| Part Ⅰ Batimastat inhibits adipocyte differentiation from MSCsObjective:(1)To explore the concentrations of Batimastat exerting inhibitory effect on MSCs proliferation;(2)To confirm the inhibitory effect of Batimastat on adipogenesis from MSCs;(3)To examine the impact of Batimastat on adipocyte-specific genes expressions during adipocyte differentiation from MSCs.Methods:(1)The MSCs were collected from of tibia and femur bone marrow of 6-8 weeks old C57BL/6 male mice.(2)CCK-8 assay was applied to detect the cell proliferation of MSCs treated with gradient concentrations from 0 to 200 μm of BB-94 after 48,72 and 96 h culture;(3)Oil red and Bodipy/DAPI staining and following quantitative analysis were used to evaluate the inhibitory effect of BB-94 on adipocyte differentiation and determine the optimal effective concentration;(4)The mRNA level expressions of adipocyte-specific genes including Pparg,Cebpa,Cebpb,Cebpd,Srebf1,Plin1,Adipoq,Lpl,Cd36,Fabp4,Cidec,Scd1,Fasn,Acaca,Lipe and Pnpla2 were detected by qPCR,using the cDNA from the control group and 10 μM BB-94 treated group after 0,2,4,8 and 12 d adipocyte differentiationResults:(1)MSCs proliferation was inhibited after 150 and 200 μM BB-94 treatment for 72 h and 200 μM BB-94 treatment for 48 h;(2)Oil red staining showed that BB-94 suppressed adipocyte differentiation from MSCs dose-dependently.Furthermore,there was no significant difference between 10 and 20 μM BB-94 treatment groups;(3)Bodipy/DAPI staining demonstrated that BB-94 hindered adipocyte differentiation from MSCs in a dosedependent manner.Further fluorescent quantitative analysis showed that there was no significant difference between 10 and 20 μM BB-94 treatment groups;(4)10 μM BB-94 inhibited adipocyte-specific genes expressions including Pparg,Cebpb,Srebf1,Plin1,Adipoq,Lpl,Cd36,Fabp4,Cidec,Scd1,Fasn,Acaca,Lipe and Pnpla2 significantly during adipogenesis.Conclusion:Batimastat exerted minor toxicity on MSCs proliferation,inhibited adipocyte differentiation and suppressed adipocyte-specific genes expressions on mRNA level notably at 10 μM.Part II The mechanism of Batimastat regulating MSCs adipogenesisObjective:To explore how Batimastat affects MSCs adipogenesis by regulating the expression of adipogenic transcription factors and signal pathway interactions.Methods:(1)The expressions of adipogenic transcription factors including PPARγ、C/EBPα、C/EBPβ and mature adipocyte marker Perilipin-1 were measured by western blot in the control and 10 μM BB-94 treated groups after adipocyte differentiation from MSCs for 0,2,4,8 and 12 d;(2)The expressions of p-Akts473,Akt,p-S6Ser240/244,and S6 were detected using western blot in the control and 10 μM BB-94 pre-treated groups with both 12 h starvation treatment after 3 μg/mL insulin stimulation at 0,5,15,30 and 60 min;(3)RNAseq was applied to analyse the transcriptional differences between the control group and 10μM BB-94 treated groups after adipocyte differentiation from MSCs for 4 d;(4)The mRNA level expressions of target genes filtered out by RNA-seq(Porcn and some downstream target genes of Wnt/β-catenin signal pathway)were verified by qRT-PCR.Furthermore,the expression of non-phospho(active)β-catenin(Ser33/37/Thr41)was detected by western blot in the control and 10 μM BB-94 treated groups after adipocyte differentiation from MSCs for 0,2,4,8 and 12 d;(5)IWP-2,an inhibitor specifically targeting Porcupine,was used to rescue the inhibitory effect of BB-94 on adipocyte differentiation.Further adipocyte formation was assessed by Oil red staining,adipocyte-specific genes expressions were detected by qRT-PCR,expression of non-phospho(active)β-catenin(Ser33/37/Thr41)was examined using western blot;(6)MK-2206,an inhibitor specifically targeting Aktl/2/3,was used to suppress PI3K/Akt signal pathway.Adipocyte formation was assessed by Oil red and Bodipy/DAPI staining,adipocyte-specific genes expressions were detected by qRT-PCR.Furthermore,expressions of Perilipin-1,p-Akts473,Akt and non-phospho(active)β-catenin(Ser33/37/Thr41)were examined using western blot to explore whether BB-94 affected Wnt/β-catenin signal pathway through suppressing Insulin/Akt activity.Results:(1)The expressions of