Cholic acid mitigates osteoarthritis by inhibiting the NF-κB/PERK/ SIRT1 signaling pathway

Introduction:Cholic acid(CA)is a natural steroid useful in treating chronic bronchitis and cholecystitis.On the other hand,its potential impact on osteoarthritis(OA)is unknown.Objective:Using an in vitro and in vivo osteoarthritis model,we sought to assess the chondroprotective properties of CA.Methods:We employed the Cell Counting Kit-8 to measure the impact of CA on chondrocyte activity to assess the toxicity of the cells.Multiple molecular biology experimental techniques were used to investigate potential signaling pathways that CA may use to prevent inflammation and give chondrocytes protection.Furthermore,how CA affects the OA model in Sprague-Dawley(SD)rats was evaluated.Results:CA significantly suppressed the up-regulation of the interleukin-1β(IL-1β),cyclooxygenase-2(COX-2),and matrix metalloproteinase 13(MMP-13)and the downregulation of aggrecan and type II collagen A1(COL2A)in chondrocytes treated tumor necrosis factor-alpha(TNF-α).Differentially expressed genes(DEG)enrichment revealed IL-17,TNF,chemokine,cytokine-cytokine receptor,toll-like receptor,and nucleotide oligomerization domain-like receptor were the primary signaling pathways.The enriched DEGs included CXCL6,CCL20,MMP3,CXCL3,CXCL11,CCL5,CXCL10,MMP9,MMP13,and CXCL2;these DEGs are involved in inflammatory responses and their expression induced by TNF-αwas reversed by CA treatment.CA inhibits p65 nuclear translocation and inhibitory subunit kappa B alpha(IκBα)phosphorylation induced by TNF-α.Furthermore,CA attenuated protein expression of protein kinase RNA-like endoplasmic reticulum kinase(PERK),inositol-requiring transmembrane kinase/endoribonuclease 1α(IRE1α),glucose regulatory protein 78(GRP78),and sirtuin 1(SIRT1),and down-regulation of phosphorylation of AMP-activated protein kinase-α(p-AMPKα)in TNF-α-treated chondrocytes.Conclusions:CA significantly ameliorated cartilage degradation in the OA rat model.CA alleviated the inflammatory response through the nuclear factor kappa B/PERK/SIRT1 axis and ameliorated cartilage degradation.

Effect of Cholic Acid on Fetal Cardiac Myocytes in Intrahepatic Choliestasis of Pregnancy

This study examined the effect of cholic acid （CA） on cultured cardiac myoeytes （CMs） from neonatal rats with an attempt to explore the possible mechanism of sudden fetal death in intra- hepatic cholestasis of pregnancy （ICP）. Inverted microscopy was performed to detect the impact of CA on the beating rates of rat CMs. MTT method was used to study the effect of CA on the viability of CMs. CMs cultured in vitro were incubated with 10 ~maol/L Ca2＋-sensitive fluorescence indicator fluo-3/AM. The fluorescence signals of free calcium induced by CA were measured under a laser scanning confocal microscope. The results showed that CA decreased the beating rates of the CMs in a dose-dependent manner. CA could suppress the activities of CMs in a time- and dose-dependent manner. CA increased the concentration of intracellular free calcium in a dose-dependent manner. Our study suggested that CA could inhibit the activity of CMs by causing calcium overload, thereby leading to the sudden fetal death in ICP.

Study on synthesis and distribution in vivo of 5-Fu-cholic acid conjugate

A series of hepatic targeting drugs, 5-Fu-cholic acid conjugate T1-T5, were synthesized. 5-Fu and cholic acid was selected as starting material, after N-hydroxymethylation, condensation, hydrogenolysis to obtain carboxylated 5-Fu 5a-e. Carboxylated 5-Fu 5a-e were condensated with intermediate 3-hydroxyethyl cholic acid benzyl ester 6 to get 7a-e, 7a-e were deprotected to get T1- T5.

