Study on Cartilage Protective Effect of Xibining on KOA Model Rats Based on UPLC-Q-Orbitrap MS/MS Technique
-
摘要:
目的 运用超高效液相色谱-四极杆-静电场轨道阱串联质谱(UPLC-Q-Orbitrap MS/MS)技术分析膝痹宁的药物活性成分, 通过干预膝骨关节炎(KOA)模型大鼠代谢因素探讨膝痹宁软骨保护效应的作用机制。 方法 制备膝痹宁水提物, UPLC-Q-Orbitrap MS/MS技术分析膝痹宁的活性成分。将大鼠分为空白组、KOA组、膝痹宁组, 提取大鼠软骨组织, HE染色、番红O-固绿染色观察组织结构形态; qPCR和Western blot法检测过氧化物酶体增殖物激活受体(PPARγ)、PPARγ辅助激活因子1α(PGC1α)及基质金属蛋白酶(MMP)3、MMP13的mRNA和蛋白表达水平; 取各组大鼠血清完成代谢组学分析。 结果 经鉴定, 膝痹宁含活性成分56种; 组织学切片提示膝痹宁具有软骨保护作用, 并能上调KOA大鼠软骨组织PPARγ、PGC1α的mRNA和蛋白表达水平(P < 0.05, P < 0.01), 降低MMP3、MMP13的mRNA和蛋白表达水平(P < 0.05, P < 0.01);此外, 代谢组学研究发现, 膝痹宁对13种KOA差异代谢物存在干预作用, 涉及10条代谢通路。 结论 膝痹宁具有多种药物活性成分, 其KOA软骨保护效应与药物活性成分对KOA的代谢调控有关。 Abstract:OBJECTIVE To analyze the active components of Xibining and its pathological mechanism of cartilage protective effect by interfering with metabolic factors in knee osteoarthritis (KOA) model rats using ultra-performance liquid chromatography-quadrupole-orbitrap mass spectrometry (UPLC-Q-Orbitrap MS/MS) technique. METHODS The water extract of Xibining was prepared and the active components were analyzed by UPLC-Q-Orbitrap MS/MS. Rats were divided into blank group, KOA group and Xibining group. Cartilage tissue was extracted, and the structure and morphology of the tissue were observed by HE staining and Safranin O staining. The qPCR and Western blot were used to detect the protein and mRNA expression of peroxisome proliferator-activated receptor-γ (PPARγ), PPARγ co-activator-1α (PGC1α) and matrix metalloproteinase (MMP) 3, MMP13, which were representative matrix metalloproteinases of cartilage degradation. Besides, the serum of each group was collected to complete the metabonomics analysis. RESULTS A total of 56 active components were identified in Xibining. Histological sections suggested that Xibining had a protective effect on cartilage, and could up-regulate the gene and protein expression levels of PPARγ and PGC1α, and decrease the gene and protein expression of MMP3 and MMP13 in cartilage tissue of KOA rats. In addition, Xibining had intervention effects on 13 different metabolites of KOA, involving 10 metabolic pathways. CONCLUSION Xibining has a cartilage protective effect of KOA, the mechanism of which may be related to the metabolic regulation of KOA by its active substance. -
Key words:
- knee osteoarthritis /
- cartilage protection /
- Xibining /
- metabonomics /
- active components
-
表 1 目的基因序列
Table 1. Target gene sequence
目的基因 前引(5'→3') 后引(5'→3') MMP3 GGGTGGATGCTGTCTTTG TGCCTTCCTTGGATCTCTT MMP13 GGGGAGCCACAGATGAG AACGCTCGCAGTGAAAG PPARγ CCTCGAGGACACCGGAGA CACGGAGCTGATCCCAAAGT PGC1α GTAGATCCTCTTCAAGATCCTG CATACAAGGGAGAATTGCGA GAPDH GTTGTGGCTCTGACATGCT CCCACGGATGCCCTTTAGT 表 2 膝痹宁的活性成分
Table 2. The active ingredient of Xibining
