Sphingosine kinase (SphK1/2) is emerging as a promising target in the field of diseases associated with excessive cell proliferation through regulation of the dynamic balance between ceramide (Cer), sphingosine (Sph), and sphingosine-1-phosphate (S1P). In this work, we synthesized six series of SphK1/2 inhibitors. 33b demonstrated high selectivity and significant inhibition towards SphK1, and showed potential in the treatment of pulmonary arterial hypertension (PAH). Meanwhile, 33i exhibited high selectivity for SphK2, effectively inhibiting the proliferation and migration of cancer cells and causing a notable arrest in the G2 phase of the cell cycle. Molecular dynamic simulations investigated the interaction of 33b with Ser254, Asp421 in SphK1 and 33i with Asp586, Ser297 and Arg321 in SphK2 is essential for their binding. Subsequently, we confirmed that 33b is a substrate competitive inhibitor of SphK1, resulting in increased Sph levels and decreased S1P levels in vitro and in vivo. Finally, 33b and 33i showed half-lives of 9.01 h and 1.89 h in liver microsomes, respectively.

Targeting Sphingosine kinase (SphK1/2) has become a novel strategy for the treatment of cancer. However, potent ATP competitive inhibitors are rare. Herein, a series of novel SphK1 and SphK2 inhibitors were identified through virtual screening and structural optimization. The structure-activity relationship revealed compound 9d had excellent inhibitory activity and selectivity on SphK1. 9d can increase the level of Sph while reducing the content of S1P in vivo and in vitro. Moreover, 9d demonstrated anti-tumor effect on various cell lines and mouse models. In addition, another compound 6a was found to have good inhibitory activity and selectivity on SphK2 and showed potent anti-proliferative activity on tumor cells. It can be studied as an inhibitor of SphK2 in the future.

Rheumatoid arthritis (RA) is a type of autoimmune disease that results in immune disorder and excessive inflammatory response due to a reduction of self-tolerance. Invariant natural killer T (iNKT) cells can effectively alleviate clinical symptoms and hyper-inflammation in RA, but their mechanism of action is not well-defined. This study aims to investigate the mechanism of iNKT cell therapy for RA. We established a DBA/1 mouse model for RA and treated it with specific iNKT cells. A cytometric bead array was used to measure the amounts of cytokines in the serum. Flow cytometry was then employed to identify different subsets of helper T cells (Th), the frequency of conventional dendritic cells (cDC), the expression of CD80, CD86, programmed cell death ligand 1 (PD-L1), and PD-L2 on cDC surfaces, and associated pathway proteins. iNKT cell treatment reduced Th1/Th2 and Th17/ regulatory T (Treg) cell ratios while increasing interleukin-4 (IL-4) and IL-10. It enhanced the generation of immature cDCs, and it upregulated the level of PD-L2 by stimulating the Signal transducer and activator of transcription 3 (STAT3) signaling pathway. Meanwhile, it activated the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and inhibited the nuclear factor kappa B (NF-κB) pathway. According to our findings, iNKT cell treatment increased the expression of phosphates STAT3 in lymph node cDC, causing them to upregulate PD-L2 molecules. While activating the ERK1/2 pathway and inhibiting the NF-κB pathway, tolerogenic cDC was produced, restoring immune homeostasis and correcting excessive inflammation. These results deliver new insights into the treatment of RA by iNKT cells.