Fine Structure of Hydrogen Bonds in Cholic Acid Revealed by 2DIR Spectroscopy

Based on cryogenic FT-IR spectroscopic studies of hydrogen bonds in cholic acid, two-dimensional FT-IR spectroscopy was applied to enhance our understanding of the hydrogen bonds of cholic acid. Fine spectral structures were revealed by asynchronous 2D FT-IR spectra. The co-relationship among various bands was discussed according to the synchronous 2D FT-IR spectrum.

SWELLING AND WETTABILITY OF LIGHT-CURED METHACRYLATE-BASED DENTAL RESINS PREPARED FROM CHOLIC ACID

2＇-methacryloxy-3α, 7α, I2α- trimethacryloyl cholic acid ethyl ester （CAGE4MA） has been prepared from cholic acid. Photo-polymeric resins were prepared from CAGE4MA. 2,2-bis[4-（2-hydroxy-3-methacrylyloxypropoxy）phenyllpropane （bis-GMA） was used for comparison, triethyleneglycol dimethacrylate （TEGDMA） was used as diluent. The polymerization was initiated by camphoroquinone （CQ）/N, N-dimethylaminoethyl methacrylate （DMAEMA） system. The conversion of CAGE4MA was 39% when the reaction time is 60s, which is lower than bis-GMA and TEGDMA. The swelling value of CAGE4MA resin was 0.41% in distilled water, which is much lower than those of bis-GMA resin （2.04%） and TEGDMA resin （4.77%） under the same conditions. Copolymers from CAGE4MA and TEGDMA have been prepared. With the increase of TEGDMA in mixture, the degree of conversion of CA GE4MA and swelling value increased. The swelling values of photocured resins in 0.1mol/L HCl were also measured.

SYNTHESIS AND THERMAL PROPERTIES OF A NEW CHOLIC ACIDCONTAINING COPOLYMER

A new copolymer was synthesized by free radical polymerization in solution from methyl 3α-methylacryloyl-7α, 12α-dihydroxy-5β-cholan-24-oate (MACAME) and maleic anhydride (MAN). The copolymer was characterized by FT-IR and functional group analysis. The reactivity ratios of the two monomers were estimated [r_1 = 11.6 (MACAME), r_2 = 0.01(MAN)] by conducting a series of copolymerizations with a variety of monomer feed compositions and analyzing thecopolymer composition. Thermogravimetric and differential scanning calorimetric analyses of the samples indicate that thecopolymer possesses good thermal stability. The temperature at which the copolymer samples experienced a 10% weight loss(T_(WL)) is over 287℃, and the T_g ranged from 174 to 185℃ for the copolymers.

The Synthesis and Anion Binding of Novel Cholic Acid-based Molecular Clefts Containing Unsymmetrically Disubstituted Urea Unit

A novel type of chiral molecular clefts consisting of a rigid deoxycholic acid methyl ester backbone and chiral unsymmetrically disubstituted urea side chain have been designed and synthesized. All these new receptors 3a^c and the corresponding key intermediates 1a^c and 2a^c are new compounds, their structures were confirmed by 1HNMR, IR, MS spectra and elemental analysis. These molecular clefts showed binding ability for halide anions.

Isotopic Shifts of the infrared Spectra of Cholic Acid and Sodium Cholate

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Study on the mechanism of cholic acid derivatives in traditional Chinese medicine based on the regulation of gene expression

