【作者】 王艳；

【导师】 田浩明； 陈香；

【作者基本信息】 四川大学 ， 内科学（内分泌代谢）， 2022， 博士

【摘要】 背景:非酒精性脂肪肝（non-alcoholic fatty liver disease,NAFLD）目前也称为代谢相关脂肪性肝病,已成为慢性肝病最主要的病因,严重威胁人类公共健康。TG及其代谢产物在肝脏沉积,导致肝细胞脂肪变性、纤维化甚至肝硬化。炎症激活和IR是NAFLD核心的病理生理机制,目前其肝外损害也逐渐引起关注。既往多数研究显示,NAFLD与骨量下降和骨折风险增加相关,IL-1、IL-6、TNF-α等炎症因子和脂联素、瘦素等脂肪因子分泌失衡,GH/IGF-1轴、Fetuin A、骨髓脂肪组织（marrow adipose tissue,MAT）异常等多种机制被发现参与其中,导致骨微结构改变及特异性骨代谢因子如OPG、RANKL、OCN、骨硬化蛋白（sclerostin,SOST）等分泌失常。但也有部分研究并未观察到NAFLD个体出现明显骨代谢异常,甚至认为NAFLD是骨骼的保护因素。因此,NAFLD对骨代谢是否存在影响及影响的具体机制仍不清楚。研究目的:1.探讨NAFLD患者与健康对照者外周血SOST水平的差异以及SOST与代谢参数指标的相关性;2.观察和比较高脂饮食（high fat diet,HFD）诱导NAFLD及HFD+阿托伐他汀钙干预小鼠较SD喂养的CON小鼠骨微结构和骨代谢改变;3.比较不同模型小鼠（NAFLD vs.CON）和阻断NF-κB通路小鼠(IL-6^-/-、TNF-α^-/-、Bay11-7082抑制)肝细胞培养条件性培养基干预BMSCs诱导成骨分化和BMCs诱导破骨分化的影响,进而明确NAFLD是否对骨代谢有直接影响及可能分子机制。材料和方法:本研究分为层次依次递进的三个部分。第一部分:临床研究我们设计了一项横断面研究,在健康体检中心纳入168名NAFLD人群和85名健康对照者（CON组）。测定所有研究对象血清SOST和代谢参数指标,并比较两组SOST水平的差异及SOST与BMI、TC、TG、LDL-C、HDL-C、肝酶、空腹胰岛素、HOMA-IR、脂肪肝指数（fatty liver index,FLI）等代谢参数指标的相关性。第二部分:动物实验12周龄雄性C57BL/6小鼠被分为SD喂养（CON）组、HFD诱导（NAFLD）组和HFD+阿托伐他汀钙干预组,每组均8只。12周造模完成后,麻醉处置小鼠,采集血样及组织样本,进行下列实验⑴.测定代谢指标:血清TC、TG、血糖、SOST水平。⑵.测定骨微结构:以μCT检测股骨微结构指标（Tb.BV/TV、Tb.Th、Tb.N、Tb.Sp和Conn.D）。⑶.组织病理学检测:肝脏组织油红“O”和HE染色;MAT行HE染色;胫骨行TRAP染色和HE染色+成骨细胞半定量计数。⑷.qRT-PCR测定基因表达水平:提取右侧股骨干骺端和MAT内RNA,转录为c DNA后qRT-PCR测定成骨和破骨相关基因表达;提取肝脏组织RNA,qRT-PCR测定脂代谢、炎症和骨代谢相关基因表达。⑸.肝脏组织转录蛋白组学检测:送相关公司测定。第三部分:体外实验（肝细胞培养条件培养基干预诱导成骨和破骨分化）,具体如下:⑴.取CON、HFD诱导NAFLD、IL-6^-/-和TNF-α^-/-小鼠,通过下腔静脉灌注、原位胶原酶消化,提取原代肝细胞并培养。HFD诱导NAFLD小鼠肝细胞再分为两组,一组为单纯肝细胞培养,另一组加入Bay11-7082（NF-κB抑制剂）共培养。收集4 h、24 h和48 h肝细胞培养上清,混匀后行检测代谢组学、测定ELISA法IL-6和TNF-α水平。⑵.肝细胞培养条件性培养基干预BMSCs诱导成骨分化和BMCs诱导破骨分化:将肝细胞上清与肝细胞基础培养基对倍稀释为50%浓度后作为新培养基,干预BMSCs成骨分化和BMCs破骨分化。在BMSCs向成骨诱导分化第7天进行ALP染色和细胞RNA提取;在BMCs向破骨诱导分化第9天进行TRAP染色和细胞RNA提取,逆转c DNA后采用qRT-PCR测定成骨和破骨相关基因表达。细胞实验重复三次。结果:本研究得出以下结果:1.临床研究:与CON组比较,NAFLD组血清SOST水平显著降低。在NAFLD组,SOST与多种代谢参数指标呈显著负相关（包括WC、尿素、肝酶、γ-GGT和TG）,而在CON组相关性不显著。在NAFLD组,血清SOST也与FLI呈显著负相关,而CON组二者相关性同样不显著。2.动物研究:⑴.与CON组比较,HFD组平均肝脏重量和血清TC水平显著升高,HFD+阿托伐他汀钙干预组体重、肝脏重量、血清TC水平较HFD组显著降低,HFD组血清TG水平与CON组无显著差异;三组血清血糖和SOST水平未见显著统计学差异。⑵.μCT扫描骨微结构分析,与CON组比较,HFD组骨微结构受损（Tb.BV/TV和Tb.Th显著降低,Tb.Sp显著增加）,HFD+阿托伐他汀钙干预组后较HFD组无显著改善。⑶.组织病理学:肝脏油红“O”染色后,可见HFD组肝小叶内脂肪沉积较CON组显著增多。HE染色可见HFD组肝细胞轻到中度空泡变性,提示脂肪变性。HFD+阿托伐他汀钙干预组肝脏病理学改变类似于HFD组,但程度较轻;骨髓MAT经HE染色后可见HFD组骨髓内脂肪细胞布满髓腔,数量显著增多,HFD+阿托伐他汀钙干预组较略有好转。胫骨石蜡包埋后切片HE染色,CON、HFD和HFD+阿托伐他汀钙干预组成骨细胞（osteoblasts,OBs）计数未见显著统计学差异;TRAP染色后,可见HFD组破骨细胞（osteoclasts,OCs）数量增多趋势,HFD+阿托伐他汀钙干预后无显著改善,OBs计数和OCs计数测量显示三组间未见显著统计学差异。⑷.组织基因表达:（1）.肝脏脂代谢相关基因相对表达:与CON组比较,HFD组与TG合成、转运和分解相关的基因Lpl、Atgl、Dgat2显著升高,与肝内新合成TG相关的Fas基因显著降低;与TG发生β氧化分解相关的Cpt2及其他TG转运和合成相关的Plin2、Hsl在两组间未见显著统计学差异。与HFD比较,HFD+阿托伐他汀钙干预组Atgl、Dgat2、Cpt2、Plin2、Hsl基因均显著降低。（2）.肝脏炎症因子相关基因相对表达:与CON组比较,HFD组Ctss、Anxa2、TNF-α和IL-6显著升高;HFD+阿托伐他汀钙干预组Ctss、Anxa2、TNF-α较HFD组显著降低。（3）.肝脏骨代谢相关基因相对表达:与CON组比较,HFD组Col1α1和Ctsk显著升高,HFD+阿托伐他汀钙干预组Ctsk较与HFD组显著降低;Rac2和Srd5α1表达量极低。（4）.