Abstract Background CMTM6 is a key regulator of PD-L1. The purpose of this study is to see whether CMTM6 impacts iNKT cells infiltration into hepatocellular carcinoma (HCC) and whether CMTM6 knockout promotes iNKT cells activation via the PD-1/PD-L1 signaling axis, hence impacting anti-HCC efficacy. Methods Immunohistochemistry and multiplex fluorescence immunohistochemistry were conducted to evaluate the expression of CMTM6, PD-1, PD-L1, and iNKT (CD3+CD56+) cells. Flow cytometry (FCM), enzyme-linked immunosorbent assay (ELISA), cell proliferation, killing, colony formation assay, and wound healing assay were applied to investigate the effect of CMTM6 knockout on the anti-HCC efficacy of iNKT cells. Results In HCC tumor tissues, there's far more CMTM6, PD-L1, and PD-1 expression. PD-L1 and PD-1 were both positively associated with CMTM6. iNKT cells infiltration was reduced, whereas PD-1 expression was increased. Infiltration of iNKT cells was reduced by increased CMTM6 expression. CMTM6 knockout inhibited the proliferation, invasion, and migration of HepG2 cells by downregulating the PD-1/PD-L1 signaling axis and promoting the secretion of IFN-γ, Perforin, and Granzyme B of iNKT cells, boosting their killing effect, and inhibiting the proliferation, invasion, and migration of HepG2 cells. Conclusions Our findings showed that CMTM6 was more abundant in HCC tumor tissues than adjacent tissues. Knocking out CMTM6 could boost iNKT cells activation and improve anti-HCC efficacy by blocking the PD-1/PD-L1 signaling axis, which may serve as a potential therapeutic target for HCC

Epigenetic regulation affects the development and differentiation of iNKT cells. Our previous study found that the number of iNKT cells in thymus of RA mice was reduced and the ratio of subsets was unbalanced, but the related mechanism remains unclear. We adopted an adoptive infusion of iNKT2 cells with specific phenotypes and functions to RA mice and used the α-Galcer treatment group as control. The findings revealed that: 1. Adoptive treatment of iNKT cells decreased the proportion of iNKT1 and iNKT17 subsets in the thymus of RA mice, and increased the proportion of iNKT2 subsets. 2. Following treatment with iNKT cells, the expression of PLZF in thymus DP T cells was increased whereas the expression of T-bet in thymus iNKT cells was decreased in RA mice. 3. Adoptive therapy reduced the modification levels of H3Kb7me3 and H3K4me3 in the promoter regions of Zbtb16 (encoding PLZF) and Tbx21 (encoding T-bet) gene in thymus DP T cells and iNKT cells, and the reduction of H3K4me3 was particularly significant in the cell treatment group. Furthermore, adoptive therapy also upregulated the expression of UTX (histone demethylase) in thymus lymphocytes of RA mice. As a result, it is hypothesized that adoptive therapy of iNKT2 cells may affect the level of histone methylation in the promoter region of important transcription factor genes for iNKT development and differentiation, thereby directly or indirectly correcting the imbalance of iNKT subsets in the thymus of RA mice. These findings offer a fresh rationale and concept for the management of RA that targets.

目的 通过采用静脉注射α-半乳糖基神经酰胺(α-GalCer),观察其对小鼠肝脏恒定性自然杀伤T细胞(iNKT)频率及亚群的影响.方法 研究时间2021年11月至2022年2月,选取C57BL/6J健康小鼠,采用随机数字表法分为观察组和对照组,观察组静脉注射α-GalCer,对照组静脉注射生理盐水,在注射后的第2、4、6、8、10天取小鼠肝脏,采用流式细胞术检测小鼠肝脏的iNKT细胞及亚群变化情况;采用酶联免疫吸附测定(ELISA)检测外周血血清中白细胞介素-4(IL-4),干扰素-γ(IFN-γ),白细胞介素-17A(IL-17A)的表达水平.结果 与对照组相比,静脉注射α-GalCer可引起肝脏iNKT细胞频率及亚群频率变化,且差异有统计学意义(P<0.05).观察组肝脏iNKT细胞频率明显升高,但随时间呈逐渐降低趋势,自第6天恢复至与正常水平相当[观察组第2、4、6、8、10天分别为(20.87±2.15)％、(5.61±0.51)％、(2.65±0.26)％、(2.62±0.64)％、(2.51±0.11)％,对照组分别为(2.72±0.19)％、(2.72±0.19)％、(2.93±0.24)％、(3.06±0.45)％、(3.14±0.35)％];注射α-GalCer后,iNKT1亚群频率低于对照组,于第10天恢复至正常水平[观察组第2、4、6、8、10天分别为(1.04±0.37)％、(0.48±0.12)％、(1.99±1.04)％、(0.60±0.11)％、(3.25±0.53)％,对照组分别为(3.68±1.02)％、(2.74±0.21)％、(2.81±0.33)％、(2.93±0.29)％、(3.12±0.11)％];iNKT2亚群频率高于对照组,后逐渐降低[观察组第2、4、6、8、10天分别为(75.13±3.48)％、(33.83±9.08)％、(17.20±5.37)％、(12.00±1.99)％、(6.26±1.28)％,对照组分别为(38.03±1.70)％、(40.13±3.20)％、(40.16±3.15)％、(38.03±1.70)％、(36.37±1.18)％];iNKT1亚群频率总体上低于iNKT2亚群;iNKT17亚群频率低于对照组,第4天亚群频率最高,后又降至正常水平以下;与对照组相比,血清中IL-4水平在注射第2天无明显变化(P>0.05),注射后第6天,IL-4水平明显降低(P<0.05);IFN-γ水平自注射α-GalCer后明显升高,第6天又降低(P<0.05),IL-17A水平均低于正常水平(P<0.05).结论 静脉注射α-GalCer可诱导小鼠肝脏iNKT细胞的增殖,影响其亚群的分化.