Objective:To investigate the pharmacological action and mechanism of cholic acid derivatives in traditional Chinese medicine(TCM)based on the regulation of gene expression.Methods:Genome-wide gene expression profiles of Michigan Cancer Foundation-7(MCF-7)cells treated with or without 4 cholic acid derivatives were detected by gene chip technology.Similarities in upregulated and downregulated genes were analyzed using the Connectivity Map(CMap)database.The affinity between cholic acid derivatives and the potential target was confirmed by molecular docking.The cholic acid derivative-regulated pathway enrichment analysis was performed by the STRING database,and the potential pathway was confirmed by in vitro experiments on MD Anderson-Metastatic Breast-231(MDA-MB-231)cells.Results:Compared with the reference genome in the CMap database,the gene expression profiles of cholic acid derivatives were similar to those of antipsychotic,anticancer,anti-inflammatory,and antiinfective drugs.Among them,4 derivatives were associated with antianxiety drugs,and molecular docking results showed that these compounds may act by binding to the ligand-binding site of gammaaminobutyric acid(GABA)receptors.Moreover,the cytoskeletal pathway is one of the pathways enriched in the derivatives.Of them,ursodeoxycholic acid showed significant inhibitory activity on the cytoskeleton formation of MDA-MB-231 cells.Conclusion:The gene expression detection method,combined with CMap and pathway enrichment analysis,could be used to study the mechanism of the active ingredients of TCM.In addition,our research showed that cholic acid derivatives have a potential affinity for membrane receptors,where they can exert anxiolytic activity by modulating opioid receptor,GABA receptor,and dopamine receptor.Moreover,ursodeoxycholic and chenodeoxycholic acid inhibit cytoskeleton formation,probably by acting on membrane proteins to activate the corresponding cytoskeletal pathways.

The Binding of Cholic Acid by Hemicellulose and Pectin of Yard-Long Bean [Vigna sesquipedalis （L.） Fruhw]

The aim of the study was to describe the binding of cholic acid by hemicellulose and pectin of yard-long bean at gastrointestinal system （in vitro）, through variation of acidity （pH 3, 5 and 7） and boiling time （raw/0, 5, 20 and 35 minutes） of yard-long bean. Two-way Anova （α= 5%） and functional groups analysis by infrared spectrophotometer were applied for the binding description. The results of the study showed： （1） The highest binding percentage of cholic acid by dietary fiber of yard-long bean at pH 3-raw treatment （76.603%）; （2） Cholic acid binding via C = O/ester or acid of hemicellulose, C-O-C/cyclic ether of pectin, while C = O/ester or acid of oectin through iron.

HPLC-MS/MS法同时测定清开灵口服液中7种成分的含量

目的 建立高效液相色谱-质谱联用(HPLC-MS/MS)法同时测定清开灵口服液中7种成分含量。方法 采用Waters ACQUITY UPLC BEH C_(18)色谱柱(2.1 mm×10 mm,1.7μm);以含10 mmol·L^(-1)乙酸铵和0.1%甲酸的水溶液(A)及甲醇(B)为流动相,梯度洗脱;电喷雾离子源,正、负离子多反应监测(MRM)模式进行定量分析。结果 腺苷、绿原酸、咖啡酸、栀子苷、黄芩苷、猪去氧胆酸和胆酸质量浓度分别在0.100 4~3.213、0.784 5~8.982、0.998~3.194、0.622 5~19.92、25.05~300.6、2.513~30.15和7.775~93.30μg·m L^(-1)范围内与峰面积呈良好线性关系(r≥0.999 0);平均回收率(n=6)分别为100.9%、98.74%、101.2%、100.2%、100.8%、99.97%和98.94%,RSD分别为1.58%、0.59%、1.78%、1.25%、0.65%、1.69%和1.07%。15批样品中腺苷、绿原酸、咖啡酸、栀子苷、黄芩苷、猪去氧胆酸和胆酸的含量范围分别为0.12~0.18、0.19~0.24、0.06~0.09、0.34~0.37、4.54~4.85、0.49~0.67和1.82~2.19 mg·m L^(-1)。15批样品测定结果较为接近。结论 该方法具有良好的灵敏度、准确性和重复性,可为清开灵口服液的质量控制和后续研究提供参考及数据支持。