编号 化合物名称 保留时间/min 测得值m/z 离子模式 分子式 主要离子碎片 1 苹果酸(Malic acid) 0.573 9 133.012 9 [M-H]- C4H6O5 71.007 6, 115.000 0 2 乌头酸(Aconitic acid) 0.582 6 173.008 0 [M-H]- C6H6O6 85.034 4, 129.027 5 3 没食子酸(Gallic acid) 0.687 3 169.013 1 [M-H]- C7H6O5 79.014 3, 107.008 5 4 右旋奎宁酸[D-(-)-quinic acid] 0.712 0 191.055 5 [M-H]- C7H12O 127.044 7, 173.050 9 5 佛手柑内酯(Bergapten) 0.720 2 215.032 1 [M-H]- C12H8O4 118.046 7, 133.050 5 6 尿囊素(Allantoin) 0.736 7 157.035 5 [M-H]- C4H6N4O3 69.935 3, 140.004 9 7 葫芦巴碱(Gynesine) 0.753 3 138.054 8 [M+H]+ C7H7NO2 65.038 9, 78.034 5, 93.057 8 8 焦儿茶酸(Pyrocatechuic acid) 0.761 5 153.018 2 [M-H]- C7H6O4 108.020 6, 153.017 8 9 龙胆二糖(Gentiobiose) 0.770 1 360.148 9 [M+NH4]+ C12H22O11 145.049 0, 163.060 1 10 麦芽三糖(Maltotriose) 0.803 0 527.158 9 [M+Na]+ C18H32O16 185.042 3, 203.051 1, 347.091 1 11 6-果糖-α-葡糖苷(Isomaltulose) 0.855 7 341.109 1 [M-H]- C12H22O11 71.012 2, 89.022 7, 113.023 3 12 烟酸(Niacin) 0.945 2 124.039 4 [M+H]+ C6H5NO2 80.049 7, 124.039 1 13 莽草酸(Shikimic acid) 0.959 2 173.044 6 [M-H]- C7H10O5 67.017 3 14 松三糖(Melezitose) 0.959 2 503.161 9 [M-H]- C18H32O16 59.012 2, 101.022 9 15 蜜二糖(Melibiose) 0.988 2 365.105 0 [M+Na]+ C12H22O11 185.042 7, 203.051 3 16 阿糖尿苷(Aburidine) 1.005 1 243.062 1 [M-H]- C9H12N2O6 110.023 9 17 次黄嘌呤(Hypoxanthine) 1.541 5 137.045 8 [M+H]+ C5H4N4O 56.943 0, 81.070 1, 90.948 0, 118.942 5 18 羟基肉桂酸(Hydroxycinnamic acid) 2.353 2 163.039 0 [M-H]- C9H8O3 119.049 3 19 腺嘌呤(Adenine) 2.609 8 134.046 1 [M-H]- C5H5N5 92.024 2, 107.034 9 20 香豆素(Cumarin) 3.396 8 147.043 9 [M+H]+ C9H6O2 91.054 2, 119.050 9 21 阿魏酸(Ferulic acid) 3.491 1 193.049 8 [M-H]- C10H10O4 134.036 6 22 猪毛菜碱(Salsoline) 3.546 7 194.117 1 [M+H]+ C11H15NO2 70.942 6, 115.054 6, 151.074 4, 177.091 2 23 泛酸(Pantothenic acid) 3.631 1 220.118 0 [M+H]+ C9H17NO5 72.045 1, 98.023 6, 124.075 9 24 山栀苷甲酯(Shanzhiside methylester) 3.843 6 405.140 5 [M-H]- C17H26O11 101.023 0, 155.034 1 25 花椒油素(Xanthoxylin) 3.854 9 197.080 5 [M+H]+ C10H12O4 79.054 4, 90.948 0, 109.065 0, 151.074 1 26 麦芽糖醇(Maltol) 4.006 7 127.039 0 [M+H]+ C6H6O3 53.039 3, 81.034 2 27 羟甲香豆素(Hymecromone) 4.025 1 177.054 4 [M+H]+ C10H8O3 77.039 2, 103.054 4 28 水杨酰胺(Salicylamide) 4.048 5 136.039 1 [M-H]- C7H7NO2 93.033 2, 108.020 5 29 水杨酸(Salicylic acid) 4.058 1 137.023 0 [M-H]- C7H6O3 81.032 9, 109.028 5 30 异甘草素(Isoliquiritigenin) 4.095 9 257.080 5 [M+H]+ C15H12O4 119.049 6 31 刺桐碱(Erysodine) 4.123 1 247.143 6 [M+H]+ C14H18N2O2 60.081 4, 115.054 3, 118.065 3 32 儿茶酸(Catechin) 4.318 4 289.072 0 [M-H]- C15H14O6 125.023 8 33 新乌宁碱(Neoline) 4.333 3 438.285 1 [M+H]+ C24H39NO6 72.081 5, 154.122 0 34 异紫堇定碱(Isocorydine) 4.379 6 342.169 5 [M+H]+ C20H23NO4 58.065 6, 219.078 9 35 高香草酸(4-Hydroxy-3-methoxyphenylacetic) 4.445 1 165.054 6 [M+H-H2O]+ C9H10O4 83.014 9, 100.024 4, 