compared with different parameters of VNS on the same side,it can be found that the HRV% is significantly higher when the current was changed than that when the voltage and frequency were changed,p<0.001.There was no significant difference between changing voltage and changing frequency of both side,the right cervical VNS p=0.969,and the left p=0.220.Conclusion: The right cervical VNS is more likely to affect cardiac function.The safe zone of each parameter as follows: voltage(0 V～10 V,2 ms,1 Hz),current(0m A～1 m A,2 ms,1 Hz),frequency(5 V,2 ms,0 Hz～3 Hz).The effect of changing voltage is safer and more stable.Part II Determination of immune threshold of vagus nerve stimulationObjective: The anti-inflammatory parameters of VNS are not standardized yet,and the stimulation parameter of VNS with the best anti-inflammatory effect needed to be selected within the safety zone.Methods: Endotoxemia was induced by intravenous injection of lipopolysaccharide(LPS)(5 mg/kg).Rats were divided into six groups as follow which depended on treatments randomly.Control(CON)group: equal volume of normal saline was injected by femoral vein without subsequent VNS.Model(LPS)group: LPS(5 mg/kg)was injected by femoral vein to build endotoxemia model without subsequent VNS.Stimulation(VNS)group 1: LPS(5 mg/kg)was injected by femoral vein to build endotoxemia model,and VNS(3 V,2 ms,1 Hz)was given for 20 minutes.VNS2 group:the same with VNS1 except the voltage of 5V.VNS3 group: the same with VNS1 except the voltage of 7V.VNS4 group: the same with VNS1 except the voltage of 9V.0.5 m L of blood was collected by arterial intubation after LPS injection and at 0 h,1 h,2 h,3h and 4 h after VNS,and numbered t,t0,t1,t2,t3,t4 sequentially.The supernatant was collected after centrifugation.The concentration of tumor necrosis factor(TNF)-α in plasma was measured by ELISA.Results: At t and t0,there was no significant difference in plasma TNF-αconcentration between the VNS group and the LPS group with different voltages,and the p values were all bigger than 0.999.At t1 the TNF-α concentration of VNS2 group and VNS4 group was significantly lower than that of LPS group,with p values of 0.020 and 0.023,respectively.Compared with the LPS group,the TNF-α concentration of VNS1 group,VNS2 group and VNS4 group was significantly lower at t2,and the p values were 0.037,0.042 and 0.022,respectively.At t3,the TNF-α concentration of the VNS2,VNS3 and VNS4 groups was significantly lower than that in the LPS group.The p value of the VNS2 group was less than 0.001,and the p values of the VNS3 and VNS4 groups were 0.002 and 0.003,respectively.The TNF-α concentration in the VNS2 group was also significantly lower than the VNS1 group at t3,p=0.030.Compared with the LPS group,the TNF-α concentration of the VNS2 group and VNS3 group were significantly lower at t4,with the p values of 0.004 and 0.037,respectively.The rest were not statistically significant.Conclusion: VNS group can reduce the peak concentration of TNF-α induced by LPS.According to the size of the reduced peak and the duration of reduction,the effect of the VNS group can be comprehensively compared,and it can be concluded that VNS2>VNS4>VNS3>VNS1.The inflammation reaches its peak at t1,the antiinflammatory effect is the most obvious at t3,and t4 is the end point.VNS(5 V,2 ms,1 Hz)has greater effects at 1 h,3 h,and 4 h.Part Ⅲ The immunoregulation of vagus nerve electrical stimulation on endotoxemia ratsObjective: The error caused by individual differences in animal models was reduced by studying simultaneously the effect of VNS on multiple factors and organs of endotoxemia rats.High throughput transcriptome sequencing(RNA-Seq)was used to obtain differential genes which is the genes with significant changes in expression patterns of control group,disease model group and VNS treatment group.The differential genes were enriched and annotated to integrate the key anti-inflammatory genes,key molecules and signal pathways,so as to find the possible therapeutic targets of endotoxemia.Methods: The rats were divided into