The Immunomodulatory Effect Of Low Dose Naltrexone On MRL/lpr Mice Through Regulating The Expression Of NF-κB Signaling Pathway

Objective:Systemic lupus erythematosus（SLE）is a complex and recurrent chronic autoimmune disease,causing multiple organ damage such as skin,joint,kidney,mental nervous system and lung,which causes a heavy burden to the patient’s family and society.At present,the etiology of SLE is not clear.It is the result of genetic,immune,environmental,endocrine and other multi factor abnormalities and interactions.It is characterized by the activation of multiple immune system pathways,and the mechanism is very complex.At present,the main treatment methods are still glucocorticoids and immunosuppressive agents,but adverse reactions are also common in the treatment process.Therefore,the research of safe and effective treatment is still the current research hotspot.The clinical manifestations of SLE are various,and the prognosis of patients is closely related to whether there is renal and nervous system involvement.Despite elaborate treatment,a large proportion of patients with lupus nephritis（LN）progress to end-stage renal disease,which has become one of the main causes of death in SLE patients.The clinical manifestations of neuropsychiatric lupus erythematosus（NPSLE）are diverse and the pathogenesis is complex.Immune,inflammatory or thrombotic pathology play an important role in the occurrence and development of NPSLE.With the deepening of research,the understanding of the pathogenesis of SLE is also progressing.As the upstream site of classical inflammatory pathway,toll like receptors 4（TLR4）is involved in the activation of innate immunity and adaptive immunity.Its mediated immune abnormalities play a very important role in the occurrence and progression of SLE.Thus,the blocking of TLR4 mediated NF-κB signal pathway can be used as one of the therapeutic targets of SLE.Naltrexone（NTX）,as a long-acting non selective opioid receptor antagonist,was initially approved for the treatment of heroin addiction and alcohol dependence.In recent years,the role of naltrexone in immune regulation has attracted expanding attention.Low dose naltrexone（LDN）can upregulate the expression of OGF-OGFr axis through the phenomenon of"opioids rebound",so as to inhibit the proliferation and activity of T cells and B cells and play an anti-inflammatory role.On the other hand,LDN can block NF-κB signal pathway mediated by TLR4,so as to alleviate the inflammatory response of autoimmune diseases,which has become the theoretical basis of its adjuvant treatment of autoimmune related diseases.At present,the research on the application of LDN is more and more extensive,especially in the treatment of autoimmune diseases,including Crohn’s disease,multiple sclerosis,fibromyalgia,etc.The Research of naltrexone abroad also involves inflammatory myopathy,psoriasis,Sjogren’s syndrome,etc.But the research on the application of LDN in SLE is still relatively limited.Therefore,we selected MRL/lpr mice as the research object and gave LDN intraperitoneal injection treatment,in order to explore the therapeutic effect of LDN on MRL/lpr mice,and preliminarily explore its mechanism,so as to provide a theoretical basis for its application in the treatment of SLE.Methods:Part I:mouse glomerular endothelial cells（MRGEC）and brain microvascular endothelial cells（BEND.3）were selected as the research objects,and the normal group did not receive drug intervention;The control group was given TNF-α0.1μg/ml after the cells adhered to the wall and then co cultured for 24 hours;The LDN group was pretreated with gradient concentration of NTX for 24 hours after the cell adhesion was stable,and then continued to be given TNF-α0.1μg/ml for 24 hours.The cell viability was tested by CCK8 to determine the optimal drug concentration.After giving the best concentration of NTX to repeat the above experiments,the m RNA expression of inflammatory cytokines and adhesion molecules was observed by RT-PCR,and Protein expression of the TLR4 mediated NF-κB signal pathway site was observed by Western blot.Part II:Twenty 10 weeks old female MRL/lpr mice were randomly divided into two groups with 10 mice in each group.One group was randomly selected as LDN group and the other group as control group.The mice in the LDN group were injected intraperitoneally with naltrexone 10mg/kg/d;the mice in the control group were injected intraperitoneally with 0.2ml/d normal saline for 8 weeks.The variation of depressive symptoms was observed by tail suspension test and forced swimming test,the variation of renal damage was observed by urinary protein/urinary creatinine value,renal pathology and immunohistochemistry,the variation of cognitive function of mental nervous system was observed by hippocampal gyrus pathology and immunohistochemistry,and the proliferation of spleen was observed by spleen pathology.Part III:The immunoglobulin and cytokines in peripheral blood was tested by ELISA;the proportion of Th1 cells,Th2 cells,Th17 cells,Treg cells and TFH cells were measured by flow cytometry to evaluate the variation of the imbalance of T lymphocyte subsets;the m RNA expressions of inflammatory cytokines,adhesion molecules and opioid receptors in target organs were observed by RT-PCR;the protein expression of the TLR4 mediated NF-κB signal pathway site was observed by Western blot.Results:1.The cell activity of NTX at the concentration of 10^-2mol/L and 10^-3mol/l was lower than that of the control group,and the cell