目的 探讨腹腔注射 α-半乳糖苷神经酰胺(α-GalCer)对小鼠肺恒定自然杀伤 T细胞(iNKT)、细胞亚群及外周血细胞因子的影响,从而为治疗相关肺部疾病提供基础研究数据.方法 60只6～8周龄雄性C57BL/6 小鼠,采用随机数字表法分为α-GalCer干预组和正常对照组,每组30只,选择不同时间点观察干预组与对照组小鼠肺iNKT 细胞、亚群及外周血细胞因子的变化.结果 组间比较发现,与对照组(3.49±0.25)%相比,腹腔注射α-GalCer可引起肺脏iNKT细胞频率及亚群的变化,差异有统计学意义(P<0.05).α-GalCer干预组肺iNKT细胞频率前2 d(6.56±0.05)%明显升高而后[d6(2.56±0.08)%、d8(2.64±0.14)%、d10(2.37±0.24)%]降低(P<0.05).iNKT 1细胞频率低于对照组且呈降低的趋势(P<0.05).iNKT 2细胞频率高于对照组在第4天肺部iNKT2细胞频率达到峰值,随后降低(P<0.05).iNKT 17细胞频率略高于对照组,差异无统计学意义(P>0.05),与第2天相比,第4、6、8、10天iNKT17细胞频率均显著升高(P<0.05).组间比较发现,干预组与对照组外周血血清IFN-γ、IL-4、IL-17A浓度比较,差异有统计学意义,α-GalCer干预组在不同时间点IFN-γ、IL-4、IL-17A 浓度比较,差异有统计学意义,与第2天相比,IFN-γ、IL-4水平于第6天、第8天均降低(P<0.05);IL-17A 水平于第6天降低(P<0.05).α-GalCer干预组的IFN-γ水平与对照组相比均有明显升高,差异有统计学意义(P<0.05);除第8天外,α-GalCer干预组的IL-4水平与对照组比较,差异有统计学意义(P<0.05);第2天、第8天α-GalCer干预组的IL-17A水平均低于对照组,差异有统计学意义(P<0.05).结论 腹腔注射α-GalCer对小鼠肺组织 iNKT细胞和亚群有不同的激活效应;iNKT细胞激活后,会由脉管系统迁入间质;腹腔注射α-GalCer可诱导血清细胞因子的变化,提示 α-GalCer注射可影响机体免疫功能.