101.025 9 36 水杨醛(Salicylaldehyde) 4.545 8 121.028 2 [M-H]- C7H6O2 61.986 7, 92.025 2 37 丁香醛(Syringaldehyde) 4.843 4 181.049 7 [M-H]- C9H10O4 123.007 5, 166.025 3 38 黑野樱素(Prunin) 4.883 6 433.113 4 [M-H]- C21H22O10 119.049 0, 271.061 0 39 羟基喹啉(4-Hydroxyquinoline) 5.197 8 144.044 1 [M-H]- C9H7NO 99.924 2 40 香草醛(Vanillin) 5.380 9 151.039 0 [M-H]- C8H8O3 136.014 8 41 滨蒿内酯(Scoparone) 5.516 5 207.065 0 [M+H]+ C11H10O4 84.959 8, 151.075 9 42 海帕乌头碱(Hipaaconitine) 6.000 0 616.311 4 [M+H]+ C33H45NO10 105.033 2, 338.171 6 43 邻甲氧基肉桂酸(o-Methoxy cinnamic acid) 6.018 9 161.059 5 [M+H-H2O]+ C10H10O3 79.054 4, 103.054 4, 105.069 9 44 甘草素(Liquiritigenin) 6.169 1 255.066 4 [M-H]- C15H12O4 135.007 9 45 肉桂醛(Cinnamyl aldehyde) 6.270 2 133.064 8 [M+H]+ C9H8O 55.018 6, 89.002 2 46 紫檀素(Angolensin) 6.344 6 271.097 6 [M-H]- C16H16O4 109.028 4, 135.043 5 47 异欧前胡素(Isoimperatorin) 6.715 4 269.082 1 [M-H]- C16H14O4 254.060 1 48 大黄素(Emodin) 6.992 4 269.045 4 [M-H]- C15H10O5 197.060 6, 225.053 5 49 十三烷酸(Tridecanoic acid) 7.973 6 213.185 3 [M-H]- C13H26O2 213.184 0 50 肉豆蔻酸(Myristic acid) 8.521 0 227.201 4 [M-H]- C14H28O2 195.570 9 51 甘草酸(Glycyrrhetic acid) 8.926 6 469.332 1 [M-H]- C30H46O4 391.236 1 52 二十二烷酸(Docosanoic acid) 9.159 6 339.326 5 [M-H]- C22H44O 183.011 2 53 棕榈酸(Palmitic acid) 9.656 4 187.096 7 [M-H]- C9H16O4 97.064 6, 125.095 4 54 十七烷酸(Heptadecanoic acid) 9.867 6 269.248 5 [M-H]- C17H34O2 59.012 6 55 硬脂酸(Stearic acid) 10.536 6 283.263 9 [M-H]- C18H36O2 62.388 1, 78.921 8, 108.558 1, 192.019 4 56 柠檬酸(Citric acid) 11.652 1 191.018 7 [M-H]- C6H8O7 85.027 7, 111.007 3 表 3 膝痹宁对大鼠血清代谢差异物的调节作用
Table 3. Regulation of Xibining on serum metabolic differences in rats
编号 名称 主要二级碎片信息 KOA组/空白组 KOA组/膝痹宁组 Fold Change 校正P值 Fold Change 校正P值 1 左旋肉碱(Levocarnitine) 57.034 1, 121.091 7 0.349 7 0.000 1 0.352 1 0.000 1 2 甘油醛(Glyceraldehyde) 52.332 4, 72.991 4 0.460 0 0.004 2 0.605 5 0.018 0 3 谷氨酸(Glutamic acid) 58.028 2, 74.588 8 0.535 1 0.000 2 0.629 7 0.004 6 4 脯氨酸(Proline) 66.033 3, 71.012 1 0.554 2 0.000 1 0.594 7 0.000 1 5 亮氨酸(Isoleucine) 69.070 5, 90.951 3 0.587 4 0.000 1 0.583 6 0.000 3 6 异亮氨酸(DL-Isoleucine) 55.935 3, 72.937 8 0.590 1 0.000 1 0.561 4 0.000 1 7 鸟嘌呤(Guanine) 56.965 4, 82.065 0 0.609 5 0.000 1 0.675 1 0.003 9 8 天冬酰胺(Asparaginate) 88.027 3, 98.025 1 0.638 5 0.000 2 0.638 5 0.000 1 9 半乳糖(Galactose) 60.026 4, 113.021 7 1.415 5 0.025 6 1.362 6 0.002 7 10 色氨酸(Tryptophan) 59.012 2, 85.027 6 1.486 1 0.023 4 1.704 7 0.000 5 11 甲基烟酰胺(Methylnicotinamide) 56.944 2, 93.069 2 1.710 0 0.013 3 1.772 6 0.001 1 12 缬氨酸(Valine) 53.041 8, 98.623 6 2.044 7 0.000 1 1.675 3 0.005 4 13 硬脂酸(Octadecanoic acid) 78.780 7, 104.813 3 2.118 7 0.041 8 1.929 6 0.001 2 表 4 代谢通路分析
Table 4. Metabolic pathway analysis