Perilipin-1 and p-Akts473 were reduced obviously after 10 μM BB-94 treatment on 4,8 and 12 d of adipogenesis.Additionally,the expressions of adipogenic transcription factors PPARγ and C/EBPα decreased after 2,4,8 and 12 d adipocyte differentiation;(2)After 3 μg/mL insulin stimulation,the expressions of p-Akts473 and p-S6Ser240/244 decreased notably at 5,15 min or 0,5,15 min in the 10 μM BB-94 pretreated group;(3)RNA-Seq results demonstrated that the expression of Porcn responsible for Wnt protein molecule secretion,maturation and palmitylation was markedly elevated in the 10 μM BB-94 treated group.Besides,the expressions of downstream target genes of Wnt/β-catenin signal pathway including Myc,Twist1,Ccnd1 and Nkd2 were increased;(4)qRT-PCR further confirmed the expression changes of Porcn and some downstream target genes of Wnt/β-catenin signal pathway,and western blot revealed that non-phospho(active)β-catenin(Ser33/37/Thr41)expression increased after BB-94 treatment during adipogenesis;(5)IWP-2 observably rescued the inhibitory effect of BB-94 on MSCs adipogenesis,remitted the changes of adipocyte-specific genes expressions and Wnt/β-catenin signal pathway activation;(6)2 μM MK-2206 suppressed adipocyte differentiation by inhibiting phosphorylation on Ser473 site,decreasing Insulin/Akt signal pathway activity and following elevating non-phospho(active)β-catenin(Ser33/37/Thr41)expression.Conclusion:Batimastat inhibits adipogenesis by down-regulating adipogenic transcription factors PPARγ and C/EBPα and restrains Insulin/Akt/mTOR signal pathway.Additionally,Batimastat stimulated Wnt/β-catenin signal pathway by increasing Porcupine expression and inhibiting Akt signal pathway to suppress adipocyte differentiation.Part Ⅲ Batimastat mitigates high fat diet-induced obesity and bone lossObjective:To explore whether Batimastat exerts mitigatory effect on high fat dietinduced obesity,glucose metabolism dysfunction and bone loss resulting from chronic inflammation state.Methods:(1)24 6-weeks old C57BL/6 male mice were divided into 3 groups,the Ctrl group was fed with purified 10%fat control diet and the HFD and HFD+BB-94 groups were fed with purified 60%fat diet;(2)After replacing the purified diets,the mice in HFD+BB94 group were injected intraperitoneally with 30 mg/kg BB-94 every other day,and the mice in Ctrl and HFD groups were injected intraperitoneally with solvent at the same dosage.The mice were weighed weekly;(3)In the 16th week after high fat diet feeding,the glucose metabolism of each mouse was examined by IPGTT and ITT;(4)In the 18th week after high fat diet feeding,the serum,liver and femur were collected,epididymo white adipose tissue,inguinal white adipose tissue,retroperitoneal white adipose tissue and interscapular brown adipose tissue were separated,weighed and fixed with 4%PFA for 2 days.Finally,the samples were stored in 75%ethanol;(5)H&E staining of white adipose tissue,brown adipose tissue and liver was performed to evaluate structural changes;(6)Micro-CT was used to assess the bone mass and trabecular bone parameters of femur;(7)H&E staining of femur was performed after 14%EDTA decalcification for 14 days;(8)The levels of serum OC and CTX-I were measured by ELISA to evaluate bone metabolic status.Results:(1)High fat diet-induced obesity in HFD group was established successfully after 18 weeks,and the mice exerted glucose metabolism dysfunction and decrease in insulin sensitivity compared with the Ctrl group.However,above situation was remitted in the HFD+BB94 group;(2)The weights of white adipose tissue and sizes of adipocytes in the HFD group increased markedly.The brown adipose tissue and livers in HFD group occurred obvious adipocyte infiltration.However,intraperitoneal injection with BB-94 might rescue those changes;(3)In obese mice in the HFD group,bone formation was decreased and bone absorption was increased,and the infiltration of adipocytes appeared in the bone marrow,leading to the decrease of bone mass,while BB-94 could significantly alleviate the loss of bone mass.Conclusion:Batimastat protects high fat diet-induced obesity,glucose metabolism dysfunction and bone loss. |
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