Objective:To investigate the mechanism of invariant natural killer T (iNKT)2 cell improving hepatic fat deposition in nonalcoholic fatty liver (NAFL).Methods:NAFL model was established by feeding C57BL/6J mice with high fat diet. The levels of serum total cholesterol, triglyceride, high density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in the peripheral blood of mice were analyzed using automatic biochemical analyzer. The pathological changes of liver were observed with HE staining. The cell frequencies of iNKT, iNKT1, and iNKT2 in liver were detected by flow cytometry. Western blotting was used to detect the expression of sterol regulatory element binding protein 1c (SREBP-1c), peroxisome proliferator activated receptor (PPAR)-α, and nuclear factor-κB (NF-κB) in liver tissues.Results:Compared with control group, the body weight of NAFL mice increased, the levels of total cholesterol, HDL-C, LDL-C, ALT, and liver fat deposition increased, the protein expression of SREBP-1c and PPAR-α in liver increased as well as the the protein phosphorylation level of NF-κB. After intraperitoneal injection of α-galactosylceramide (α-GalCer), the levels of total cholesterol, HDL-C, and LDL-C, liver fat deposition decreased, liver SREBP-1c was down-regulated, PPAR-α expression was up-regulated, and the proportion of liver iNKT2 subgroup increased in NAFL mice.Conclusion:iNKT2 cells improve NAFL liver fat deposition, which is related to the down-regulation of SREBP-1c and up-regulation of PPAR-α.

Objective:To observe the changes in percentages and subsets of invariant nature kiler T (iNKT) cells in adipose and related tissues at different stages of obesity, and analyze the role of iNKT cells during chronic inflammation in adipose tissues in a mouse model of obesity established with high-fat diet.Methods:Changes in mouse body weight, mental state, glucose tolerance and insulin tolerance were recorded. Hematoxylin and eosin (HE) staining was used to observe pathological changes in adipose tissues. Flow cytometry was performed to detect the percentages and subsets of iNKT cells as well as the percentages and subtypes of macrophages. The levels of cytokines in serum samples and the culture supernatants of lymphocytes in adipose tissues were detected with CBA. The expression of related proteins in adipose tissues was detected by Western blot.Results:(1) The volume of adipose cells increased significantly after four weeks of high-fat feeding, but the infiltration of inflammatory cells was not obvious. Significantly increased infiltration of inflammatory cells was observed after 12 weeks of high-fat feeding. (2) High-fat feeding could reduce the percentage of iNKT cells, increase the proportion of iNKT1 subgroup and decrease the proportion of iNKT10 subgroup in adipose tissues. The proportion of iNKT1 subgroup in thymus increased, but that of iNKT2 subgroup decreased. The percentage of macrophages and the proportion of M1 subgroup in adipose tissues increased, while the proportion of M2 subgroup decreased, which were more obvious after 12 weeks of high-fat feeding. (3) High-fat feeding resulted in decreased expression of E4BP4 and arginase-1 (Arg-1) in adipose tissues and increased expression of inducible nitric oxide synthase (iNOS). (4) High-fat feeding significantly increased the pro-inflammatory cytokines and decreased the anti-inflammatory cytokines in mouse serum and culture supernatants of lymphocytes in adipose tissues with more significant changes observed after 12 weeks of high-fat feeding.Conclusions:Increased iNKT1 and decreased iNKT10 in obese adipose tissues might be closely related to the increased M1 polarization and the imbalance of iNKT subsets might be involved in the progression of chronic inflammation in obese adipose tissues.

The rates of invariant natural killer T (iNKT) cells in vivo are very low, and the amounts of cells obtained directly from the body are hard enough to fulfill their potential in clinical application. To overcome this problem, we subcutaneously injected alpha-galactosylceramide (α-GalCer) into DBA/1 mice and thymic single cells were isolated and cultured in vitro. Fluorescence-activated cell sorting was used to detect the iNKT cells and their subsets in the thymus after the injection of α-GalCer by different methods. In addition, in vitro changes of single-cell suspensions and their cytokines in culture supernatants were assessed. Compared with the α-GalCer multiple subcutaneous injection group, the rates of iNKT cells in the α-GalCer single subcutaneous injection group were markedly higher at each time point, while the highest levels of iNKT1 and iNKT2 cells were observed on day 4 and 8, respectively. In α-GalCer single subcutaneous injection for 8 days and thymic mononuclear cell cultured for 14 days group, the expansion rate of iNKT cells was significantly faster than the other groups, while it reached a peak for iNKT1 cells. Interferon-gamma was consistent with the development of iNKT1 cells, however no difference was found between the cultured iNKT cells in vitro and the natural iNKT cells in vivo in terms of cytokine production. Herein, we introduced a method in which antigenic stimulation in vivo and directed induction in vitro yielded high levels of iNKT cells with specific functions.

