Honokiol Induce The Differentiation Of 3T3-L1 Pre-adipocyte Via Regulating PPARγ Expression And Activation

Objective:Recently, the morbidities of obesity and diabetes keep increasing. The differentiation of pre-adipocytes is relative to the generation and development of obesity, insulin resistance, type 2 diabetes and cardiovascular diseases. So that adipocyte differentiation and its target therapy is a hot spot. Chinese medicine has great effect on the adipocytes differentiation, which plays an important role in controlling and reducing of obesity, insulin resistance, type 2 diabetes and cardiovascular diseases. There are some researches about honokiol, the extraction of Houpo, has obvious therapeutic effects on obesity and type2 diabetes. While the mechanism is still under making clear. Peroxisome proliferator-activated receptors (PPAR), members of the nuclear receptor transcription factor superfamily, was firstly discovered by the English scientists Issemann and Green. A large number of studies have shown that PPARs closely relate to obesity, cardiovascular diseases and tumors, and made clear that PPARs is the new drug target of diseases mentioned before. PPAR has three subtypes, which are α,β, γ. All three PPAR subtypes are expressed in various location and their functions are different. PPARγ distributed mainly in the fat, spleen, kidney and colon, it modulates the process of fat synthesis and decomposition.3T3-L1 pre-adipocyte is a kind of fibroblast cells of Swiss mice, which can differentiate to adipocyte. The 3T3-L1 cell is the ideal cell model in case of studying fat and its metabolism in vitro for its characteristics are very similar with vivo cells, such as morphological changes, the expression of a variety of fat metabolism enzymes and lipid accumulation. Honokiol, the main active constituent of Houpo, which has the functions of resolving dampness to move qi, warming meridian/channel and relieving pain, ownbearing counterflow to suppress cough and to calm panting, the earliest study discovered it had free radical and lipid peroxidation function, and then there are many research shown that honokiol had anti-inflammation, anti-tumor, anti-bacterial, recuperation intestines and stomach, anti-diabetes and cardiovascular protection and so on. It is necessary to delve into the mechanism of honokiol modulate the lipid metabolism not only for that the great importance given to the honokiol treat the diabetes but also for that it is need to find new targets for the treatment of diabetes… We found that PPARs could be activated by many kinds of phenolic compounds from others studies, while there is no relative studies reported that honokiol is PPARγ ligand-binding, whether it modulates adipocytes differentiation through activating PPARγ signaling pathway.This project put forward the subje’ct that whether honokiol modulate 3T3-L1 pre-adipocytes differentiation through binding and activating PPARγ. The first part demonstrated honokiol induced 3T3-L1 pre-adipocytes differentiation through activating PI3K/Akt signaling pathway. The second part detected the effect of honokiol on PPRE and PPARγ promoter, whether the inducing effect of honokiol impaired after inhibiting PPARγ.Methods1. The effect of honokiol on 3T3-L1 pre-adipocytes.3T3-L1 pre-adocytes differentiation:the group of inducing cells with differentiation cocktail was positive control, honokiol group was treated with 15mM honokiol. MTT assay to make sure the suitable concentration of honokiol. Oil red 0 staining to detect the degree of differentiation. Triglyceride assay to measure the triglyceride concentration of each group. RT-PCR to determine the relative target genes of adipocytes differentiation.2. The mechanism of honokiol induced 3T3-L1 pre-adipocytes differentiation Cell transfection:transfected the PPRE-Luc plasmid and PPARγ promoter into the 3T3-L1 cells, pRL-Lus plasmid was used as internal control. Different treatments were applied after 24 hours.Luciferase assay of PPRE-Luc to determine the PPRE activity:the effect of honokiol on Luciferase activity, Rosiglitazone (PPARγ ligand) as the positive control.Luciferase assay of PPARγ to determine the PPARγ promoter activity:the effect of honokiol on PPARγ promoter activity, Rosiglitazone (PPARγ ligand) as the positive control.The degree of differentiation after inhibiting PPARγ was measured by Oil red 0 staining and triglyceride assay.QRT-PCR is to determine the relative target genes of adipocytes differentiation after honokiol treatment. Results1. MTT assay result shows that there wasn’t significant impact on the livability using 1-15 μM honokiol treatment.25 μM honokiol inhibited the cells proliferation.2. Oil red 0 staining and Triglyceride assay shows that honokiol induced the lipid accumulation, RT-PCR result showed that honokiol increased the marker genes of adipocytes like aP2, PPARγ, C/EBPα and Glut4mRNA.3. Luciferase assay shows that honokiol enhanced the PPRE and PPAR γ promoter activities.4. The effect of honokiol enhanced 3T3-L1 pre-adipocytes differentiation reduced by the GW9662(The PPARγ inhibitor).5. QRT-PCR and Western Blot results show that the expressions of relative genes decreased after inhibiting PPAR y. Conclusion1. The reliable concentration of honokiol is 15μM.2. Honokiol induced the pre-adipocytes differentiation, enhanced the glucose transport, increase the expressions of adipocytes differentiate relative genes.3. GW9662 inhibited the 3T3-L1 pre-adipocytes differentiation after honkiol treatment. Combined with the results of PPRE and PPARγ Luciferase assay, it could made a conclusion that honokiol induce pre-adipocytes differentiation through directly binding to the PPARγ and activating PPARγ.4. Honokiol modulate the PI3K/Akt signaling pathway, increase the AKT phosphorylation to induce adipocytes differentiation.