代谢通路 P值 FDR Impact 嘌呤代谢(Purine metabolism) 0.000 1 0.000 1 0.012 8 缬氨酸、亮氨酸和异亮氨酸降解(Valine, leucine and isoleucine degradation) 0.000 1 0.000 1 0.000 1 缬氨酸、亮氨酸和异亮氨酸合成(Valine, leucine and isoleucine biosynthesis) 0.000 1 0.000 1 0.000 1 氨酰-tRNA生物合成(Aminoacyl-tRNA biosynthesis) 0.000 1 0.000 1 0.000 1 甘油磷脂代谢(Glycerophospholipid metabolism) 0.000 3 0.000 7 0.000 1 烟酸盐和烟酰胺代谢(Nicotinate and nicotinamide metabolism) 0.013 3 0.021 0 0.138 1 甘油酯代谢(Glycerolipid metabolism) 0.021 0 0.021 0 0.093 5 乙醛酸和二羧酸的代谢(Glyoxylate and dicarboxylate metabolism) 0.021 0 0.021 0 0.079 4 甘氨酸、丝氨酸和苏氨酸代谢(Glycine, serine and threonine metabolism) 0.021 0 0.021 0 0.024 2 磷酸戊糖途径(Pentose phosphate pathway) 0.021 0 0.021 0 0.000 1 -
[1] KATZ JN, ARANT KR, LOESER RF. Diagnosis and treatment of hip and knee osteoarthritis[J]. JAMA, 2021, 325(6): 568. doi: 10.1001/jama.2020.22171 [2] 陈卫衡. 膝骨关节炎中医诊疗指南(2020年版)[J]. 中医正骨, 2020, 32(10): 1-14. doi: 10.3969/j.issn.1001-6015.2020.10.001CHEN WH. Guidelines for TCM diagnosis and treatment of knee osteoarthritis(2020 edition)[J]. J Tradit Chin Orthop Traumatol, 2020, 32(10): 1-14. doi: 10.3969/j.issn.1001-6015.2020.10.001 [3] 王培民. 王培民效方治验: 膝痹宁方[J]. 江苏中医药, 2021, 53(2): 5-6. https://www.cnki.com.cn/Article/CJFDTOTAL-JSZY202102005.htmWANG PM. Xibi ning prescription-A prescription made and proved effective by Wang Peimin[J]. Jiangsu J Tradit Chin Med, 2021, 53(2): 5-6. https://www.cnki.com.cn/Article/CJFDTOTAL-JSZY202102005.htm [4] 王培民, 夏岩, 许建安, 等. 膝宁方治疗膝关节骨性关节炎平地行走疼痛的临床观察[J]. 中国骨伤, 2005, 18(4): 240-241. doi: 10.3969/j.issn.1003-0034.2005.04.021WANG PM, XIA Y, XU JN, et al. Treatment of walking pain caused by osteoarthritis of knee joint with XiNing decoction[J]. China J Orthop Traumatol, 2005, 18(4): 240-241. doi: 10.3969/j.issn.1003-0034.2005.04.021 [5] 王宽. 膝痹宁治疗寒湿痹阻型膝骨关节炎的临床疗效观察[D]. 南京: 南京中医药大学, 2021.WANG K. Clinical study on the treatment of knee osteoarthritis with cold and dampness obstruction[D]. Nanjing: Nanjing University of Chinese Medicine, 2021. [6] 张力, 张立, 邢润麟, 等. 基于缺氧组织中NLRP3炎症小体的活化研究膝痹宁减轻KOA滑膜炎症的效应机制[J]. 南京中医药大学学报, 2020, 36(1): 68-72. http://xb.njucm.edu.cn/article/id/zr20200114ZHANG L, ZHANG L, XING RL, et al. Mitigation effect of xibining on synovitis of knee osteoarthritis based on the activation of NLRP3 inflammasomes in hypoxic tissues[J]. J Nanjing Univ Tradit Chin Med, 2020, 36(1): 68-72. http://xb.njucm.edu.cn/article/id/zr20200114 [7] WOODELL-MAY JE, SOMMERFELD SD. Role of inflammation and the immune system in the progression of osteoarthritis[J]. J Orthop Res, 2020, 38(2): 253-257. doi: 10.1002/jor.24457 [8] ZHENG LL, et al. The role of metabolism in chondrocyte dysfunction and the progression of osteoarthritis[J]. Ageing Res Rev, 2021, 66: 101249. doi: 10.1016/j.arr.2020.101249 [9] MALEMUD CJ. Inhibition of MMPs and adam/adamts[J]. Biochem Pharmacol, 2019, 165: 33-40. doi: 10.1016/j.bcp.2019.02.033 [10] ZHU XB, CHEN F, LU K, et al. PPARγ preservation via promoter demethylation alleviates osteoarthritis in mice[J]. Ann Rheum Dis, 2019, 78(10): 1420-1429. doi: 10.1136/annrheumdis-2018-214940 [11] 张改君, 苗静, 郭丽颖, 等. 