Herein, the migration distribution and safety of specific phenotypic and functionally identified spleen-derived invariant natural killer T2 (iNKT2) cells after adoptive infusion in mice were studied. The proliferation and differentiation of iNKT cells were induced by intraperitoneal injection of α-galactosylceramide (α-GalCer) in vivo. Mouse spleens were isolated in a sterile environment. iNKT cells were isolated by magnetic-activated cell sorting columns (MS columns). Cytometric bead array (CBA) assay was used to detect cytokine secretion in the supernatant stimulated by iNKT cells. The basic life status of the mice was observed, and systematic quantitative scoring was conducted after injecting spleen-derived iNKT cells through the tail vein. An in vivo imaging system was used to trace the migration and distribution of iNKT cells in DBA mice. The percentage of the iNKT2 subgroup was the highest in 3 days after intraperitoneal injection of α-GalCer, and iNKT2 subsets accounted for more than 92% after separation and purification by magnetic-activated cell sorting (MACS). Anti-inflammatory cytokine IL-4 was mainly found in the supernatant of cell cultures. The adoptive infusion of iNKT cells into healthy mice resulted in no significant change in the basic life status of mice compared with the noninjected group. iNKT cells were detected in the lung, spleen, and liver, but no fluorescence was detected in lymph nodes and thymus. After dissecting the mice, it was found that there were no significant abnormalities in the relevant immune organs, brain, heart, kidney, lung, and other organs. Intraperitoneal injection of α-GalCer results in a large number of iNKT2 cells, mainly secreting anti-inflammatory cytokine IL-4, from the spleen of mice. After adoptive infusion, the iNKT2 cells mainly settled in the liver and spleen of mice with a satisfactory safety profile.

探讨了腹腔注射α-GalCer对非酒精性脂肪性肝病(NAFLD)的影响,为以iNKT细胞为靶点的NAFLD免疫学防治奠定基础.利用高脂饲料或蛋氨酸/胆碱缺乏饲料分别喂养C57BL/6J小鼠,建立非酒精性脂肪肝(NAFL)模型和非酒精性脂肪性肝炎(NASH)模型.全自动生化分析仪分析小鼠外周血中TCH、TG、HDL、LDL、ALT及AST水平;H&E染色观察肝脏病理变化;流式细胞术检测肝脏iNKT、iNKT1、iNKT2细胞频率.与对照组相比,NAFL小鼠体质量升高,血清TCH、HDL、LDL及ALT水平升高,肝脏脂肪沉积增加;而NASH小鼠体质量降低,血清TCH、TG及LDL水平降低,肝脏脂肪沉积增加并伴有炎细胞浸润.腹腔注射α-GalCer后,NAFL小鼠血清TCH、HDL及LDL水平降低,肝脏脂肪沉积减少,肝脏iNKT2亚群比例增加;NASH小鼠血清ALT和AST水平升高,肝脏脂肪沉积减少,炎细胞浸润增加,肝脏iNKT1、iNKT2亚群比例增加,其中以iNKT1亚群增加为主.腹腔注射α-GalCer可以改善NAFL和NASH小鼠肝脏脂肪沉积;小鼠NAFLD发病不同阶段(NAFL或NASH)肝脏局部微环境的改变可能影响α-GalCer对肝脏iNKT细胞不同亚群的激活,为进一步研究NAFLD的发病机制及免疫学治疗策略提供了有价值的实验数据.

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T (iNKT) cells. Patients with active RA present fewer iNKT cells, defective cell function, and excessive polarization of Th1. In this study, an RA animal model was established using a mixture of hGPI325-339 and hGPI469-483 peptides. The iNKT cells were obtained by in vivo induction and in vitro purification, followed by infusion into RA mice for adoptive immunotherapy. The in vivo imaging system (IVIS) tracking revealed that iNKT cells were mainly distributed in the spleen and liver. On day 12 after cell therapy, the disease progression slowed down significantly, the clinical symptoms were alleviated, the abundance of iNKT cells in the thymus increased, the proportion of iNKT1 in the thymus decreased, and the levels of TNF-α, IFN-γ, and IL-6 in the serum decreased. Adoptive immunotherapy of iNKT cells restored the balance of immune cells and corrected the excessive inflammation of the body.