activity of NTX at the concentration of10^-4mol/L and below was higher than that of the control group.Among them,the difference between the cell activity of 10^-4mol/L drug concentration and the control group was the most obvious,which was the best concentration.After induced by TNF-αand pretreatment with NTX,TNF-α,IL-6 and BAFF m RNA levels of LDN group and control group in mouse brain microvascular endothelial cells and glomerular endothelial cells were significantly higher than those in the normal group,and the caspase-1 m RNA levels of LDN group and control group in mouse brain microvascular endothelial cells was higher than that in the normal group;in the LDN group of two kinds of cells,the m RNA expression levels of TNF-α、IL-6 and BAFF were lower than those in the control group,and the expression level of Caspase-1 m RNA in mouse brain microvascular endothelial cells was lower than that in the control group;in mouse glomerular endothelial cells,the expression level of Caspase-1 m RNA in normal group and LDN group was low,which was meaningless.The m RNA expressions of P-selectin and VCAM-1 in LDN group and control group were significantly higher than those in normal group;the m RNA expressions of P-selectin and VCAM-1 in LDN group was lower than that of control group.And after co culture with TNF-α,the protein expressions of TLR4,NF-кB,Iккαβand phosphorylated NF-кB in the LDN group and control group were higher than that in the normal group,while the protein expression of IкBαwas lowerer than that of normal group,suggesting NF-кB pathway is activative.After LDN pretreatment,The protein expression of TLR4,NF-кB,Iккαβand phosphorylated NF-кB in LDN group was lower than that of the control group,while the IкBαincreased expression.It suggests that NTX downregulates the activation of NF-кB signal pathway.2.The immobility time of LDN group mice in tail suspension test and forced swimming test was less than that of the control group,suggesting that the depressive behavior of LDN group mice was better than that of the control group.The urinary protein/urinary creatinine values of mice in the two groups increased continuously from the first week to the fifth week,the growth of mice in LDN group slowed down from the sixth weeks,and the urinary protein/urinary creatinine values of mice in LDN group were lower than those in the control group from the seventh week to the eithth week.The results were statistically significant.The spleen conefficient of LDN group was lower than that of the control group.Pathology showed that the number of spleen germinal centers and Ig G deposition were lower than those of the control group;Pathology and immunohistochemistry showed that the proliferation of mesangial cells,endothelial cells and inflammatory cell infiltration in the kidney of LDN group were lighter than those in the control group.The number of Nissl bodies in LDN group was more than that in the control group,which was more orderly than that in the control group,suggesting that the damage and loss of neuron cells in hippocampal gyrus were improved;The Ig G deposition in kidney and hippocampal gyrus in LDN group was lighter than that in control group.3.The levels of Ig G1 and Ig G2 in peripheral blood of mice in LDN group were lower than those in control group,and there was no significant difference of Ig G3 level between the two groups.The level of IL-10 in peripheral blood of mice in LDN group was lower than that in control group.The levels of IL-17,TNF-αand BAFF in peripheral blood between the two groups were not statistically significant.Flow cytometry showed that the ratio of Th1/Th2 cells in LDN group was higher than that in control group.In LDN group,the proportion of Th17 cells was lower,the proportion of Treg cells was higher,and the proportion of TFH cells was lower.RT-PCR results showed that in LDN group the expressions of opioid receptors m RNA except KOR in spleen,kidney and brain increased,and the m RNA of inflammatory cytokines and adhesion factors in kidney and brain of LDN group were lower than those of control group.WB results suggest that that among the sites related the TLR4-NF-кB signaling pathway,the protein expression of TLR4,NF-кB,Iккαβ,phosphorylated NF-кB,phosphorylated Iккαβand phosphorylated IкBαin LDN group were lower than those of control group,while the protein expression of IкBαIncreased.Conclusion:1.NTX may alleviates the injury of brain microvascular endothelial cells and glomerular endothelial cells induced by TNF-αthrough downregulating the activation of NF-κB signaling pathway mediated by TLR4.2.LDN can alleviate the depressive behavior in neuropsychiatric damage and alleviate the abnormal cognitive function of mice.LDN can reduce proteinuria and the pathological damage of kidney in MRL/lpr mice,alleviate the proliferation of spleen,and reduce the deposition of Ig G in kidney,hippocampal gyrus and spleen.3.LDN reduces the production of inflammatory cytokines and Ig G in peripheral blood,alleviates the imbalance of the proportion of T cell subsets in the spleen,upregulates the transcriptions of opioid receptor m RNA,downregulates the transcriptions of inflammatory cytokines and adhesion factors m RNA in the brain and kidney,and the expressions of proteins related to TLR4-NF-κB signaling pathway were downregulated as well.It is suggested that LDN may plays a therapeutic role in MRL/lpr mice through upregulating the expression of opioid receptors and downregulating the activation of NF-κB signaling pathway mediated by TLR4.