胆酸和去氧胆酸抗惊厥作用的血清代谢组学研究

目的:探究胆汁酸单体化合物胆酸(cholic acid,CA)和去氧胆酸(deoxycholic acid,DCA)的抗惊厥作用机制。方法:将3周龄雄性SD大鼠随机分为对照组、模型组、丙戊酸钠组(sodium valproate/valproic acid,VPA,189 mg/kg)、CA组(60 mg/kg)和DCA组(60 mg/kg),每组9只,对照组及模型组为假给药,各给药组于造模前1 h预给药,连续给药16 d。采用(45.0±0.5)℃水浴建立惊厥大鼠模型,每隔1 d水浴1次,共计8次,观察记录模型组及各给药组大鼠惊厥发作时间、惊厥结束时间,对大鼠惊厥发作行为的严重程度进行评分。检测大鼠血清及海马组织中白介素1β(interleukin 1β,IL-1β)、肿瘤坏死因子α(tumor necrosis factor α,TNF-α)和IL-6含量及海马组织中谷氨酸(glutamic acid,Glu)和γ-氨基丁酸(γ-aminobutyric acid,GABA)含量,苏木精-伊红(hematoxylin-eosin,HE)染色观察海马神经元病理损伤。超高效液相色谱仪串联高分辨质谱技术对大鼠血清进行代谢组学分析。结果:与模型组相比,各给药组大鼠惊厥潜伏期均显著延长,惊厥持续时间显著缩短(P均<0.001);VPA组和DCA组惊厥发作等级显著降低(P <0.001,P <0.01),CA组差异无统计学意义。与对照组相比,模型组血清和海马组织中TNF-α,IL-6和IL-1β含量均显著升高(P <0.001),海马组织中Glu和GABA含量均显著升高(P <0.001);与模型组相比,DCA组和VPA组各项生化指标水平均显著降低(P <0.001),而与模型组相比,除血清中IL-1β和海马组织IL-6的水平外,CA组其他各项指标均显著降低(P <0.01)。海马组织HE染色结果显示,与对照组相比,模型组大鼠海马组织中锥体细胞胞体紧缩,体积变小,染色加深,嗜碱性增强,胞质胞核分界不清;与模型组相比,各给药组大鼠海马神经元细胞形态得到明显改善。其中,DCA组大鼠海马神经元细胞形态与VPA组相近。血清代谢组学分析结果经过数据处理、文献及数据库比对,共鉴定出312个差异化合物。通过主成分分析(principal component analysis,PCA)及正交偏最小二乘法判别分析,共筛选出9个差异化合物;代谢通路富集结果显示,CA和DCA的抗惊厥作用主要涉及柠檬酸循环、氨基酸代谢和丁酸代谢通路。结论:CA和DCA对热性惊厥大鼠的行为学和生化指标等均具有一定的改善作用,其作用机制可能与惊厥过程中的能量代谢、氨基酸代谢和丁酸等短链脂肪酸代谢有关。

糖皮质激素联合腺苷蛋氨酸、熊去氧胆酸治疗药物性急性重度胆汁淤积性肝病的临床疗效

目的探讨糖皮质激素联合腺苷蛋氨酸(S-adenosyl-L-methionine,SAMe)、熊去氧胆酸(Ursodeoxycholic acid,UDCA)治疗药物性急性重度胆汁淤积性肝病(Drug-induced cholestatic liver disease,DICLD)的效果。方法选取2018年1月至2022年12月青海省第四人民医院收治的106例急性重度DICLD患者,采用随机数字表法将其分为两组,各53例。对照组给予SAMe、UDCA治疗,观察组给予SAMe、UDCA联合糖皮质激素(甲泼尼龙)治疗。治疗2周后检测并比较两组患者肝功能指标[碱性磷酸酶(Alkaline phosphatase,ALP)、总胆汁酸(Total bile acid,TBA)、γ-谷氨酰转移酶(γ-glutamyltransferase,γ-GT)及血清胆红素(Total bilirubin,TBil)]、生化指标[前白蛋白(Prealbumin,PA)、白蛋白(Albumin,ALB)]及免疫功能指标[可溶性黏附分子1(Soluble vascular cell adhesion molecule-1,sVCAM-1)、干扰素γ(Interferon-γ,IFN-γ)]水平并评估两组患者临床疗效。采用Spearman相关性建议分析临床疗效与生化、免疫功能指标的相关性,并观察两组治疗方案的安全性。结果观察组治疗总有效率为90.57%,高于对照组的73.58%(P<0.05)。治疗后,两组ALP、TBA、γ-GT、TBil、PA、ALB、sVCAM-1、IFN-γ水平均较治疗前降低(P<0.05),且观察组ALP、TBA、γ-GT、TBil、PA、ALB、sVCAM-1、IFN-γ水平均低于对照组(P<0.05)。Spearman相关性分析显示,患者临床疗效与PA、ALB水平呈正相关,与sVCAM-1、IFN-γ水平呈负相关(P均<0.05)。观察组、对照组药物不良反应率分别为18.87%、11.32%,两组比较差异无统计学意义(P>0.05)。结论糖皮质激素联合SAMe、UDCA能够提高急性重度DICLD患者疗效,改善肝功能及炎症因子水平,提高机体免疫功能,且安全性良好。