骨组织骨代谢相关基因相对表达:与CON组比较,HFD组Col1α1和Sost显著降低;HFD+阿托伐他汀钙干预组Sost较HFD组显著升高;Ctsk在三组间未见显著统计学差异。（5）.MAT脂代谢相关基因相对表达:与CON组比较,HFD组Plin2显著升高,而HFD+阿托伐他汀钙干预组较HFD组显著降低。⑸.肝脏转录蛋白组学分析共检出2745个差异表达蛋白,CON组与HFD组、HFD+阿托伐他汀钙干预组PCA分析提示蛋白表达谱具有显著差异。其中CON组和HFD组蛋白KEGG通路富集分析到与多条炎症通路如NF-κB通路有关联;与CON组比较,发现在HFD组肝脏组织高表达（比值≥1.5）与骨代谢相关的基因（Ctss、Anxa2、Rac2、Srd5α1和Col1α1）。3.体外实验:⑴.CON、HFD、IL-6^-/-、TNF-α^-/-及HFD+Bay11-7082小鼠肝细胞培养上清IL-6水平分别为145.51±2.40 pg/m L、167.35±2.40 pg/m L、87.76±1.15 pg/m L、112.82±1.51 pg/m L和114.84±1.48 pg/m L;TNF-α水平分别为5.73±0.12 pg/m L、7.85±0.16 pg/m L、2.46±0.09 pg/m L、0.41±0.12 pg/m L和2.20±0.13 pg/m L。⑵.代谢组学分析提示CON和NAFLD小鼠肝细胞培养上清共检出193个差异代谢物,PCA分析可见组间代谢物表达谱有显著差异。两组差异代谢主要集中在氨基酸代谢、脂代谢、核苷酸代谢和膜转运通路。通过差异代谢物KEGG通路富集分析提示两组肝细胞培养上清液m TOR信号通路、cAMP信号通路、ABC转运体及多种糖、脂类和氨基酸代谢中存在显著差异,同时自噬和凋亡相关通路也存在显著差异。⑶.肝细胞培养条件性培养基干预BMSCs诱导成骨分化和BMCs诱导破骨分化:（1）.与CON比较,HFD诱导NAFLD小鼠肝细胞培养条件性培养基显著抑制BMSCs成骨分化,促进BMCs破骨分化。（2）.与HFD相比,阻断NF-κB后各组促BMSCs成骨分化均显著增加,促成骨分化能力依次为IL-6^-/->TNF-α^-/->HFD+Bay11-7082。（3）.与HFD相比,阻断NF-κB后HFD+Bay11-7082和IL-6^-/-促破骨分化能力反而增加,其中HFD+Bay11-7082最强,IL-6^-/-其次,TNF-α^-/-略下降。结论:1.NAFLD组血清SOST水平较CON组显著降低,且与多种代谢参数指标如WC、尿素、肝酶、γ-GGT、TG、FLI呈显著负相关,提示NAFLD可能对骨代谢有不利影响。2.HFD诱导NAFLD小鼠导致骨组织微结构受损和Sost表达减少,Sost减少可能反映NAFLD小鼠骨量和骨细胞数量减少。3.HFD诱导NAFLD小鼠肝细胞培养条件性培养基干预BMSCs和BMCs,成骨相关基因表达降低,破骨相关基因表达增加,提示NAFLD肝脏直接影响骨代谢,抑制成骨分化,促进破骨分化。4.IL-6、TNF-α是NAFLD肝脏对骨代谢作用的重要介质,且IL-6的作用可能呈双相性,尤其是对破骨分化。5.NF-κB炎症通路并非NAFLD对骨代谢产生直接影响的关键通路,FFA代谢异常诱导的细胞凋亡和自噬可能是另一个重要机制,且对破骨的影响大于成骨。更多还原

【Abstract】 Background:NAFLD,also known as metabolic associated fatty liver disease（MAFLD）,has become the major cause of chronic liver disease and poses a great threat to public health.Deposition of TG and its metabolites in the liver leads to detrimental hepatocyte steatosis fibrosis and even cirrhosis.Since inflammatory activation and insulin resistance are the core pathophysiological mechanisms for NAFLD,extra-hepatic damage in NAFLD has begun to draw attention.Most previous studies have shown that NAFLD is associated with decreased bone mass and fracture risk;a variety of mechanisms including imbalance of secretion of inflammatory factors such as IL-1,IL-6,TNF-α,and adipokines including adiponectin and leptin,and increase of GH/IGF-1 axis,Fetuin A,and MAT are found to be involved,leading to alterations in skeletal microstructure and abnormalities in secretion of specific bone metabolic factors such as OPG,RANKL,OCN,sclerostin（SOST）.However,some studies did not observe significant bone metabolism abnormalities in individuals with NAFLD,and even considered NAFLD as a protective factor for the bone.Therefore,it remains unclear whether NAFLD affects bone metabolism and the specific mechanism underlying the effect.Objectives:1.To investigate the discrepancy in circulating SOST levels between NAFLD and healthy controls,and the correlation with metabolic parameters.2.To observe and compare the changes in bone microstructure and bone metabolism