Regulation Function Of Heptamethine Cyanine Small-molecule IR-61 On Chronic Inflammation In Obese Mice And A Preliminary Study On Colitis-associated Tumorigenesis

Background:Obesity and obesity-associated metabolic syndromes represent a global health problem.According the report of the World Health Organization in 2014,there are 1.9 billion overweight adults and 600 million obese adults in the world,accounting for 39%and 13%of the global adult population respectively.Obesity increases the risk of various metabolic diseases,including type 2 diabetes and hepatic steatosis.Moreover,a recent study estimated that 3.6 percent of new cancer cases worldwide are caused by obesity.Among them,colorectal cancer(CRC)is closely related to obesity,and about 63.6%of the incidence of CRC is attributed to obesity.A growing number of findings have shown that systemic chronic inflammation is an important feature of obesity.Persistent chronic inflammation also leads to obesity and obesity-related metabolic diseases.Inflammation is an important component of tumor initiation and development,and obesity-induced inflammation carries an additional risk of cancer beyond obesity itself.Anti-inflammatory agents are considered as a new treatment strategy for obesity-related disorders and have been tested in animal models and human clinical trials for more than a decade.However,these therapies have not shown significant progress in either model system and result in side effects causing by the distribution in off-target tissue,such as Cushing's syndrome,gastrointestinal bleeding and immunosuppression.The lack of more selective and effective anti-inflammatory agents for obesity treatment highlights the need to develop novel therapeutic targets.Adipose tissue is the warehouse of long-term energy storage of human beings.It stores energy in the form of lipid to maintain physiological homeostasis.Adipose tissue is considered to be the largest endocrine organ of human body,which can secrete more than 50different adipokines,cytokines and chemokines,and is at the intersection of metabolism and immunity.Visceral adipose tissue(VAT)is the main tissue that produces pro-inflammatory cytokines in obesity.Over-expanded VAT causes fat dysfunction and inflammation and increases the level of pro-inflammatory factors throughout the body,thus plays an important role in systemic inflammation and obesity-related diseases.Adipose tissue macrophages(ATMs)activation plays a decisive role in adipose tissue inflammation.Macrophages account for 10-15%of the cell population in normal adipose tissue and mainly present with an anti-inflammatory phenotype(M2-like).However,in the state of obesity,macrophages constitute approximately 40-50%of adipose tissue cells and display a pro-inflammatory phenotype(M1-like).These pro-inflammatory ATMs secrete a range of pro-inflammatory cytokines,causing both local and systemic inflammation.Moreover,due to the critical role of inflammation in tumorigenesis,ATMs may be a novel target for suppressing obesity-associated CRC tumorigenesis.Mitochondria have long been considered as organelles that only produce energy,but in the past two decades,a lot of studies have shown that mitochondria are also signaling organelles,which play a vital role in cell proliferation,death and differentiation.Recent studies have suggested that mitochondrial metabolism plays an important role in maintaining and changing the inflammatory phenotype of macrophages.M1 macrophages undergo metabolic changes that drive them toward glycolysis and exhibit minimal reliance on mitochondrial oxidative phosphorylation.In contrast,M2 macrophages mainly depend on oxidative phosphorylation and have increased oxygen consumption.Previous research has shown that reduced oxidative phosphorylation of macrophages caused exacerbated inflammation and insulin resistance in obese mice.Therefore,targeting oxidative phosphorylation in macrophages holds promise for therapeutic treatment of obesity-related disorders.However,due to the lack of methodology or agents capable of simultaneously targeting ATMs and their oxidative phosphorylation,treatment is still challenged by potential off-target effects.Furthermore,it is not clear if potentiating the oxidative function of ATMs would improve obesity-related disorders.As a potential therapeutic target for obesity-associated disorders,ATMs have attracted extensive attention.Nanomedicine currently offers a potential solution for the off-target effects of traditional drugs.Tissue-specific agent delivery can be realized through molecular and cellular targeting,thereby reducing off-target side effects.To obtain therapeutic agents targeting ATMs,current strategies mainly explore chemical drugs conjugated to various