| Background:In addition to the bone homeostasis disorder coupled with osteoblasts and osteoclasts,bone lipid imbalance mediated by bone marrow mesenchymal stem cells(BMSC)is also an important cause of osteoporosis.In the early stage,our research under the guidance of"collateral disease" theory confirmed that Wenshen Tongluo Zhitong Decoction can improve bone and lipid metabolism and delay the process of osteoporosis,but its mechanism of action is still not clear.This project intends to verify and reveal the regulating effect and mechanism of Wenshen Tongluo Zhitong Decoction on bone lipid balance through in vivo and in vitro experiments,and provide new ideas for the prevention and treatment of osteoporosis.Objective:Observe the effect of adipocyte-derived exosomes on the osteogenic and adipogenic differentiation of BMSCs and the intervention effect of Wenshen Tongluo Zhitong Decoction,and clarify the specific molecular mechanism of Wenshen Tongluo Zhitong Decoction affecting fat cell-derived exosomes and regulating bone lipid balance.Methods:(1)Prepare the freeze-dried powder of the decoction of Wenshen Tongluo Zhitong Recipe,and establish a fingerprint by high performance liquid chromatography(HPLC)technology combined with standard product comparison to identify the effective ingredients of the compound,and pass the liquid chromatography-triple quadruple bar Mass spectrometry(LC/MS)was used to verify and quantify the content of some compounds.(2)Prepare the medicated serum of Wenshen Tongluo Zhitong Recipe,and use medicated serum and blank serum to intervene 3T3-L1 adipocytes.After the intervention,the exosomes are extracted by ultracentrifugation;the exosomes are extracted by transmission electron microscope.Observation of morphology,Nanosight300 particle size detection,Western Blot(WB)detection of exosomes showed that the markers CD9 and CD63 were identified;then the miRNAs in exosomes were extracted through the kit,and the differential miRNAs were screened by high-throughput sequencing.All target genes of differential miRNAs were enriched in GO and KEGG signaling pathways.(3)Construct a mouse primary BMSC culture system in vitro,and identify the surface markers CD90,CD29,CD45,and CD34 from morphological observation and flow cytometry,and induce the potential of three-line differentiation(osteogenesis,adipogenesis,and cartilage)Characterization was completed from three perspectives;after purification,the exosomes were confirmed by PKH-67 immunofluorescence labeling to verify that they can be taken up by BMSC;the co-culture system of BMSC and adipocytes and the co-culture system of BMSC and adipocyte-derived exosomes were constructed respectively,and stained by ALP,Alizarin red staining,and oil red staining to observe the osteogenesis and adipogenic differentiation in the two systems.Detect the osteogenic markers RUNX2,Osterix and the adipogenic marker CEBP-? in the two systems by q-PCR and WB methods,PPAR?2 mRNA and protein expression.(4)q-PCR to verify the expression of differential miRNA in exosomes;after confirming the effector miRNA,construct the corresponding analogue sequence,inhibitor sequence and corresponding negative sequence and then carry out group transfection;and use ALP staining and alizarin Red staining and oil red staining were used to observe the effect of the miRNA on osteogenic and adipogenic differentiation;then q-PCR was used to detect the effect of the miRNA on the mRNA expression of osteogenic markers RUNX2,Osterix and adipogenic markers CEBP-? and PPAR?2 Impact;Three online prediction websites of TargetScan,miRDB,and miRTarbase are used to predict the target genes that the effector miRNA may bind to,and use q-PCR to verify;further through the luciferase reporter assay and RNA immunoprecipitation experiment(RIP-QPCR)to verify the binding of the effector miRNA to the target gene;construct the overexpression plasmid and knockdown plasmid of the target gene to co-transfect BMSC with miRNA sequences,and observe the osteogenesis of each group by ALP staining,alizarin red staining,and oil red staining.Lipogenesis staining,the expression of SPRY2 in each group was detected by WB,and the mRNA expression of osteogenic markers RUNX2,Osterix and adipogenic markers CEBP-?and PPARy2 were detected again by q-PCR.(5)Prepare a mouse model of ovariectomized osteoporosis,and randomly divide 80 mice into a sham operation group(Sham,100?l/mouse,normal saline),a model group(OVX,100?l/mouse,normal saline),and exocrine Body group(EXO,30?g(100?l)/only,exosomes),traditional Chinese medicine intervention exosomes group(EXO+WSTLZTF,30?g(100?l)/only,traditional Chinese medicine intervention exosomes),respectively,once a week as required Tail vein injection of corresponding drug dose;ELISA method to detect serum bone metabolism index B-ALP,OCN,TRAP,IL-6 levels;automatic biochemical detector to detect serum lipid metabolism index total cholesterol(CHOL),triglyceride(TG),low-density lipoprotein(LDL)and high-density lipoprotein(HDL)expression levels;HE staining and TRAP staining to observe the histopathological changes and bone resorption of bone tissue in mice;Micro-CT was used to detect bone microscopic