three different treatment groups randomly:control group(Con): equal volume of 0.9% sodium chloride solution was injected into femoral vein with VNS false stimulation;endotoxemia model group(LPS): LPS(5 mg/ kg)was injected into femoral vein with VNS false stimulation;VNS treatment group(VNS): LPS(5 mg / kg)was injected into femoral vein with VNS(5 V,2 ms,1 Hz)for20 min.Then 3 m L whole blood and ileum,liver,spleen,kidney,thymus,heart and lung were collected at T0(1 h after VNS/ false stimulation),T1(3 h after VNS/ VNS false stimulation)and T2(4 h of VN/ false stimulation).RNA was extracted from 2 m L whole blood with Trizol for RNA-Seq.1 m L whole blood was centrifuged to take the supernatant for detecting the concentrations of TNF-α,IL-18,MIP-1 α,IFN γ,IL-1 β,IL-6,CINC-1,RANTES,IL-12p70,IP-10 and IL-10 in plasma by Luminex.Organs were fixed,embedded,sliced and stained with HE.Results: Compared with the LPS group,the thymus cortex was thickened and the spleen periarterial lymphatic sheath increased in the VNS group at T0,and cell necrosis and inflammatory infiltration of the other organs were reduced over time.The expression levels of TNF-α and IL-18 continue to decrease;the chemokine MIP-1αdecreased only at T0(1 h after VNS)in the early stage of inflammation,but there was no significant difference at T1 and T2(3 h and 4 h after VNS)in the middle and late stages,but it was significantly higher than that in Con group;IFNγ,IL-1β,IL-6,CINC-1,RANTES were just the opposite,there was no statistical difference between the two groups and the Con group in the early stage,while the concentrations of these cytokines in the middle and late stages were significantly reduced,although they were still higher than those in the Con group The concentration of IL-12p70 and IP-10 was significantly lower,although it was still higher than that of the Con group;IL-12p70 and IP-10 also had no statistical difference in the early stage,but significantly increased in the middle and late stages.The level of anti-inflammatory cytokine IL-10 continued to increase.LPS can be transmitted to CD14 by lipopolysaccharide-binding protein in serum,which enhances the sensitivity of phagocytes to LPS;it can also be recognized by TLR(mainly TLR4)on the cell membrane directly or by NLR in the cell indirectly.TLR recognized ligands and activated downstream signaling pathways through My D88-dependent pathway or My D88-independent pathway;After binding with ligands,NLR interacted with downstream RIP2 to activate downstream pathways such as NF-κB,MAPKs and IRFs,and promoted the transcription and expression of MHC and different cytokines such as IL-1β,TNF-α,IL-6,IL-18,IFN,CXCL,IP-10 and MIP-α.Under the synergistic effect of these cytokines,MHC II induced lymphocyte proliferation and differentiation in immune organs such as thymus and spleen,and mainly induced T cells to differentiate into different subsets of CD4+ T cells(Helper T cells,Th).MHC I induced differentiation of CD8+ T cells(Cytotoxic T cells,Tc)and activation of NK cells.Different Th release different cytokines and further promote the activation of other immune cells and cytokines release.The interaction between cytokines and receptors activated downstream signaling pathways such as NF-κB and MAPK,up-regulated the gene expression of various cytokines,and promoted the activation and chemotaxis of other immune cells.Tc and NK cells mediated apoptosis through TNF-α,Fas and other pathways.Without intervention,the cascade reaction of cytokines activated immune cells persistently,and white blood cells migrated to various tissues,which caused pyroptosis of cells in the tissues,tissue damage and organ dysfunction further.Conclusion: Short-term stimulation of VN can inhibit inflammation in a long time,make immune response faster by identifying pathogens earlier,inhibit NF-κB,JAK/STAT,MAPK and other signaling pathways,inhibit sequentially the expression of pro-inflammatory cytokines and promote the expression of anti-inflammatory cytokines,and reduce the tissue damage and dysfunction of various organs ultimately.