| In recent years,people have begun to pay attention to the relationship between inflammation and tumor.Chronic inflammation is often closely related to the occurrence and development of tumors.Many factors can cause the body to produce varying degrees of chronic inflammation,such as chemical stimulation,viral infection and autoimmune injury,and so on.Inflammation can lead to DNA damage in normal cells,genomic instability,proto-oncogene mutations and migration of cancer cells.Therefore,anti-inflammatory therapy is essential in the prevention and treatment of cancer.Currently,the steroidal and non-steroidal anti-inflammatory drugs were used in clinical practice have great side effects while relieving inflammation.Chinese traditional medicine may play an immune intervention role in the treatment of inflammation,because it has various biological effects including antioxidant,bacteriostasis,sterilization,and the lower level of endotoxin in blood and organs.The combination of traditional Chinese and Western medicine has broad prospect as a new method of treatment.Ginkgo,a traditional Chinese medicine,has a history of treating diseases for thousands of years.Ginkgol C17:1 is one of the monomers of Ginkgo.Some studies have confirmed that Ginkgol C17:1 had the higher anti-tumor activity,but its anti-inflammatory and mechanism have not been reported.In present years,many researches have shown that autophagy was involved in inflammatory diseases.Autophagy can inhibit the synthesis of pro-inflammatory complex directly and remove the damaged organelles or intracellular pathogens indirectly.Thus,we mainly used lipopolysaccharide(LPS)-induced macrophages as the inflammatory model to observe the changes of inflammatory cytokine,oxidative stress and autophagy.The aim was to elucidate the protective effect of Ginkgol C17:1 on inflammation from the perspective of autophagy,so as to provide a theoretical basis for clinical treatment of inflammation and expand the pharmacological effect of Ginkgo phenol.Experimental methods and contents of the study:(1)Detected the viability of macrophage cells treatment with Ginkgol C17:1 by MTT assay.(2)Enzyme-linked immunosorbent assay(ELISA)was used to detect the concentration of inflammatory factors IL-6 and TNF-? in macrophage supernatants,determine the optimal concentration and time of LPS,and establish a model of cellular inflammation.(3)Real-time quantitative PCR(q RT-pcr)and enzyme-linked immunosorbent assay(ELISA)were used to detect the m RNA expression levels of inflammatory cytokines IL-6 and TNF-? and their contents in cell supernatants.Western Blot was used to detect the expression of NLRP3 and IL-1? protein in LPS-induced macrophage cells,and to analyze the effect of Ginkgol C17:1 on inflammasome and inflammatory factors.(4)Fluorescence microscopy was used to observe the effect of Ginkgol C17:1 on the expression of reactive oxygen species(ROS)in LPS-induced macrophage cells.(5)Western Blot was used to detect the expression of TLR4,My D88,NF-?B,Phos NF-?B,ERK,Phos-ERK,STAT3 and Phos-STAT3 proteins in LPS-induced macrophage cells,and to analyze the effects of Ginkgol C17:1 on TLR4 signaling pathways.(6)To analyze the effect of Ginkgol C17:1 on the cell autophagy,we used western blot to detect the expression of the autophagy-related proteins(Beclin1,LC3,P62),and the autophagy flux by infecting the double-labeled(RFP-GFP-LC3)adenovirus treatment with Ginkgol C17:1 in LPS-induced macrophage cells.(7)To confirm the role of autophagy in inhibiting inflammation,the expression of autophagy-related proteins(LC3,P62),inflammatory factors IL-6 and TNF-?were detected again after treatment with autophagy inhibitor 3-methyladenine(3-MA)in LPS-induced macrophage cells.Experimental results of the study:(1)MTT results showed that there are no significant effects of Ginkgol C17:1 on macrophages viability in the concentration of 0-80 ?g/ml for 24 hours.(2)The ELISA results showed that the optimal concentration of LPS treatment was100 ng/ml,and the optimal time was 12 h.(3)After induction with 100 ng/ml LPS for 12 hours,the m RNA levels of IL-6 and TNF-? as well as their concentration in the medium of macrophages were decreased by treatment with 40 ug/ml Ginkgol C17:1 for 24 hours(P < 0.01 vs LPS).Western blot results showed that Ginkgol C17:1 inhibited the expression levels of NLRP3 and IL-1? proteins in a dose-dependent manner.It is suggested that Ginkgol C17:1 has an inhibitory effect on inflammatory cytokines secretion.(4)Fluorescence microscopy showed that Ginkgol C17:1 inhibited LPS-induced ROS production in macrophage cells(P<0.01 vs LPS),indicating that Ginkgol C17:1interfered with the secretion function of free radicals.(5)Western blot results showed that LPS could activate TLR4 and its downstream proteins in macrophages.Ginkgol C17:1 decreased the expression of TLR4,My D88,NF-?B,Phos NF-?B,ERK,Phos-ERK,STAT3,Phos-STAT3 proteins in a dose-dependent manner(P<0.01 vs LPS),suggesting that inhibition of TLR4,NF-?B/ERK,STAT3 signaling pathways could inhibit the secretion of inflammatory factors in LPS-induced macrophage cells.(6)Western blot results showed that the expression of autophagy-associated proteins(Beclin1,LC3)were slightly lower than that of the control group,and the autophagy downstream protein P62 was increased.However,the expression of proteins(Beclin1,LC3)increased,and the expression of P62 decreased in a concentration-dependent manner after adding different concentrations of Ginkgol C17:1.Similarly,the immunofluorescence results showed that the aggregation of autophagy-specific protein LC3 particles increased after the addition of Ginkgol C17:1,indicating that Ginkgol C17:1 can up-regulate autophagy in LPS-induced macrophage cells.(7)After the addition of the autophagy inhibitor 3-methyladenine(3-MA),the autophagy-associated protein LC3 II was reduced and P62 was accumulated.Meanwhile,the results of real-time quantitative PCR showed that the expression of inflammatory factors IL-6 and TNF-? were increased(P<0.01 vs control).It is suggested that LPS stimulation can weaken the autophagy ability of macrophages,while Ginkgol C17:1 can up-regulate autophagy in macrophages under the state of inflammation.After the inhibition of autophagy,the expression of inflammatory factors increased,further indicating that autophagy plays an important role in the anti-inflammatory process of Ginkgol C17:1.Conclusion:LPS may induce inflammation,activate the TLR4 signaling pathways and inhibit autophagy.However,Ginkgol C17:1 had the opposite effects in inflammatory environment.So,Ginkgol C17:1 can fight inflammation by inhibiting TLR4 signaling pathways and inducing autophagy.This anti-inflammatory effect may be associated with inhibition of TLR4,NF-?B/ERK,STAT3 signaling pathways and up-regulated autophagy in LPS-induced macrophage cells. |
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