Structure and distribution of fat around the bone;bone biomechanics-three-point bending test to detect the biomechanical properties of the femur;immunohistochemistry(IHC)to detect osteogenic markers RUNX2,Osterix and adipogenic markers CEBP-?,PPAR?2 in bone tissue The expression and distribution of the protein;the mRNA expression of osteogenic markers RUNX2,Osterix and adipogenic markers CEBP-? and PPAR?2 in bone marrow tissues were detected by q-PCR.The rat bone marrow monocytes to establish induced osteoclast culture system:using a special device for separating bone marrow monocytes extracted bone marrow monocytes;morphologic observation of bone marrow mononuclear cell growth;CCK-8 was adopted to observe the growth characteristics of bone marrow monocytes;mononuclear cell purity by flow cytometry method validation get through this method;staining by tartrate resistant acid phosphatase(TRAP)and calcitonin receptor(CTR)immunofluorescence staining was used to identify osteoclasts;construct induced osteoclast culture system by mouse RAW264.7 cell lines:To observe the morphological characteristics of the growth of RAW264.7 cells;identification of osteoclasts by tartrate resistant acid phosphatase staining;Results:(1)The fingerprint of the decoction of Wenshen Tongluo Zhitong Decoction was initially established,and icariin,loganin,naringin,osthole,benzoyl aconitine,and benzoyl aconitine were identified.There are 10 kinds of effective ingredients in Noumenonine,Benzoyl Hypoconitine,Isoperatorin,Gastrodin,and Protocatechin.And completed the 7 effective ingredients of icariin,loganin,naringin,osthole,benzoyl aconitine,benzoyl aconitine,and benzoyl aconitine.Quantitative testing.(2)The exosomes of the blank group and the drug-containing serum group were successfully extracted by ultracentrifugation,and successfully identified by transmission electron microscopy,particle size detection,and surface markers CD9 and CD63 expression detection;the exosomes were extracted after miRNA High-throughput sequencing found differential miRNAs such as miR-122-5p,miR-27b-5p,miR-16-1-3p,miR-322-3p,miR-3963,etc.The predicted target genes of differential miRNAs were enriched in GO analysis The positive regulation of the MAPK signaling pathway and other biological processes are enriched in the post-synaptic,cell front and other cellular components,and enriched in molecular functions such as GTPase activation activity;KEGG analysis shows that the signaling pathway may be involved in the signal pathway:Ras signaling pathway,Signal pathways that regulate the pluripotency of stem cells and other signal pathways.(3)Successfully extract mouse primary BMSCs,flow cytometry to detect high expression of CD29(99.37%)and CD90(99.85%),low expression of CD45(0.04%)and CD34(5.66%),and stain by Alizarin Red,Oil red staining,and toluidine blue staining successfully identified the differentiation potential of its three lines,combined with its morphological characteristics,conformed to the biological characteristics of animal-derived MSCs;successfully verified by immunofluorescence labeling that fat-derived exosomes can be taken up by BMSCs;In the co-culture system of BMSC and adipocytes,it can be found that the co-culture system itself can promote the osteogenic differentiation of BMSCs and inhibit adipogenic differentiation,and the medicated serum of Wenshen Tongluo Zhitong Decoction can further promote osteogenesis and inhibit Adipogenesis,but when the exosome inhibitor GW4869 is added to the co-culture system,this trend can be significantly reversed.The results of the above groups are significantly different from those of the control group(P<0.05).For BMSCs in the system The results of PCR and WB detection of osteogenic markers and adipogenic markers also verified this effect;in the co-culture system of BMSCs and adipocytes,we found that exosomes can significantly improve the osteogenic effects of BMSCs and inhibit Adipogenic differentiation,and Wenshen Tongluo Zhitong Recipe can further amplify this regulatory effect.The results of the above groups are statistically different from those of the control group(P<0.05).For the osteogenic markers and osteogenic markers of BMSC in the system The PCR and WB detection results of lipid markers also verified this effect.(4)The effector miRNA was selected by q-PCR to be miR-122-5p;after BMSCs transfected with NC mimic,miR-122-5p mimic,NC inhibitor,and miR-122-5p inhibitor were induced to osteize and adipate Characteristic staining showed that the osteogenic differentiation was enhanced in the miR-122-5p mimic group,and there was no significant change in the adipogenic differentiation compared with the other groups,while the adipogenic differentiation in the miR-122-5p inhibitor group was enhanced,and the osteogenic differentiation was higher than the others in the miR-122-5p inhibitor group.There was no significant change in the group;q-PCR experiments also found that miR-122-5p mimic can increase the expression of osteogenic differentiation marker genes RUNX2 and Osterix(P<0.05),while inhibiting the expression of adipogenic differentiation marker genes CEBP-? and PPARy2(P<0.05),while the results