The Effects And Mechanisms Of Lagerstroemia Speciosa Total Flavonoids On The Inflammatory Response And Insulin Resistance In 3T3-L1 Adipocytes

Lagerstroemia speciosa,a kind of traditional herbal medicine,mainly distributed in southeast asia,has been used for thousands of years for prevention and treatment of diabetes.The previous studies have confirmed that the Lagerstroemia specious leaves have multi-pharmacological effects such as hypoglycemic,weight loss,antioxidant,anti-tumor,et al,and have not toxic and side-effect.The majority research works are focused on the corosolic acid,which commonly known as "plant insulin",whereas the biological activities of total flavonoids in Lagerstroemia specious leaves have not been reported yet.The extract process of Lagerstroemia specious total flavonids(LTF)were firstly optimized with single factor test and orthogonal test,and the extract was further purified by macroporous adsorptive resins and dialysis method subsequently.The single factor test showed that the extraction condition was solid-liquid ration 1:40,60% ethanol,60 ℃extract for 0.5h.The orthogonal test indicated that the optimum extraction condition was solid-liquid ration 1:40,50% ethanol,60 extract for ℃ 1.5h.The total flavonids with purity of 90.7% was obtained.In order to investigate the effects and mechanisms of the LTF on adipocyte inflammation reaction,3T3-L1 adipocytes were induced inflammatory response by TNF-α and administered with 50,5,0.5 μg/m L LTF(named as H group,M group and L group respectively).Supermatant were collected at different times and the IL-6 concentration was detected by ELISA.The IL-6,MCP-1 and ADPN m RNA expression level were detected by Q-PCR.The phosphorylation p38,JNK and ERK signal pathways of MAPK and the key protein factor Iκβ of NF-κB of the cells were determined by Western-blot to explore the molecular mechanism of LTF on the inflammatory adipocytes.The results showed that IL-6 secretion,IL-6 and MCP-1 m RNA expression in the inflammatory 3T3-L1 cells induced by TNF-α were significantly elevated(p<0.01),whereas the expression of ADPN m RNA was significantly decreased(p<0.01).When treated with different concentrations of LTF,the IL-6 and MCP-1 m RNA expression levels were significantly down-regulated,and the ADPN m RNA expression level was significantly up-regulated.Moreover,the IL-6 concentrations in all experimental groups were decreased compared with that of model group,i.e,the IL-6 concentrations of M group were significantly lower(p<0.05),H group were significantly lower(p<0.01)than that of the inflammatory 3T3-L1 adipocytes,indicating that the LTF can inhibit 3T3-L1 adipocytes inflammatory response with a dose-effect manner.The Western-blot detection results showed that the phosphorylation of p38,JNK and ERK of MAPK signal pathways and Iκβ of NF-кB signal pathway in the inflammatory adipocytes were significantly inhibited by LTF treatment(p<0.01).It suggested that LTF ameliorate adipocyte inflammatory response through inhibition MAPK and NF-кB signaling pathways.The TNF-α induced 3T3-L1 adipocytes were employed to construct the insulin resistant celluar mode,and the glucose intake of the cells was detected to evaluate the insulin resistance status,subsequently the phosphorylation of IRS1(Ser307)and PI3K/AKt signaling pathway were detected by Western-blot to elucidate the possible molecular mechanisms of LIF on insulin resistant 3T3-L1 cells.The results indicated that the 3T3-L1 adipocytes were induced insulin resistance by TNF-α,which showed significant lower glucose intake than that of control,the insulin resistant of cells were significantly improved with addition of LTF and insulin in the medium,and the glucose intake was approximately the same as that of control group.Furthermore,the significant inhibition effect of the LTF on IRS1(Ser307)phosphorylation in 3T3-L1 adipocytes which had insulin resistance induced by TNF-α was observed(p<0.01),and the phosphorylation of PI3K/AKt in the cells was significant promoted(p<0.01)by the LTF addition.It can be inferred that the improvement effect of LTF on insulin resistance of the adipocytes mainly due to inhibition of Ser 307 phosphorylation of IRS1 and promotion of AKt phosphorylation,the downstream effector of insulin signal pathway.In a word,we conclude that LTF can inhibit 3T3-L1 adipocytes inflammatory response and insulin resistance,and may be great useful for clinical applications.