in high fat diet（HFD）induced NAFLD mice and HFD-and-atorvastatin-intervened mice compared to SD induced CON.3.To observe the effects of mice hepatocyte culture supernatant of different intervening methods（NAFLD vs.CON）and inhibited NF-κB pathway(IL-6^-/-,TNF-α^-/-,Bay11-7082)on bone marrow mesenchymal cells（BMSCs）into osteoblasts（OBs）and bone marrow cells（BMCs）differentiation into osteoclast（OCs）,and thus clarify on whether and potentially how NAFLD directly affects bone metabolism.Materials and methods:This study was divided into three sequential parts.Part Ⅰ:Clinical studyA cross-sectional study was designed to include 168 NAFLD subjects and 85healthy CON.Serum SOST levels and metabolic parameters were measured in all subjects,and differences in SOST level between the two groups were compared.Differences in the correlation between SOST and BMI,TC,TG,LDL-C,HDL-C,liver enzymes,fasting insulin,HOMA-IR,fatty liver index（FLI）were also evaluated.Part Ⅱ:Animal experiment.12-week-old male C57BL/6 mice were divided into SD-fed CON group,HFD-induced NAFLD group and HFD-induced with atorvastatin intervention group,each group with 8 mice.After 12 weeks of modeling,mice were dissected for blood and tissue sampling after excessive anesthesia.The following experiments were carried out:⑴.After centrifugation,serum was used to determine TC,TG,SOST and blood glucose levels by ELISA.⑵.Femur bone mineral density（BMD）and microstructure were evaluated by microcomputed tomography（μCT）,on parameters including Tb.BV/TV,Tb.Th,Tb.N,Tb.SP and Conn.D.⑶.Histopathological examinations:liver tissue was stained with oil red O and HE;tibial TRAP staining and HE staining,and semi-quantitative counting of osteoblasts were conducted.⑷.Gene expression:metaphyseal RNA and bone marrow adipose tissue（MAT）RNA were extracted from the right femur for reverse transcription into c DNA,which was then used to measure gene expressions associated with osteogenesis and osteoclastogenesis by qRT-PCR fluorescence quantification;RNA extracted from liver tissue was used to measure gene expressions associated with lipid metabolism,inflammation and bone metabolism,along with liver tissue transcription protein expression.⑸.Some liver tissues were sent to relevant companies for proteomic studies.Part Ⅲ:Cell culture and intervention experiments in vitro.Liver perfusion followed by collagenase digestion was performed on.⑴.Primary hepatocyte culture from CON,HFD-induced NAFLD,IL-6^-/-and TNF-α^-/-mice to extract hepatocytes for in vitro cell culture,HFD-induced NAFLD mouse hepatocytes were subdivided into two groups,in which one group was added Bay11-7082（NF-κB inhibitor）as intervention,and the other uninterrupted.primary hepatocyte culture was carried out from hepatocytes extracted through liver perfusion from inferior vena cava and collagenase digestion.Supernatants of hepatocyte cultures at 4 h,24 h,and 48 h were collected and subjected to metabolomic assay and ELISA to measure levels of IL-6 and TNF-α.