of the miR-122-5p inhibitor group were just the opposite(P<0.05);Shengxin predicted and suggested through q-PCR experiments that SPRY2 may be the target gene of miR-122-5p,which was tested by Luciferase reporter assay and RIP-QPCR further verified the combination of the two;in the Luciferase reporter assay,in the SPRY2-wild type group,the luciferase activity was significantly reduced after miR-122-5p mimic was transfected(P<0.01);miR-122 was transfected After-5p inhibitor,luciferase activity increased significantly(P<0.01).In the SPRY2-mutant group,no matter the miR-122-5p mimic or miR-122-5p inhibitor was transfected,the luciferase activity did not change significantly(P>0.05);while in RIP-QPCR,it was found that miR was overexpressed After-122-5p,the level of SPRY2 mRNA in the immune and silent complexes increased(P<0.01);by further constructing SPRY2 overexpression and knockdown plasmids and co-transfecting BMSCs,after induction of osteogenesis and adipogenesis,observe The differentiation of each group and the expression of osteogenic adipogenic marker genes were examined,and the protein expression of SPRY2 was detected.The relevant results confirmed that miR-122-5p can regulate the balance of osteolipid differentiation of BMSCs by targeting SPRY2.The results suggest that miR-122-5p can promote osteogenic differentiation and inhibit adipogenic differentiation of BMSCs by targeting SPRY2;detect the expression of MAPK-related signal proteins in each group,and find p-JNK/JNK,p-p38/p38,p-ERK1/2 The three relative ratios of/ERK1/2 were significantly increased in the miR-122-5p mimic group(P<0.01),but were significantly down-regulated in the miR-122-5p inhibitor group(P<0.05),The relative protein expression level of p-ERK1/2/ERK1/2 in the miR-5p+SPRY2 KD group was higher than that in the NC mimic group(P<0.01).(5)Successfully constructed a mouse ovariectomized osteoporosis model;the test results of serum bone metabolism index showed that the two bone formation indexes in the drug intervention group were increased to different degrees,which was statistically significant compared with the OVX group(P<0.01).However,the contents of the two bone resorption indexes slightly increased or decreased compared with the OXV group,and the contents did not change significantly compared with the OVX group(P>0.05);the results of the serum lipid metabolism test showed that the serum total cholesterol(CHOL),the OVX group Triglycerides(TG)and low-density lipoprotein(LDL)were significantly increased,and high-density lipoprotein(HDL)was significantly decreased.The lipid metabolism level of mice in the model group was significantly worsened(P<0.05),while the use of exosomes The disorder trend in the two groups was significantly improved(P<0.05),and the effect of traditional Chinese medicine in regulating the exosomes group was more significant(P<0.05);HE staining found that exosomes can improve the bone microstructure to a certain extent and inhibit fat Vacuoles are produced,and the effect of traditional Chinese medicine intervention in the exosomes group is more significant;Micro-CT observations show that the exosomes can be significantly injected and can significantly improve the three bone cancellous bones except for the trabecular bone thickness(Tb.Th)Parameters:three-dimensional bone density(3D BMD),bone volume fraction(BV/TV),bone trabecular separation(Tb.Sp)(P<0.01),and the effect of traditional Chinese medicine in regulating exosomes is slightly better than exosomes Group(P<0.05).In addition,injection of exosomes can also significantly improve the accumulation of tibia-regulated bone marrow adipose tissue(rMAT)caused by ovariectomy(P<0.05),and the effect of traditional Chinese medicine in regulating exosomes group is slightly better than that of exosomes group(P<0.01).),but the adjustment of exosomes to the tibia basic bone marrow adipose tissue(cMAT)is not significant(P>0.05);the three-point bending test indicates that exosomes injection can significantly improve the mechanical properties of the femur(P<0.05),and traditional Chinese medicine The effect of regulating exosomes is more obvious,but the difference between the two exosomes groups is not statistically significant(P>0.05);through q-PCR and IHC detection,it is found that fat-derived exosomes can increase the significant decrease in the OVX group The expression of osteogenic markers can also reverse the significantly increased expression of lipid markers in the OVX group,and the effect of traditional Chinese medicine in regulating the exosomes group is better than that of the exosomes group,and the difference is statistically significant compared with the OVX group(P<0.01).Conclusion:Adipocyte-derived exosomes can regulate the SPRY2-mediated MAPK signaling pathway through the effector miRNA-122-5p carried by them,thereby affecting the osteogenic and adipogenic differentiation of BMSCs in the bone marrow,and ultimately regulating the bone-fat balance.Wen Shen Tong Luo Zhi Tong Decoction can promote the secretion of exosomes from fat cells and exert the anti-osteoporosis effect through the above process. |
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