Regulation Of Pro-inflammatory Cytokine Generation By Allicin In Mouse Adipose Tissue

ObjectivePathophysiology of obesity-induced insulin resistance is associated with the activation of inflammatory signaling pathway and the release of inflammatory cytokines in adipose tissue.Allicin can decrease the blood lipid level,reducing the risk of type 2 diabetes.The objective of the present study was to uncover the molecular mechanism underlying the anti-inflammatory action of allicin,providing new ideas and methods for clinical application of allicin and medical therapy of IR-related diseases.An immunological stress model and an obesity model were employed,respectively,by in vitro and in vivo administration of lipopolysaccharide（LPS）in mice 3T3-L1 adipose cells or fat tissue,and feeding high-fat diets.The expression of a serial of key proteins involved in NF-κB and MAPK signaling pathway and the production of pro-inflammatory cytokines,such as TNF-αand IL-6,were detected with single or combined treatment of allicin.MethodsTrial 1 was conducted to investigate the effect of in vitro allicin administration on LPS-induced pro-inflammatory cytokine generation in mice adipose cells.3T3-L1pre-adipocytes were successfully differentiated into the mature adipocytes,and were randomly subjected to one of the following treatments:1)controls（+DMSO）;2)LPS（100ng/mL）;3)allicin（dissolved in DMSO,100μg/mL）;and 4)LPS+allicin（the cultured cells were preincubated with allicin for 2h,followed by a 1h incubation in the presence of LPS）.The culture supernatant was sampled,and total RNA and different protein fractions（cytoplasmic and nuclear protein extraction）were isolated.Trial 2 was carried out to determine the effect of in vivo allicin administration on LPS-induced pro-inflammatory cytokine generation in mice adipose tissue.A total of 40male Kunming mice（6-week-old）were randomly assigned to 4 groups:1)controls（injected with saline）;2)LPS（an intraperitoneal injection of 10mg/kg BW,once a day）;3)allicin（30mg/kg BW）;and 4)LPS+allicin（a combination of the 2）.There were 10 mice in each treatment,and the experiment lasted for 14 days.At the age of 8 week,mice were blood sampled,then killed.The epididymal and subcutaneous adipose tissue were rapidly removed and frozen in liquid nitrogen.Trial 3 was performed to examine the effect of in vivo allicin administration on pro-inflammatory cytokine generation in adipose tissue of obese mice.A total of 40 male Kunming mice（6-week-old）were randomly divided into 4 groups:1)low-fat diets（5%CF）;2)high-fat diets（20%CF）;3)low-fat diets+allicin（an intraperitoneal injection of30mg/kg BW,once a day;4）high-fat diets+allicin.Ten mice were included in each treatment,and the experimental duration was 3 weeks.At 9 week of age,mice were blood sampled,then killed.The epididymal and subcutaneous adipose tissue were rapidly removed and frozen in liquid nitrogen.ResultsTrial 1 showed that LPS increased concentrations of TNF-αand IL-6 in cell supernatant（P<0.05）,which returned to the control level with the combined effect of allicin.LPS upregulated（P<0.01）protein expression of p-ERK1/2(Thr²⁰²/Tyr²⁰⁴)and p-JNK(Thr¹⁸³/Tyr¹⁸⁵),but allicin suppressed the stimulation achieved by LPS.LPS increased cytoplasmic abundance of p-IκBα(Ser³²/Ser³⁶)protein,promoting NF-κBp65nuclear translocation（P<0.05）.The stimulatory effect of LPS was blocked by allicin.Trial 2 suggested that LPS exerted an inhibitory effect on BW at 7 and 8 week of age,which was offset by allicin（P<0.05）.LPS enhanced mRNA abundance of TNF-αand IL-6in epididymal and subcutaneous adipose tissue,increasing their concentrations in the serum（P<0.01）.Allicin suppressed LSP-stimulated gene expression and circulating level of inflammatory cytokines.LPS increased（P<0.05）protein abundance of p38MAPK,p-p38MAPK(Thr¹⁸⁰/Tyr¹⁸²),ERK1/2,p-ERK1/2(Thr²⁰²/Tyr²⁰⁴),JNK and p-JNK(Thr¹⁸³/Tyr¹⁸⁵),but allicin counteracted the activation of LPS on these signaling proteins in MAPK pathway.Nuclear NF-κB activation and cytoplasmic IκBαdegradation were hardly affected by LPS and allicin.Trial 3 inidcated that BW was significantly increased by high-fat diets（P<0.05）,and allicin had no anti-obesity effect.In terms of glucose（GTT）and insulin tolerance tests（ITT）,high-fat diets increased blood glucose level（P<0.05）,and allicin improved insulin sensitivity,alleviating obesity-induced hyperglycemia.High-fat diets elevated gene transcripts of TNF-αand IL-6 in epididymal and subcutaneous adipose tissue,promoting their secretion into the circulation（P<0.01）.Allicin inhibited these inflammatory responses induced by high-fat diets.Protein expression of p-p38MAPK(Thr¹⁸⁰/Tyr¹⁸²),p-ERK1/2(Thr²⁰²/Tyr²⁰⁴),and p-JNK(Thr¹⁸³/Tyr¹⁸⁵)was enhanced by feeding high-fat diets in both epididymal and subcutaneous adipose tissue（P<0.05）,a process that could be stopped by allicin.In epididymal adipose tissue,high-fat diets and allicin had a marginal influcence on NF-κBp65 nuclear translocation.ConclusionAll above results allow concluding that allicin can reduce inflammation of adipose tissue induced by LPS and high-fat diets,via depressing the activation of MAPK signaling pathway.The inconsistent activation of NF-κB pathway between in vitro and in vivo conditions needs further investigation.