Whether different injection modes of α-galactosylceramide (α-GalCer) affect the activation of different subsets of invariant natural killer T (iNKT) cells in different tissues and organs of mice is unclear. This study included healthy control, subcutaneous injection, and intraperitoneal injection groups (n=10 in each group). The subcutaneous and intraperitoneal injection groups were injected with α-Galcer (0.1 mg/kg weight), and then the changes in thymus, spleen, and liver iNKT cell frequencies and subsets were observed. The intraperitoneal injection of α-GalCer could increase the frequency of splenic iNKT cells, but the subcutaneous injection did not affect the frequency. Neither injection had any effect on the frequency of iNKT cells in the thymus and liver. The subcutaneous injection of α-GalCer increased the rate of iNKT2 subsets in the thymus but did not affect the rate of iNKT1 subsets. However, the intraperitoneal injection of α-GalCer did not affect thymus iNKT1 and iNKT2 subsets. Interestingly, the subcutaneous injection of α-GalCer significantly increased the proportion of iNKT1 in the spleen and liver but did not significantly change the proportion of iNKT2. The intraperitoneal injection of α-GalCer significantly increased the rate of iNKT2 in spleen and liver but decreased the rate of iNKT1. Subsets of iNKT1 or iNKT2 cells in the spleen and liver were selectively activated by the subcutaneous or intraperitoneal injection of α-GalCer. It provides a valuable means for treating tumors and certain autoimmune diseases. Further exploration of the activation mechanism may provide new ideas about the development of related vaccines.

Epigenetic modifications have been shown to be important for immune cell differentiation by regulating gene transcription. However, the role and mechanism of histone methylation in the development and differentiation of iNKT cells in rheumatoid arthritis (RA) mice have yet to be deciphered. The DBA/1 mouse RA model was established by using a modified GPI mixed peptide. We demonstrated that total peripheral blood, thymus, and spleen iNKT cells in RA mice decreased significantly, while iNKT1 in the thymus and spleen was increased significantly. PLZF protein and PLZF mRNA levels were significantly decreased in thymus DP T cells, while T-bet protein and mRNA were significantly increased in thymus iNKT cells. We found a marked accumulation in H3K27me3 around the promoter regions of the signature gene Zbtb16 in RA mice thymus DP T cells, and an accumulation of H3K4me3 around the promoters of the Tbx21 gene in iNKT cells. The expression levels of UTX in the thymus of RA mice were significantly reduced. The changes in the above indicators were particularly significant in the progressive phase of inflammation (11 days after modeling) and the peak phase of inflammation (14 days after modeling) in RA mice. Developmental and differentiation defects of iNKT cells in RA mice were associated with abnormal methylation levels (H3K27me3 and H3K4me3) in the promoters of key genes Zbtb16 (encoding PLZF) and Tbx21 (encoding T-bet). Decreased UTX of thymus histone demethylase levels resulted in the accumulation of H3K27me3 modification.

ObjectiveThe role of iNKT cells was investigated in chronic adipose tissue inflammation in obese mice after administration of α-GalCer in different pathways.MethodsC57BL/6J mice were fed high-fat diet (HFD) for 12 weeks to establish the obese mouse model. The pathology of adipose tissue was observed by H&E staining. The rates of iNKT cells, macrophages and cell subsets in adipose tissue were detected by FCM. Cytokine levels in serum and adipose tissue lymphocyte-stimulated supernatants were assessed with the CBA kit. The expression levels of related transcription factor in adipose tissue were detected by Western blot.ResultsThe proportions of iNKT cells, iNKT10 cells and M2 macrophages were decreased, while those of iNKT1 and M1 macrophages were increased in adipose tissue of HFD-fed mice. The expression levels of the related transcriptional proteins E4BP4 and Arg-1 were decreased while iNOS expression was increased in adipose tissue. Administration of α-GalCer by subcutaneous injection resulted in increased rates of iNKT10 cells and M2 macrophages, and decreased amounts of M1 macrophages in adipose tissue of HFD-fed mice. The expression of E4BP4 and Arg-1 were up-regulated, but iNOS was down-regulated. Meanwhile, infiltration of inflammatory cells into adipose tissue was further reduced.ConclusionThe imbalance between the proportions of iNKT1 and iNKT10 cells may be involved in the development of chronic inflammation in obese adipose tissue. Administration of α-GalCer by subcutaneous injection in HFD-fed mice activates adipose tissue iNKT10 cells, which promote M2 macrophage polarization and improve chronic inflammation in obese adipose tissue.

