| ObjectiveIL-37 is a new member of the IL-1 family and was first discovered in 2000.Unlike the pro-inflammatory effects of other members of the IL-1 family,IL-37 exerts an anti-inflammatory effect.IL-37 gene is located in the cluster region of the IL-1 family which on the long arm of human chromosome 2,and there are 6 exons.Exons 4,5 and 6 encode 12 β-helices to form the IL-1 family unique beta clover structure,which is required for IL-37 to function biologically.According to the different exons of composition,IL-37 is divided into five subtypes a,b,c,d,and e.Among them,IL-37b is composed of exons 1,2,4,5,and 6 and is the longest functional subtype;IL-37d consists of exons 1,4,5,and 6 and only exon 2 is less than IL-37b.The current research is mainly focused on IL-37b,and there is still little research on IL-37d.Our laboratory uses IL-37d transgenic mice prepared by ourselves and firstly proved that IL-37d is a functional subtype in the world.IL-37d has the same anti-inflammatory effect as IL-37b,but its molecular pathway is not identical and unique.However,the other functions and detailed mechanisms of IL-37d are unclear.It has been reported that overexpression of IL-37b can reduce the phosphorylation level of rapamycin target protein(mTOR)by protein microarray,suggesting that IL-37 can inhibit mTOR activity,but the detailed mechanism of IL-37b on mTOR and the meaning of the function is not clear.mTOR belongs to the PI3K-associated protein kinase(PIKK)family and is a serine/threonine kinase.mTOR exists as a catalytic subunit in the form of two different complexes-mTORC1 and mTORC2,of which mTORC1 has more wide functions and received widespread attention.Since the mTOR signaling pathway can sense signals such as energy,nutrition,stress and growth factors,which plays an important role in many processes such as cell growth and metabolism,suggesting that IL-37 plays a role in cell growth and metabolism in addition to anti-inflammatory effects.So,whether IL-37d have a role in regulating the mTORCl signaling pathway?What is the regulatory mechanism and what is the role in cell metabolism,especially lipid metabolism?It is a scientific issue that worth exploring.The research objectives of this paper include the following three aspects:1.Using mTORC1 as the study point,determine the regulation of IL-37d on mTOR signaling pathway;2.Explore the specific molecular mechanism of IL-37d regulating mTORCl signaling pathway;3.Using IL-37d transgenic mice and liver lipid accumulation model,the regulation of IL-37d on mTORC1 signaling pathway and lipid accumulation in liver was studied in vivo.Further reveal the function and mechanism of IL-37 and provide a new experimental basis for the future clinical application of IL-37.MethodsⅠ.Effect of IL-37d on mTORCl signaling pathway1.A549 cells were infected IL-37d or NC lentivirus,respectively,and cell size was detected by flow cytometry.2.A549 cells were infected IL-37d or NC lentivirus,respectively,and the phosphorylation of molecule S6 downstream of mTORCl signaling pathway was detected by flow cytometry and western blot in basal state,starvation state or starvation reactivation state.3.A549 cells were infected IL-37d or NC lentivirus,respectively,and the phosphorylation of molecule S6 was detected by flow cytometry in the starvation by using serum-free medium,glutamine-free medium or EBSS starvation,respectively,followed by normal medium containing serum activation.4.IL-37 was silenced in HepG2 cells and the level of p-S6 was detected by immunofluorescence.5.IL-37 was silenced in HepG2 cells and the level of p-S6 and p-4eBP1 were detected by Western blot in the basal state,starvation state or starvation reactivation state.Ⅱ.Molecular mechanism of IL-37d regulating mTORCl signaling pathway1.Co-IP experiments was used to investigate whether IL-37d interacts with Rheb.2.Immunofluorescence experiments was used to investigate whether IL-37d co-localized with Rheb.3.A549 cells were infected IL-37d or NC lentivirus,respectively,and the form of GTP-Rheb was detected by immunofluorescence.4.The IL-37d plasmid with different concentration gradients was overexpressed in the HEK-293T cells,and the interaction of exogenous Rheb to endogenous mTOR was detected by immunoprecipitation.5.The cells were tansfected with IL-37d on Rheb plasmids and the level of p-S6 was detected by flow cytometry and western blot in basal state.6.The cells were tansfected with IL-37d and active Rheb plasmids and the cells were starvated by using serum-free,arginine-free and leucine free medium for 2 hours and the level of p-S6 was detected by flow cytometry and western blot.7.The cells were tansfected with IL-37d and active Rheb plasmids and the cells were starvated by using EBSS buffer for 2 hours and the level of p-S6 was detected by flow cytometry.