functional nanocarriers that are recognized by macrophage receptors.However,the wide application of these targeted nanocarriers is usually limited by high cost,instability,toxic effects and so on.Therefore,it is of great significance to design and develop structure-inherent small molecules targeting ATMs for obesity treatment.A lipophilic cationic near-infrared(NIR)fluorescence heptamethine cyanine dye termed IR-61 has been identified with the structure-inherent characteristic of mitochondria targeting.More importantly,previous study has demonstrated that IR-61 significantly alleviates cell damage from acute oxidative stress by activating the cellular antioxidant system and possesses potential anti-inflammatory properties.Here,we find that IR-61 has the inherent properties of ATMs targeting and suppressing the pro-inflammatory activation of macrophages.Basing on these special properties,we explore its possible regulatory mechanism of suppressing inflammation and study the role of IR-61 on obesity-related inflammation and colitis-associated tumorigenesis in obese mice,which provides a reference for the development of new strategies for the treatment of obesity-related disorders and tumorigenesis of CRC.Methods and Results:1 The distribution of IR-61 in mice and identification its ATMs targeting characteristics,and study subcellular localization and uptake pathways of IR-61 in macrophages.1.1 IR-61 was injected into obese mice via intraperitoneal administration.We found that IR-61 predominantly accumulated in VAT.Results of flow cytometry and immune fluorescence indicated that IR-61 was highly internalized by ATMs.1.2 IR-61 was further indicated to target mitochondria of macrophages.To determine the specific endocytotic pathway involved in IR-61 internalization,we performed a series of IR-61 uptake assays in the presence of biochemical inhibitors to block specific pathways.The results showed that IR-61 was internalized via macropinocytosis,clathrin-dependent or caveolae-dependent pathways.2.Identification the effect of IR-61 on macrophage pro-inflammatory activation and exploration the potential influence of IR-61 on mitochondrial function of macrophages.2.1 To investigate the role of IR-61 in macrophage activation,we treated RAW264.7cells with IR-61 and performed gene microarray analysis,which indicated that IR-61 reduced the expression of a panel of pro-inflammatory molecules.We also detected these genes in IR-61-treated bone marrow derived macrophages(BMDMs)using quantitative reverse transcription-polymerase chain reaction(q RT-PCR)and obtained the same results as the gene microassay.2.2 Moreover,we demonstrated that IR-61 reduced the expression and secretion of pro-inflammatory cytokines in M1 macrophages induced by Lipopolysaccharide(LPS)using q RT-PCR and Enzyme-linked immunosorbent assay(ELISA).Western-blot was carried out to confirm the effect of IR-61 suppressing the phosphorylation of classical pro-inflammatory signaling molecules(p65,JNK)of LPS-stimulated macrophages.Moreover,IR-61 did not induce cytotoxicity or cell apoptosis/necrosis,which excluded cell death as an explanation for the effects observed on inflammatory-related gene expression.2.3 IR-61 was confirmed to enhance mitochondrial function and increase ATP production of macrophages.Furthermore,we pretreated macrophages with FCCP,a potent uncoupler of mitochondrial oxidative phosphorylation.We demonstrated that the IR-61-induced reduction in pro-inflammatory cytokine in macrophages was blocked by FCCP.3.Study the potential mechanism of IR-61 regulating mitochondrial function in macrophages.3.1 In order to explore the mechanism of IR-61 enhancing mitochondrial function,macrophages were treated with IR-61,and then q RT-PCR was used to detect the expression of genes related to mitochondrial generation and mitochondrial DNA copy number in macrophages.Flow cytometry was used to detect the mitochondrial content after Mitochracker Green staining.All these results showed that IR-61 did not affect the mitochondrial mass and content of macrophages.The content of mitochondrial complex and supercomplex was detected by western-blot and blue native polyacrylamide gel electrophoresis(BN-PAGE),and these results showed that IR-61 increased the content of both mitochondrial complex and supercomplex in macrophages.Mitochondrial complex activity assay kit was used to detect complex activity,and we found that IR-61 increased the activity of mitochondrial complex I and IV in macrophages.3.2 We next explored the upstream pathway of IR-61 regulating mitochondrial complex content and activity.Since both Akt and PKA could promote the phosphorylation of Acly at Ser⁴⁵⁵,the phosphorylation level of Acly in macrophages pretreated with