The existence of association between the subpopulation of iNKT cells with different functions and nonalcoholic fatty liver disease has not been confirmed. To investigative the role of iNKT cells in the pathogenesis of nonalcoholic fatty liver disease, we established a non-alcoholic fatty liver model by feeding C57BL/6J mice for 12 weeks with a high-fat diet and injecting α-GalCer through different routes to activate hepatic iNKT cells. The liver of the mice fed a high-fat diet (HFD) had severe hepatic steatosis appearance, elevated pro-inflammatory cytokines and reduced anti-inflammatory cytokines in the liver, and high serum levels of TC, LDL, HDL, and ALT. Our results showed that the percentage of iNKT cells in the liver of the HFD-fed mice was lower than that of the control mice. The expression levels of the related transcription factor of T-bet increased but that of GATA-3 decreased in the HFD-fed mice. The administration of α-GalCer by intraperitoneal injection resulted in increasing of hepatic iNKT and iNKT2 cells but decreasing of hepatic iNKT1 cells, and the expression of GATA-3 and anti-inflammatory cytokine (IL-4) was increased in the liver, and hepatic steatosis was ameliorated in the HFD-fed mice. The administration of α-GalCer by subcutaneous injection resulted in a decrease in hepatic iNKT and iNKT2 and an augmentation of hepatic iNKT1 cells. However, hepatic steatosis was not significantly improved. We concluded that the intraperitoneal injection with α-GalCer effectively improved hepatic steatosis, according to increasing the number of hepatic iNKT2 cells. The precise mechanism requires further exploration.

Background: The metabolic enzyme isocitrate dehydrogenase 1 (IDH1) belonging to β-decarboxylase dehydrogenase family has been identified as a tumor suppressor. Withaferin A (WA), a bioactive compound derived from Withania somnifera , has the anti-tumor activity. Based on the data set that WA inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced IDH1 inactivation and mitochondrial dysfunction, we focused on how WA suppressed the skin carcinogenesis mediated by IDH1. Methods: The mRNA levels of IDH1 were measured after treated with TPA and/or WA. The expression of IDH1, lactate dehydrogenase (LDH) involved in glycolysis, hypoxia inducible factor-1α (HIF-1α) and its target gene glucose transporter-1 (Glut1) were detected. The activities of proteasome and the mitochondrial complex I related to mitochondrial functions were determined. The enzymatic activities of LDH, proline hydroxylase (PHD) and vascular endothelial growth factor (VEGF) were analyzed. Results: The qPCR data have shown the mRNA levels of IDH1 were no difference with TPA and/or WA treatment. Next, data demonstrated that WA could stabilize IDH1 by inhibiting the ubiquitin-proteasome pathway (UPP). Followed by illuminating the mechanism of IDH1 inhibiting tumorigenesis, the results mirrored that upregulated IDH1 suppressed LDH activity whereas increased mitochondrial complex I activity. Furthermore, via its product α-KG, upregulated IDH1 activated PHD, and inhibited HIF-1α and its downstream signaling pathway. Conclusions: Our results indicate that WA inhibits tumor promotion partially via stabilizing IDH1, leading to inactivating the HIF-1α signaling.