Ⅲ.IL-37d inhibits liver lipid accumulation mediated by mTORCl signaling pathway(Ⅰ).IL-37d inhibits high fat diet induced activation of mTORCl signaling pathway and liver lipid accumulationSix to eight weeks male IL-37d transgenic mice and WT mice were selected for high-fat diet(60%fat)for 18 weeks and then the mice were dissected.1.Mice body weight and liver weight were detected.2.The liver tissue sections of the mice were taken and HE staining was performed to observe the morphology.3.Liver tissue paraffin sections of mice were taken and the expression of p-S6 was detected by immunohistochemical staining.4.Mice liver tissue proteins were extracted and the expression of p-S6 was detected by Western-blot.5.The mice liver tissue homogenate was taken to detect the content of triglyceride(TG),total cholesterol(TC)and free fatty acid(FFA).(Ⅱ).IL-37d inhibits activation of mTORCl signaling pathway and short-term lipid accumulation in liver induced by fasting for 16 hoursSix to eight weeks male IL-37d transgenic and WT mice were selected for fasting 16 hours and the mice were dissected immediately after then.1.Mice body weight and liver weight were detected.2.The liver tissue sections of the mice were taken and HE staining was performed to observe the morphology.3.Liver tissue paraffin sections of mice were taken and the expression of p-S6 was detected by immunohistochemical staining.4.Mice liver tissue proteins were extracted and the expression of p-S6 was detected by Western-blot.5.The mice liver tissue homogenate was taken to detect the content of triglyceride(TG),total cholesterol(TC)and free fatty acid(FFA).ResultsⅠ.IL-37d inhibits mTORCl signaling pathway(Ⅰ).IL-37d inhibits cell sizeThe mTORCl signaling pathway is known to control cell growth and cell size.To explore the effect of IL-37d on the mTORC1 signaling pathway,we first examined the effect of overexpression of IL-37d on cell size.A549 cells were infected with IL-37d and control lentivirus,respectively,and the changes of cell size in basal state,starvation state and starvation reactivation state were detected by flow cytometry.We found that the cell size of IL-37d lentivirus infected group was smaller than the NC group in the basal state and the hunger reactivation state and that was more obvious in the hunger reactivation state.It was confirmed that IL-37d inhibited cell size and suggested that IL-37d might inhibit the activation of mTORC1 signaling pathway.In order to clarify the inhibitory effect of IL-37d on the mTORCl signaling pathway,we used the overexpression system to detect the effect of IL-37d on the phosphorylation of mTORCl downstream effector S6 by flow cytometry and Western-blot.The results of flow cytometry showed that the phosphorylation of the molecule S6 downstream of the mTORC1 signaling pathway was significantly inhibited in the cells infected with IL-37d lentivirus under the basal state and starvation reactivation,and this phenomenon in the starvation reactivation state was more obvious.The same results were obtained by Western-blot method.Further,we compared the function of IL-37d on mTORC1 signaling pathway in three different hunger modes(F12K medium without FBS,F12K medium without FBS glutamine-free and HBSS starvation)reactivation(activation of F12K medium containing 10%FBS).We found IL-37d can inhibit the expression of p-S6 in the above three conditions,and then determine the inhibitory effect of overexpression of IL-37d on mTORCl signaling pathway.(Ⅲ).Silence of IL-37 upregulates of mTORCl signaling pathwayTo further determine the inhibitory effect of IL-37d on the activation of the mTORC1 signaling pathway,we silenced IL-37 expression in HepG2 cells,and then detected changes of mTORC1 pathway activity by Western-blot and immunofluorescence.The results show that silencing IL-37 promotes the activation of the mTORCl signaling pathway,and demonstrates the inhibitory effect of endogenous IL-37 on the mTORCl signaling pathway in another aspect.Ⅱ.Molecular mechanism of IL-37d regulating mTORCl signaling pathway(Ⅰ).IL-37d interacts with RhebRas-related GTPase(Rheb)is known to be a key molecule for the activation of mTORC1.Our previous study found that IL-37b binds to another member of the Ras family,Racl,and inhibits its activity,so we hypothesized that IL-37 may be regulated by Rheb and then affects mTORC1 activity.In order to verify our hypothesis,the relationship between IL-37d and Rheb was investigated by immunofluorescence and co-immunoprecipitation.The results of immunofluorescence showed that IL-37d and Rheb had obvious co-localization in the cytoplasm and the co-immunoprecipitation experiment further demonstrated that IL-37d can bind to Rheb.It is suggested that IL-37d can regulate mTORCl signaling pathway by binding to Rheb.(Ⅱ).IL-37d inhibits the activity of RhebIn order to investigate whether the binding of IL-37d to Rheb affects the activity of Rheb,we used immunofluorescence to detect the effect of overexpression of IL-37d on the expression of the Rheb active form GTP-Rheb,and the results showed that the expression of GTP-Rheb was weak after infected by IL-37d lentivirus,indicating that IL-37d can inhibit the activity of Rheb.