⑵.The supernatant was used as a new conditioned culture medium after partially diluting with hepatocyte basal medium to 50%concentration to interfere with induction of osteogenic differentiation of BMSCs and induction of osteoclast differentiation of BMCs.ALP staining and RNA extraction were performed on day 7of osteogenic differentiation in BMSCs;TRAP staining and RNA extraction were performed on day 9 of osteoclast differentiation in BMCs.qRT-PCR was used to quantify gene expressions related to osteogenic and osteoclast differentiation.Cell experiments were repeated three times.Results:1.In the clinical study,serum SOST levels were significantly lower in the NAFLD group compared to the CON group.In the NAFLD group,SOST level was negatively correlated with several metabolic parameters,including WC,urea,liver enzymes,γ-GGT and TG,while this correlation was not significant in the CON group.Serum SOST level was also negatively correlated with fatty liver index（FLI）in the NAFLD group,with no suggested correlation in the CON group.2.In the animal study,⑴.Compared to the CON group,TC levels and liver weight were significantly higher in the HFD group,while TG level showed no signifiacnt difference;compared to the HFD group,mice in the HFD+atorvastatin intervention group had significantly lower TC levels and liver weight.No statistically significant difference on blood glucose and serum SOST levels was observed among the three groups.⑵.μCT analysis showed that the bone microstructure of HFD group was significantly reduced（decreased Tb.BV/TV and Tb.Th and Tb.Sp increased significantly）,while the HFD with atorvastatin intervention group had no significant improvement compared with HFD group.⑶.Histopathological examinations:deposition of oil red O in hepatic lobules in HFD group was significantly higher than that in CON group.HE staining showed mild to moderate vacuolar degeneration of hepatocytes in HFD group,suggesting steatosis.The pathological changes of liver in HFD with atorvastatin intervention group were similar to those in the HFD group,but to a lesser extent.HE staining of MAT showed that the adipocytes in the bone marrow of HFD group was full of bone marrow cavities,and the number was significantly increased;the situation was slightly better with atorvastatin intervention.HE staining of paraffin-embedded tibia sections revealed that the number of OBs decreased in HFD group with no significant improvement in HFD with atorvastatin intervention group.TRAP staining showed the number of OCs in HFD group increased,and there was no significant improvement with atorvastatin intervention.Overall measurements of OBs count and OCs count showed no significant statistical difference among the three groups.