PI3K/Akt inhibitor(LY294002)and PKA inhibitor(H89)was detected.These results showed that IR-61promoted Acly phosphorylation through Akt.Flow cytometry analysis showed that IR-61transiently and mildly promoted mitochondrial ROS elevation.Then macrophages were pretreated with intracellular ROS inhibitor(NAC)and mitochondrial ROS inhibitor(Mn TMPYP,SS-31),respectively.Western-blot results showed that IR-61 promoted the phosphorylation of Akt-Acly through ROS.3.3 Acly in macrophages was knocked down to explore whether Acly mediated the function of IR-61 regulating mitochondrial function and suppressing macrophage pro-inflammatory activation.Mitochondrial complex content was detected by western-blot,mitochondrial complex activity assay kit was used to detect the complex activity,and the mitochondrial oxidative phosphorylation level was detected by seahorse.These results indicated that IR-61 modulated the mitochondrial function of macrophages through Acly.The results of q RT-PCR indicated that IR-61 inhibited the pro-inflammatory activation of macrophages through Acly.4.Study the anti-inflammatory effect and the improvement of obesity-associated disorders of IR-61 in obese mice.And further study the effect of IR-61 on colitis-associated CRC tumorigenesis in obese mice.4.1 Male C57 mice were divided into four groups.They were given normal chow diet(NCD)+PBS,NCD+IR-61,high-fat diet(HFD)+PBS and HFD+IR-61,respectively.We found that IR-61 suppressed inflammation in VAT and plasma of obese mice.4.2 Weight of mice in each group was measured and recorded weekly,and we found that IR-61 inhibited weight gain of obese mice.After the mice were sacrificed,the weight of vital organs was measured,and the level of triglyceride(TG)and total cholesterol(TC)in serum was detected.These results indicated that IR-61 reduced VAT and liver weight and decreased the level of TG and TC in obese mice.In order to clarify the mechanism of IR-61 to reduce the weight of obese mice,the food intake of each mouse per week was monitored.Energy expenditure and activity of mice were measured using the CLAMS(Columbus Instruments)open-circuit indirect calorimetry system.We found that IR-61 inhibited weight gain of obese mice by increasing energy expenditure.4.3 The effect of IR-61 on insulin sensitivity of mice was detected by glucose tolerance test(GTT)and insulin tolerance test(ITT).After intraperitoneal injection of insulin,VAT,liver and skeletal muscle of obese mice were collected to detect the phosphorylation level of Akt using western-blot.These results showed that IR-61 improved insulin resistance in obese mice.4.4 The mice were treated with azoxymethane(AOM)+1.5%dextran sulfate(DSS)to induce colitis-associated CRC,and the effect of IR-61 on colon inflammation and CRC tumorigenesis was detected.IR-61 was demonstrated to suppress pro-inflammatory cytokines expression of colon and prevent CRC tumorigenesis in obese mice.Conclusion:1 In vivo distribution studies showed that IR-61 targeted to VAT in obese mice after intraperitoneal injection and was preferentially accumulated in ATMs.IR-61 was internalized via macropinocytosis and clathrin-dependent or caveolae-dependent pathways and exclusively accumulated in the mitochondria of macrophages.2 The effect of IR-61 on suppressing the pro-inflammatory activation of macrophages was clarified.We demonstrated that IR-61 enhanced the mitochondrial function and promoted ATP production in macrophages.3 IR-61 increased the content of mitochondrial complex and supercomplex and promoted the activity of mitochondrial complex I and IV in macrophages.Mechanism study showed that IR-61 temporarily and mildly increased mitochondrial ROS and thus promoted Akt-Acly phosphorylation.Acly mediated the role of IR-61 in regulating mitochondrial complex and inhibiting the pro-inflammatory activation of macrophages.4 In vivo studies showed that IR-61 inhibited VAT and systemic inflammation in obese mice.IR-61 inhibited weight gain by promoting energy expenditure in obese mice.GTT,ITT and acute insulin tests showed that IR-61 improved insulin resistance.Furthermore,IR-61alleviated AOM/DSS-induced CRC tumorigenesis and colon inflammation in obese mice.In conclusion,we found that IR-61 possessed inherent ATMs-targeting property,which regulated the content and activity of mitochondrial complex through the ROS-Akt-Acly pathway,promoted oxidative phosphorylation function,and thereby inhibited the pro-inflammatory activation of macrophages.In vivo studies showed that IR-61 reduced pro-inflammatory cytokines levels locally and systemically and improved obesity-associated disorders in obese mice.Meanwhile,IR-61 alleviated colon inflammation and inhibited the colitis-associated tumorigenesis in obese mice.