(Ⅲ).IL-37d can not competitively inhibit the combination of Rheb and mTORIn order to clarify whether IL-37d can competitively inhibit the binding of Rheb to mTOR,we overexpressed different concentrations of IL-37d,and analysis different expression levels of IL-37d on the binding of Rheb to mTOR.IL-37d was found to have no significant effect on the binding of Rheb to mTOR,suggesting that IL-37d does not exert an effect by competitively inhibiting the binding of Rheb and mTOR.(Ⅳ).IL-37d inhibits mTORCl signaling pathway through RhebTo further investigate whether IL-37d inhibits Rheb and down-regulates the mTORCl signaling pathway,IL-37d and Rheb plasmids were transfected firstly and detecting p-S6 by Western-blot and flow cytometry under basal conditions.The expression of p-S6 was up-regulated after overexpression of Rheb,indicating that overexpression of Rheb can activate the mTORC1 pathway;however,after overexpression of IL-37d,this inhibition was reversed,indicating that IL-37 can down-regulate mTORC1 pathway by inhibiting Rheb.To further determine the role of IL-37d,the cells were transfected with IL-37d and active Rheb plasmids and the expression of p-S6 was examined by Western-blot and flow cytometry under the condition of starvation for two hours in a FBS-free,arginine-free and leucine-free medium and the results showed that IL-37d can reverse the activation of p-S6 by active Rheb.It was demonstrated that IL-37d down-regulates the mTORC1 signaling pathway by inhibiting the activity of Rheb.Ⅲ.IL-37d inhibits liver lipid accumulation mediated by mTORCl signaling pathwayIt is known that activation of mTORCl signaling pathway plays a role in promoting lipid accumulation in the liver.In order to study the effects of IL-37d on the mTORC1 signaling pathway and its role in lipid accumulation in liver,liver lipid accumulation model induced by long-term high-fat diet and short-term lipid accumulation model induced by starvation for 16 hours were used.(Ⅰ)IL-37d inhibits high-fat-induced activation of mTORCl signaling pathway and liver lipid accumulationSix to eight weeks male IL-37d transgenic and WT mice were selected for high-fat diet(60%fat)for 18 weeks and then dissected.The body weight and liver weight of IL-37d transgenic mice were found to be significantly reduced.Immunohistochemistry and Western-blot assay showed that the expression of p-S6 in IL-37d transgenic mice was significantly lower than that in WT mice,indicating that IL-37d inhibits the activation of mTORCl signaling pathway induced by high fat.HE staining and intrahepatic lipid content detection showed that lipid droplets in liver cells of IL-37d transgenic mice were decreased compared with WT mice,and intrahepatic triglyceride levels were significantly decreased,indicating that IL-37d inhibits liver lipid accumulation mediated by mTORCl signaling pathway.(Ⅱ).IL-37d inhibits activation of mTORCl signaling pathway induced by fasting for 16 hours and short-term lipid accumulation in liverSix to eight weeks male IL-37d transgenic and WT mice were fasted for 16 hours to build the model of short-term lipid accumulation in the liver.Immunohistochemistry and Western-blot assay showed that the expression of p-S6 in IL-37d transgenic mice was significantly lower than that in WT mice,indicating that IL-37d inhibits the activation of mTORCl signaling pathway induced by fasting for 16 hours.Intrahepatic lipid content assay showed that IL-37d transgenic mice had lower triglyceride content than WT mice,indicating that IL-37d inhibits short-term liver lipid accumulation mediated by inhibiting mTORC1 signaling pathway.Conclusion1.IL-37d inhibits the mTORC1 signaling pathway.2.IL-37d inhibits the mTORC1 signaling pathway through Rheb.3.IL-37d inhibits lipid accumulation in the liver and alleviates fatty liver disease caused by excessive lipid synthesis.Innovation and significance1.To propose a new mechanism of IL-37d as an endogenous "Rapamycin"inhibition of mTORCl pathway:IL-37d was found to inhibit mTORCl signaling pathway and demonstrate that IL-37d down-regulates mTORC1 signaling pathway by inhi’biting Rheb activity,enriched the understanding of the function and mechanism of IL-37 and the mechanisms of mTORCl regulation.2.In addition to anti-inflammatory effects,IL-37 is also involved in lipid metabolism and other processes:This study found that IL-37 can inhibit liver lipid accumulation mediated by mTORC1 signaling pathway and relieves the fatty liver phenomenon caused by excessive lipid accumulation,providing a basis for the treatment of IL-37 for metabolic diseases.Limitation1.The mechanism’by which IL-37d regulates the mTORCl signaling pathway is not sufficient and in-depth.2.Need to explore the role of IL-37d in regulating the mTORC1 signaling pathway in a variety of disease models. |
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