⑷.Tissue gene expression:（1）.Relative expression of genes related to lipid metabolism in liver tissue:compared with the CON group,the HFD group showed remarkably higher expression of Lpl,Atgl,Dgat2,Fas were significantly lower;expression levels of Cpt2,Plin2,and Hsl were not statistically significantly different between the two groups.Compared to level of expression of the HFD group,Atgl,Dgat2,Cpt2,Plin2,and Hsl all showed a significantly reduced relative expression in the HFD with atorvastatin intervention group.（2）.Relative expression of inflammatory factor-related genes in liver tissue:compared to the CON group,the relative expression of Ctss,Anxa2,TNF-αand IL-6 were significantly higher in the HFD group;compared to the HFD group,expressions of Ctss,Anxa2,and TNF-αwere significantly lower in the HFD with atorvastatin intervention group.（3）.Relative expression of genes related to bone metabolism in liver tissue:levels of Col1α1 and Ctsk were significantly higher in the HFD group compared to the CON group;Ctsk level was significantly lower in the HFD with atorvastatin intervention group compared to the HFD group.qRT-PCR results indicated that Rac2 and Srd5α1 expressions were extremely low.（4）.Relative expression of bone metabolism-related genes in bone tissue:Col1α1 and Sost were significantly lower in the HFD group than in the CON group;Sost level in the HFD with atorvastatin intervention group was significantly higher than in the HFD group;there was no statistically significant difference in Ctsk level among the three groups.（5）.Relative expression of lipid metabolism-related genes in MAT:Plin2 level was significantly higher in the HFD group compared to the CON group.At the same time,Plin2 level was significantly lower in the HFD+atorvastatin intervention group compared to the HFD group;it was also significantly lower than in the CON group.⑸.A total of 2745 differentially expressed proteins were detected by hepatic transcriptional proteomics analysis,and PCA in the CON group,HFD groups and the HFD with atorvastatin intervention group suggested significant differences in protein expression profiles.KEGG pathway enrichment analysis of the proteins in the CON and HFD groups revealed associations with several inflammatory pathways such as the NF-κB pathway,and several genes related to bone metabolism were found to be highly expressed in the liver tissue of the HFD group compared to the CON group.Such genes include Ctss,Anxa2,Rac2,Srd5α1 and Col1α1.3.In the in vitro cell culture study,⑴.IL-6 and TNF-αlevels of hepatocyte culture supernatant from five mice models were measured by ELISA.The levels of IL-6 were 145.51±2.40 pg/ml,167.35±2.40 pg/ml,87.76±1.15 pg/ml,112.82±1.51 pg/ml and 114.84±1.48 pg/ml,and the levels TNF-αwere were 5.73±0.12 pg/ml,7.85±0.16 pg/ml,2.46±0.09 pg/ml,0.41±0.12 pg/ml and 2.20±0.13 pg/ml in CON,HFD,IL-6^-/-,TNF-α^-/-and HFD+Bay11-7082 group respectively.