多组学联用在中药作用机制研究中的应用[J]. 中草药, 2021, 52(10): 3112-3120. https://www.cnki.com.cn/Article/CJFDTOTAL-ZCYO202110030.htmZHANG GJ, MIAO J, GUO LY, et al. Application of multi-omics combination in mechanism studies of traditional Chinese medicine[J]. Chin Tradit Herb Drugs, 2021, 52(10): 3112-3120. https://www.cnki.com.cn/Article/CJFDTOTAL-ZCYO202110030.htm [12] 杨楠, 刘子修, 茆军, 等. 三色散凝胶贴膏剂的HPLC指纹图谱研究和多元成分含量测定[J]. 南京中医药大学学报, 2021, 37(4): 548-553. doi: 10.14148/j.issn.1672-0482.2021.0548YANG N, LIU ZX, MAO J, et al. HPLC fingerprint analysis and multiple ingredients determination of sanse powder gel paste[J]. J Nanjing Univ Tradit Chin Med, 2021, 37(4): 548-553. doi: 10.14148/j.issn.1672-0482.2021.0548 [13] 任亚硕, 许凤清, 马凤爱. 山茱萸果肉化学成分研究[J]. 广州化工, 2016, 44(8): 76-77. doi: 10.3969/j.issn.1001-9677.2016.08.028REN YS, XU FQ, MA FG. Study on the chemical constituents from the fruit of Cornus officinalis[J]. Guangzhou Chem Ind, 2016, 44(8): 76-77. doi: 10.3969/j.issn.1001-9677.2016.08.028 [14] 冯芮, 郭力, 蒲忠慧, 等. 川芎化学成分及其抑制子宫平滑肌收缩作用[J]. 中成药, 2020, 42(7): 1781-1785. https://www.cnki.com.cn/Article/CJFDTOTAL-ZCYA202007020.htmFENG R, GUO L, PU ZH, et al. Chemical constituents from Ligusticum striatum and their inhibitory effects on uterine smooth muscle contraction[J]. Chin Tradit Pat Med, 2020, 42(7): 1781-1785. https://www.cnki.com.cn/Article/CJFDTOTAL-ZCYA202007020.htm [15] 吴学峰, 周熙, 黄晓兰, 等. 基于UPLC-Q-TOF MS的广佛手指纹图谱建立及炮制前后成分差异分析[J]. 分析测试学报, 2022, 41(3): 299-308. https://www.cnki.com.cn/Article/CJFDTOTAL-TEST202203001.htmWU XF, ZHOU X, HUANG XL, et al. Construction of UPLC-Q-TOF MS fingerprints for fingered citron and its ingredient differences analysis before and after processing[J]. J Instrum Anal, 2022, 41(3): 299-308. https://www.cnki.com.cn/Article/CJFDTOTAL-TEST202203001.htm [16] 何秋梅, 高慧, 白燕远, 等. 越南金花茶化学成分差异及基于表型性状的亲缘关系分析[J]. 中草药, 2022, 53(2): 557-568. https://www.cnki.com.cn/Article/CJFDTOTAL-ZCYO202202025.htmHE QM, GAO H, BAI YY, et al. Analysis of difference in chemical composition and phenotypic characteristicsrelated genetic relationships of Camellia insularis samples[J]. Chin Tradit Herb Drugs, 2022, 53(2): 557-568. https://www.cnki.com.cn/Article/CJFDTOTAL-ZCYO202202025.htm [17] 石琳, 王泽, 崔涛, 等. 肉碱/有机阳离子转运体对元胡止痛方中主要药性成分的转运机制研究[J]. 中草药, 2021, 52(8): 2384-2391. https://www.cnki.com.cn/Article/CJFDTOTAL-ZCYO202108022.htmSHI L, WANG Z, CUI T, et al. Mechanism of transmembrane transport of main medicinal components in Yuanhu Zhitong Prescription by OCTNs[J]. Chin Tradit Herb Drugs, 2021, 52(8): 2384-2391. https://www.cnki.com.cn/Article/CJFDTOTAL-ZCYO202108022.htm [18] 王启隆, 刘超, 权鹏, 等. 