目的:通过对NOD/Ltj小鼠在未发病、发病初期与发病末期不同组织器官中CD4+T、CD8+T细胞,Th1、Th2、Th17亚群,iNKT细胞频率及亚群,细胞因子、相关转录因子进行观察分析,进一步了解NOD/Ltj小鼠Ⅰ型糖尿病不同发病阶段细胞免疫功能状态.方法:选用雌性NOD/Ltj小鼠为实验对象.血糖仪检测小鼠空腹血糖值,根据尿糖阳性且连续2次≥11.1 mmol/L作为T1D发病标准将动物分为未发病组、发病初期组、发病末期组.流式细胞技术(FCM)检测各组小鼠外周血、胸腺、脾脏、肝脏中CD4+T、CD8+T细胞,Th1、Th2、Th17亚群,iNKT细胞频率及亚群比例以及腹股沟淋巴结CD4+T、CD8+T细胞;CBA检测IFN-γ、TNF-α、IL-2、IL-6、IL-17A、IL-4、IL-10;WB检测PLZF、T-bet、GATA-3、ROR-γt.结果:①与未发病组比较,发病初期组CD4+、CD8+T细胞频率在脾脏、肝脏、胸腺、腹股沟淋巴结中均显著增加(P<0.05);与发病初期组比较,发病末期CD4+T细胞频率在肝脏、胸腺、腹股沟淋巴结及外周血中均显著降低(P<0.05).②在脾脏、肝脏中,与未发病组和发病初期组比较,发病末期组Th1亚群比例显著增加(P<0.05);在肝脏中,与发病初期组比较,发病末期组Th2、Th17亚群水平显著升高(P<0.05).③与未发病组比较,发病初期组肝脏、腹股沟淋巴结中iNKT细胞频率均显著增高(P<0.05);与发病初期组比较,发病末期组外周血、肝脏中iNKT细胞频率显著降低(P<0.05);与未发病组比较,发病初期组和发病末期组胸腺iNKT1亚群比例均显著增加,iNKT2亚群比例均显著降低(P<0.05),脾脏、肝脏、腹股沟淋巴结iNKT1及iNKT2亚群比例三组两两比较均差异均无统计学意义(P>0.05).④在脾脏和腹股沟淋巴结中致炎性细胞因子和抑炎性细胞因子水平在发病初期较未发病组和发病末期组均显著升高(P<0.05);在肝脏中致炎性细胞因子水平随小鼠病情进展逐渐升高,两两比较差异均有统计学意义(P<0.05);抑炎性细胞因子水平在发病初期最高,发病末期显著降低(P<0.05).⑤胸腺PLZF相对表达量,三组两两比较差异均无统计学意义(P>0.05);脾脏和肝,与未发病组和发病初期组比较,发病末期组T-bet相对表达量显著增加(P<0.05).结论:①发病初期CD4+T和CD8+T细胞的增加,特别是CD4+T细胞的增加以及Th亚群的失衡是导致胰岛炎重要的免疫基础;②发病初期iNKT细胞频率的增加以及胸腺iNKT1/iNKT2亚群比例的翻转,提示了iNKT细胞在NOD/Ltj小鼠发病初期可能参与了T1 D的发生.

肥胖症是一种多因素引起的慢性代谢性功能紊乱疾病,炎症在肥胖和代谢综合征中起重要的作用.肥胖所伴随的低度慢性炎症,与冠心病、 高血压、 糖尿病等紧密相关.近来研究发现恒定自然杀伤T(invariant natural killer T,iNKT)细胞在脂肪组织炎症中发挥重要作用,并一度被认为是肥胖脂肪组织炎症的主要参与者,其在慢性炎症中的研究也越来越受到人们的关注.在脂肪组织中,激活iNKT是通过脂肪细胞上面的CD1d来呈递脂质抗原,同时iNKT细胞产生的细胞因子又会调节脂肪细胞的功能,以此来改变抗炎脂肪因子的表达以及炎症性和趋化因子的产生.因此,脂肪细胞与iNKT细胞之间的相互作用可能不仅影响肥胖,还会影响肥胖相关疾病的发展,但iNKT细胞到底是促进还是抑制肥胖脂肪组织炎症的发生发展呢?目前具体作用机制还没有详细阐明.在该综述中,总结并讨论了iNKT细胞在肥胖脂肪组织慢性炎症中的最新研究进展,从而为后续肥胖及肥胖相关性疾病研究做理论支撑和基础准备.