Effect Of Shikonin On Exosome Palmitoleic Acid Mediated Osteoclastic Differentiation And Its Mechanism

Background:In previous studies,through the metabolomic analysis of bone exosomes in patients with osteoporosis and non osteoporosis,it was found that the expression of palmitoleic acid was significantly different.It was speculated that metabolite molecules may play a role in the process of osteoclast differentiation through the way of exosome.Studies had shown that,shikonin,a traditional Chinese medicine component,may play a role in biological processes by affecting the secretion of exosomes and the expression of carrying factors.Traditional Chinese medicine believes that osteoporosis mostly belongs to the category of"bone flaccidity".Kidney deficiency is the root cause of the disease.The kidney is the congenital foundation and the main bone generates marrow.At the same time,a large number of clinical practices have also found that while treating from the kidney,combined with the methods of dredging collaterals,strengthening the spleen and soothing the liver,it will achieve better curative effects.The liver stores blood to drain the tendons,and the kidney stores essence to seal the bones.The"essential reading of medical school"mentioned that"the liver and kidney share the same origin".The liver stores blood,the kidney stores essence,the liver is the son of the kidney,the liver blood nourishes the kidney essence,and the purple grass returns to the heart and liver meridian,which can cool and activate blood,combined with the function of exosomes and dredging waterways,we can better supplement the essence and Qi in the kidney and fill lean marrow to treat bone flaccidity.Objective:To explore the effect of palmitoleic acid on osteoclast differentiation mediated by exosomes,to explore the effect of SCD1 gene knockout on mouse bone phenotype,and to clarify the mechanism of shikonin on exosome formation and osteoclast differentiation.Methods:（1）The osteogenic differentiation model of ST2 cells in vitro was established.The osteogenic ability was identified by alizarin red staining,ALP enzyme activity and the expression of osteogenic marker genes;The exosomes derived from ST2 cells supernatantat at the mature stage of osteogenic differentiation were extracted and identified for morphology,particle size and expression of marker proteins;The exosomes derived from the supernatant of ST2 cell culture medium on day 0,3,7 and 14 of osteogenesis were analyzed by targeted metabolomics of palmitoleic acid;（2）The CRSPR/cas9 technology was used to construct the knockout mouse model of SCD1,the key enzyme of palmitoleic acid synthesis.After the homozygous were identified,they was bred.The bone morphology of wild-type and knockout mice were detected by micro CT method,and BMD,BV/TV,BS/BV,BS/TV and TB.Sp、Tb.Th were deteced;After 6 weeks of shikonin gavage,the changes of bone parameters were detected by micro CT,and wild-type and knockout mice bone tissue sections were prepared for HE and TRAP staining;The exosomes extracted from bone tissue of wild-type and knockout mice were detected by metabonomics;BMDM cells from wild-type and knockout mice were extracted and added with M-CSF and RANKL for osteoclast induction.The expression of osteoclast marker genes was detected by TRAP staining and RT-q PCR,and the change of expression trend after adding shikonin was observed;（3）The stable transformation of lentivirus ST2 cell line was constructed.The detections were used by Alizarin Red staining,ALP enzyme activity detection and RT-q PCR.The effect of down-regulated SCD1 gene expression on cell osteogenic differentiation and the change trend after shikonin addition was detected;The exosomes derived from wild-type ST2 cells,stable transformation ST2 cells and the culture system was added into shikonin stable ST2 cells were co-cultured with osteoclasts.The expression of osteoclast marker genes were detected by TRAP staining and RT-q PCR,and its effect on osteoclast differentiation was observed;（4）NTA was used to detect the effect of shikonin on the secretion concentration of exosomes in stable cell line ST2 and RAW264.7 cells;The stable cell lines ST2and the RAW264.7 cells extracted with or without shikonin interference and four kinds of exosomes were detected and analyzed by mass spectrometry,and the differentially expressed proteins were analyzed by cluster analysis;（5）Rab11a,a protein differentially expressed in exosomes after the addition of shikonin,was screened.Rab11a activator was used in osteoclast differentiation culture system to detect its effect on osteoclast differentiation and the role of shikonin in this process.Results:（1）By adding 50μg/ml ascorbic acid,10 mmβ-glycerol phosphate and10nm dexamethasone in the culture system,the osteogenic differentiation model of ST2 cells in vitro was successfully established.It was detected on the 0,3,7 and 14days of osteogenic differentiation.With the extension of induction time,the production of calcium nodules gradually increased,the activity of ALP enzyme increased firstly and then decreased,and the expression of Runx2 and ALP genes increased firstly and then decreased