炮制对草乌中乌头类生物碱经皮吸收的影响及其贴剂的设计与评价[J]. 中草药, 2021, 52(11): 3201-3208. https://www.cnki.com.cn/Article/CJFDTOTAL-ZCYO202111006.htmWANG QL, LIU C, QUAN P, et al. Effect of processing on transdermal absorption of alkaloids from Aconiti Kusnezoffii Radix and development and evaluation of their patches[J]. Chin Tradit Herb Drugs, 2021, 52(11): 3201-3208. https://www.cnki.com.cn/Article/CJFDTOTAL-ZCYO202111006.htm [19] 李晓强, 谭余庆, 李慧杰, 等. 欧前胡素药理作用及机制研究进展[J]. 中国实验方剂学杂志, 2020, 26(18): 196-201. https://www.cnki.com.cn/Article/CJFDTOTAL-ZSFX202018028.htmLI XQ, TAN YQ, LI HJ, et al. Research progress on pharmacological effect and mechanism of imperatorin[J]. Chin J Exp Tradit Med Formulae, 2020, 26(18): 196-201. https://www.cnki.com.cn/Article/CJFDTOTAL-ZSFX202018028.htm [20] 黎忠大, 刘东, 王晓, 等. 葫芦巴碱调节肾移植术后代谢紊乱的应用前景[J]. 器官移植, 2021, 12(3): 363-368. https://www.cnki.com.cn/Article/CJFDTOTAL-QGYZ202103017.htmLI ZD, LIU D, WANG X, et al. Application prospects of trigonelline in regulating metabolic disorders after renal transplantation[J]. Organ Transplant, 2021, 12(3): 363-368. https://www.cnki.com.cn/Article/CJFDTOTAL-QGYZ202103017.htm [21] LIAN CJ, WANG XD, QIU XJ, et al. Collagen type Ⅱ suppresses articular chondrocyte hypertrophy and osteoarthritis progression by promoting integrin β1-SMAD1 interaction[J]. Bone Res, 2019, 7: 8. [22] STEGEN S, LAPERRE K, EELEN G, et al. HIF-1α metabolically controls collagen synthesis and modification in chondrocytes[J]. Nature, 2019, 565(7740): 511-515. [23] KARSDAL MA, DETLEFSEN S, DANIELS SJ, et al. Is the total amount as important as localization and type of collagen in liver fibrosis attributable to steatohepatitis?[J]. Hepatology, 2020, 71(1): 346-351. [24] ZHANG WD, LIKHODⅡ S, ZHANG YH, et al. Classification of osteoarthritis phenotypes by metabolomics analysis[J]. BMJ Open, 2014, 4(11): e006286. [25] MALEK MAHDAVI A, MAHDAVI R, KOLAHI S. Effects of l-carnitine supplementation on serum inflammatory factors and matrix metalloproteinase enzymes in females with knee osteoarthritis: A randomized, double-blind, placebo-controlled pilot study[J]. J Am Coll Nutr, 2016, 35(7): 597-603. [26] KHODIR SA, AL-GHOLAM MA, SALEM HR. L-Carnitine potentiates the anti-inflammatory and antinociceptive effects of diclofenac sodium in an experimentally-induced knee osteoarthritis rat model[J]. Iran J Basic Med Sci, 2020, 23(8): 1035-1044. [27] HAGHIGHATDOOST F, JABBARI M, HARIRI M. The effect of L-carnitine on inflammatory mediators: A systematic review and meta-analysis of randomized clinical trials[J]. Eur J Clin Pharmacol, 2019, 75(8): 1037-1046.