⑵.Metabolomic analysis of primary hepatocyte culture supernatants detected a total of 193 differential metabolites between CON and NAFLD mice,and PCA analysis showcased significant differences in metabolite expression profiles between them.The differential metabolism between the two groups primarily concentrated on amino acid metabolism,lipid metabolism,nucleotide metabolism and membrane transport pathway.KEGG pathway enrichment analysis of the differential metabolites from the supernatants of the two groups suggested significant differences in the m TOR signaling pathway,cAMP signaling pathway,ABC transporter and various glycolipid,lipid metabolism and amino acid metabolism.In the meantime,significant difference was also observed in autophagy and apoptosis-related pathways.⑶.Effects of conditioned medium on osteogenesis and osteoclastogenesis of primary bone marrow cells:（1）Compared with CON,HFD induced NAFLD mice hepatocyte culture conditioned medium significantly inhibited osteogenesis of BMSCs and promoted osteoclastogenesis of BMCs.（2）.Compared with CON,The effect on osteogenesis of conditioned medium of hepatocyte culture which NF-κB pathway was inhibited increased significantly,and the ability sequence was IL-6^-/->TNF-α^-/->HFD+Bay11-7082.（3）.Compared with CON,The effect on osteogenesis of HFD+Bay11-7082 and IL-6^-/-were increased significantly,HFD+Bay11-7082 had the greatest effect on the differentiation,followed by IL-6^-/-,TNF-α^-/-was decreased slightly.Conclusion:1.Serum SOST levels were significantly lower in the NAFLD group compared to the CON group and were negatively correlated with multiple metabolic parameters such as WC,urea,liver enzymes,γ-GGT,TG,and FLI,suggesting that NAFLD impacts bone metabolism.2.HFD-induced NAFLD mice showed reduced BMD and Sost expression in bone tissue,and decreased Sost level may reflect reduced bone volume and osteoblast count in NAFLD.3.By using hepatocyte culture supernatant from HFD-induced NAFLD mice to intervene BMSCs and BMCs,it was found that expression of osteogenesis-related genes decreased while expression of osteoclastogenesis-related genes increased,suggesting NAFLD liver directly affects bone metabolism by inhibiting osteogenic differentiation and promoting osteoclastogenic differentiation.4.IL-6 and TNF-αare crucial mediators of the effects of NAFLD liver on bone metabolism,and the effects of IL-6 may be biphasic,especially on osteoclastogenic differentiation.5.The NF-κB inflammatory pathway is not the only critical pathway from which NAFLD directly affects bone metabolism.Cell apoptosis and autophagy induced by abnormal FFA metabolism may be another important mechanism,and the effects on osteogenesis are greater than those on osteoclastogenesis.更多还原