slightly,the genes expression of COL1A1 and OCN showed a gradual upward trend,which was consistent with the results of Alizarin Red staining and ALP enzyme activity detection.We used Exo Quick ^TMreagent to extract the exosome precipitation from the supernatant of the culture medium of ST2 osteogenic differentiation for 14 days.Through transmission electron microscopy,it was found that the diameter of the exosome was about 30-400 nm.It was a double-layer membrane coated vesicle,and expressed membrane marker proteins CD81,CD9 and HSP70.The concentration of the exosome was 1.5×10¹²/ml detected by NTA,with a diameter range of about 50-500nm;GC-MS analysis of free fatty acids in exosomes of ST2 culture medium on days 0,3,7 and 14 showed that the expression of palmitoleic acid increased gradually with the increase of osteogenic induction days of ST2 cells,suggesting that it may play an important role in the later stage of osteogenic differentiation.（2）The results of mouse tail identification showed that homozygous mice had only one band at 452bp.Compared with wild-type mice,SCD1^-/-mice had obvious body length,light weight and sparse hair;The results of micro CT detection of knockout mice,OVX mice and wild-type mice showed that compared with wild-type mice,BMD of knockout mice decreased significantly,and BV/TV,BS/BV and BS/TV showed a significant downward trend,TB/SP increases,TB/Th decreased;The results of HE staining showed that compared with wild-type mice,SCD1 knockout mice had sparse trabecular bone structure,reduced bone mineral density and increased bone marrow cavity vacuoles.After shikonin administration,the bone trabecular structure of knockout mice was greatly improved,bone mineral density increased,and the number of bone marrow cavity vacuoles was relatively small,indicating that shikonin had a good alleviating effect on the bone phenotype of knockout mice.Trap staining showed that the number of osteoclasts in knockout mice was significantly higher than that in wild-type mice.After shikonin administration,the number of osteoclasts in knockout mice was greatly reduced,indicating that shikonin had a good inhibitory effect on the differentiation and maturation of osteoclasts;After identification of exosomes from bone tissue of knockout mice and wild-type mice,metabonomics analysis based on LC-MS/MS was carried out to screen out significantly different metabolites.KEGG annotation results of different metabolites showed that the different metabolites were mostly concentrated in metabolic pathway,fatty acid biosynthesis,unsaturated fatty acid biosynthesis,glycerol lipid metabolism Among the signal pathways such as c AMP signal pathway,IPA analysis also showed that the differential expression of lipid metabolism signal pathway was obvious,which was basically consistent with the previous results of human bone tissue derived exosome metabolism group;The gene expression of NFATc1,TRAP and CSTK in knockout mice was higher than that in wild-type group.After 6 weeks of shikonin administration,the gene expression showed a downward trend.（3）Sh RNA was transferred into ST2 cells.After puromycin screening,the virus transfection efficiency was more than 85%.A stable ST2 cell line was established.The expression of SCD1 gene and protein decreased significantly.Sh RNA interference ST2 cell line was successfully constructed;The toxic effects of different shikonin concentrations on ST2 cells were detected by CCK8 method.The detection analysis found that 0.5μM concentration is the best;Osteoblasts were induced by stable cell lines.The results of alizarin red staining showed that the production of calcium nodules in the interference group decreased significantly,and after 0.5μM shikonin was added,the red stained by Alizarin Red increased slightly,but there was no significant difference;The detection results of osteogenic marker gene expression showed that the expression of Runx2,ALP,COL1A1 and OCN genes in the interference group decreased.After shikonin intervention,the expression of idling group and interference group increased to a certain extent.The detection results of ALP activity were consistent with the results of RT-q PCR,which showed that the interference of SCD1 gene expression had a significant effect on the osteogenic differentiation ability of cells;In the interference group,ST2 cell exosomes could enter RAW264.7 cells;Exosomes derived from cell lines in the idling group,those derived from cell lines in the interfering group and those derived from cell lines in the interfering group intervened with shikonin were co cultured with RAW264.7 cell osteoclast differentiation system respectively.Trap staining results showed that the number of osteoclasts increased slightly due to exosomes derived from the interfering group,and the trend was not significant.RT-q PCR results showed that,The secretion from the interference group significantly increased the expression of bone breaking marker genes NFATc1,trap and ctsk,while the shikonin intervention group showed a downward trend with statistical difference,and down regulated to a level close to that of the control group.