基于UPLC-Q-Orbitrap MS/MS技术研究膝痹宁对KOA模型大鼠的软骨保护效应

张力; 刘子修; 廖太阳; 吴鹏; 李晓辰; 梅伟; 茆军; 王培民; 张立

doi:10.14148/j.issn.1672-0482.2023.0032

基于UPLC-Q-Orbitrap MS/MS技术研究膝痹宁对KOA模型大鼠的软骨保护效应

doi: 10.14148/j.issn.1672-0482.2023.0032

南京中医药大学附属医院, 江苏南京 210029

基金项目:

国家自然科学基金青年科学基金项目 81904224

江苏省领军人才培养资助项目 SLJ0207

江苏省研究生培养创新工程资助项目 KYCX21_1675

详细信息

作者简介:
张力, 男, 博士研究生, E-mail: 183718670@qq.com

通讯作者:
张立, 男, 主治中医师, 主要从事中医药防治骨关节病的临床与基础研究, E-mail: fsyy00725@njucm.edu.cn

中图分类号: R285.5
计量
- 文章访问数: 186
- HTML全文浏览量: 23
- PDF下载量: 9
- 被引次数: 0
出版历程
- 收稿日期: 2022-05-10
- 网络出版日期: 2023-01-18
- 发布日期: 2023-01-10

Study on Cartilage Protective Effect of Xibining on KOA Model Rats Based on UPLC-Q-Orbitrap MS/MS Technique

The Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing 210029, China

摘要

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。
- 膝骨关节炎 /
- 软骨保护 /
- 膝痹宁 /
- 代谢组学 /
- 活性成分
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.
- knee osteoarthritis /
- cartilage protection /
- Xibining /
- metabonomics /
- active components

HTML全文

图 1 膝痹宁药液的总离子流图

Figure 1. Total ion current diagram of Xibining

下载: 全尺寸图片幻灯片

图 2 各组大鼠软骨组织代表性HE染色、番红O-固绿染色

Figure 2. The representative HE and Safranin O-Fast Green staining on cartilage tissue of each group

下载: 全尺寸图片幻灯片

图 3 各组大鼠软骨组织MMP3、MMP13 mRNA和蛋白相对表达水平比较

注: A. MMP3、MMP13的相对mRNA表达; B. MMP3、MMP13的代表性蛋白条带; C. MMP3、MMP13的相对蛋白表达; 与空白组比较, ^**P < 0.01;与KOA组比较, ^#P < 0.05, ^##P < 0.01。x±s, n=5。

Figure 3. The relative expression levels of MMP3 and MMP13 in cartilage of rats in each group

下载: 全尺寸图片幻灯片

图 4 各组大鼠软骨组织PPARγ、PGC1α mRNA和蛋白相对表达水平比较

注: A. PPARγ、PGC1α的相对mRNA表达; B. PPARγ、PGC1α的代表性蛋白条带; C. PPARγ、PGC1α的相对蛋白表达; 与空白组比较, ^**P < 0.01;与KOA组比较, ^#P < 0.05, ^##P < 0.01。x±s, n=5。

Figure 4. The relative expression levels of PPARγ and PGC1α in cartilage of rats in each group

下载: 全尺寸图片幻灯片

图 5 代表性大鼠血清色谱图

Figure 5. The representative chromatogram of rat serum

下载: 全尺寸图片幻灯片

图 6 各组大鼠血清的PCA分析

注: K.KOA组; N.空白组; XBN.膝痹宁组; QC.质控样本

Figure 6. PCA analysis of serum in each group

下载: 全尺寸图片幻灯片

图 7 OPLS-DA得分图和Permutation test置换检验

注: K.KOA组; N.空白组

Figure 7. OPLS-DA score map and Permutation test

下载: 全尺寸图片幻灯片

图 8 代谢差异物的富集分析

Figure 8. Enrichment analysis of metabolic difference

下载: 全尺寸图片幻灯片

图 9 代谢通路分析

注: A.烟酸盐和烟酰胺代谢; B.甘油酯代谢; C.乙醛酸和二羧酸的代谢; D.甘氨酸、丝氨酸和苏氨酸代谢、磷酸戊糖途径; E.甘油磷脂代谢; F.氨酰-tRNA生物合成; G.缬氨酸、亮氨酸和异亮氨酸降解、合成; H.嘌呤代谢

Figure 9. Metabolic pathway analysis

下载: 全尺寸图片幻灯片

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

下载: 导出CSV

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

下载: 导出CSV

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
编号	名称	主要二级碎片信息	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

下载: 导出CSV

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

下载: 导出CSV

参考文献(27)

[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.1001-6015.2020.10.001 CHEN 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.htm WANG 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.1003-0034.2005.04.021 WANG 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/zr20200114 ZHANG 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.htm ZHANG 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.1672-0482.2021.0548 YANG 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.1001-9677.2016.08.028 REN 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.htm FENG 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.htm WU 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.htm HE 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.htm SHI 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.htm WANG 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.htm LI 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.htm LI 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.