（4）0.5μM shikonin was added to the osteogenic differentiation system of sh RNA interference ST2 cells,extract the exosomes of extracellular matrix and culture medium supernatant respectively.In the group without shikonin,the concentration of exosomes derived from extracellular matrix was 1.3×10¹¹/ml,the peak value of exosome diameter distribution is about 400-500nm.In the shikonin group,the concentration of exosomes from extracellular matrix is 1.2×10¹¹/ml.In the group without shikonin,the concentration of exosomes from the supernatant of culture medium is 3.1×10¹¹/ml;In the shikonin group,the concentration of exosomes from the supernatant of culture medium was 8.0×10¹⁰/ml,and the concentration of ST2 exosomes changes little after the addition of shikonin;0.5μM shikonin was added to RAW264.7 cell osteoclast induction system,extract the exosomes from the supernatant of cell culture medium.RAW264.7 cells were induced to break bones for2 days.The concentration of exosomes from the supernatant of culture medium without shikonin was 6.0×10¹²/ml,the peak value of exosome diameter distribution is about 400nm.The concentration of exosomes from the supernatant of RAW264.7 cell osteoclast induced by shikonin group for 2 days is 1.7×10¹¹/ml,the peak value of secrete diameter distribution was about 500nm.RAW264.7 cells were induced to osteoclast for 6 days.The concentration of secrete from the supernatant of culture medium without shikonin was 1.8×10¹²/ml,the peak value of exosome diameter distribution is about 300-1000nm.The concentration of exosomes from the supernatant of the culture medium of RAW264.7 cells plus shikonin group is after osteoclast induction for 6 days 2.5×10¹⁰/ml,the peak value of diameter distribution is about 300-1000nm.After the addition of shikonin,the concentration of exosomes decreased significantly,and the particle size distribution of exosomes also decreased slightly;Extract（excluding）0.5μM shikonin respectively interfered with the exosomes of the supernatant of ST2 cells and RAW264.7 cells.Four groups of exosomes were detected by 4D label free proteome quantitative technology,which showed that the proteins in the exosomes were distributed in all molecular weights,and the distribution was relatively uniform.Mass spectrometry analysis found that the shikonin treatment group interfered with the exosomes of ST2 cells had 130 down regulated proteins and 80 up-regulated proteins compared with the control group,Compared with the control group,there were 49 down regulated proteins and 174 up regulated proteins in RAW264.7 cell exosome shikonin treatment group.The results of subcellular structure annotation classification showed that most of the components of RAW264.7 cell-derived exosome proteins came from cytoplasmic proteins,followed by extracellular proteins;Most of the proteins interfering with ST2cell-derived exosomes come from extracellular proteins,followed by cytoplasmic proteins.The subcellular sources of exosomes from the two sources are similar;The results of cluster analysis of the enrichment of differential protein biological processes show that there are significant differences in proteins involved in the regulation of extracellular matrix tissue,collagen fiber tissue and fatty acid biosynthesis,suggesting that shikonin affects the function of osteoblast and osteoclast derived exosomes,which may play a role through fatty acid metabolism or extracellular matrix protein function.（5）After adding Rab11a activator QX77 to the RAW264.7 cells osteoclast induction culture system,the expression of osteoclast marker genes NFATc1,trap and CSTK showed an upward trend.TRAP staining experiment also showed that the formation of osteoclast multinucleated cells increased,and the number of multinucleated cells decreased significantly after adding shikonin,indicating that shikonin can inhibit osteoclast differentiation by inhibiting the effect of Rab11a.Conclusion:The expression of exosomal palmitoleic acid was up-regulated during osteogenic differentiation;SCD1^-/-mice showed a certain degree of osteoporosis phenotype,and shikonin could correct the bone phenotype of SCD1^-/-mice;The osteogenic ability of stably transformed ST2 cells was decreased,the inhibitory effect of exosomes on the differentiation of RAW264.7 cells decreased,and shikonin could be effectively reversed after intervention;Shikonin could inhibit the secretion of exosomes from RAW264.7 cells and affect the proteins expression profile of exosomes from stably transformed ST2 cells and RAW264.7 cells.There is a significant difference in the expression of Rab11a,and Rab11a also plays an important role in the formation of exosomes;Activation of Rab11a expression could promote osteoclastic differentiation of RAW264.7 cells,and shikonin could inhibit the above effects.In conclusion,metabolites may play a role in RAW264.7 osteoclast differentiation in an exosome mediated manner.The knockout of SCD1 gene would significantly affect the bone phenotype of mice;Shikonin treatment can affect the secretion of exosomes and change the protein mass spectrum.It may also inhibit the secretion of osteoclast exosomes and osteoclast differentiation through Rab11a pathway.