壳聚糖抑脂作用的研究

目的 : 观察壳聚糖抑制脂肪吸收的效果和机制 ,为发展营养健康的快餐食品提供科学依据。方法 : 1 .体外消化实验 磷钼酸比色法 ;2 .体内实验 将小鼠 2 4 0只随机分为对照(A)、高脂 (B)、壳聚糖 (C)、高脂加壳聚糖 (D) 4组 ,各组饲料脂肪和壳聚糖含量 (% )为 A(1 0 ,0 ) ,B(6 0 ,0 ) ,C(8,2 0 ) ,D(3 4 .3 ,1 4.8) ,饲养 45 d。测定血脂各项指标和粪中脂肪排出量 ,观察体重增长和抗应激能力的变化。结果 : 1 .模拟体外消化 ,壳聚糖与胆汁酸作用 ,在比例为 1∶ 8时 ,壳聚糖可以较好的络合胆汁酸 ,干扰机体对脂肪的吸收 ,使摄入的脂肪排出体外。2 .冰水游泳 ,证明食用适量壳聚糖对体质有增强和改善作用。 3 .与高脂组 (B)相比 ,C和 D的血脂、体重均无显著增加 ,脂肪排出量则显著增加。

脑微血管内皮细胞体外缺血再灌注模型的建立及牛胆酸的保护作用

目的:建立脑微血管内皮细胞体外缺血再灌注模型,并观察了牛胆酸对其影响。方法:体外培养大鼠脑微血管内皮细胞,以氧糖剥夺(Krebs液)再复氧复糖模型模拟缺血再灌注损伤,并用牛胆酸进行了干预,用MTT比色法测定A值代表细胞生存活性。结果:氧糖剥夺4h再复氧复糖12h可造成培养大鼠脑微血管内皮细胞损伤,牛胆酸干预后A值明显高于模型组(P<0.01)。结论:牛胆酸可显著减轻脑微血管内皮细胞体外模拟缺血再灌注细胞损伤。

中药复方“清开灵”注射液中胆酸类物质的液相色谱/质谱/质谱分析

采用液相色谱／质谱／质谱法，通过保留时间、分子量和二级质谱的信息对复方“清开灵”注射液中的胆酸、去氧胆酸和鹅去氧腿酸进行了定性；建立了内标法对复方中相对含量较大的胆酸的定量分析方法；结果表明胆酸在８７５ ｎｇ／Ｌ－１４０μｇ／Ｌ范围内线性良好，线性相关系数Ｒ２＝０．９９９９；ＲＳＤ＝２．４％。该方法样品处理简单，选择性好，灵敏度高。对中药的质量控制具有重要的意义。

清开灵注射液中三种胆酸含量测定方法比较

目的 :建立同时测定清开灵注射液中 3种胆酸 (牛胆酸 ,猪去氧胆酸和去氧胆酸 )含量的 2种高效液相色谱法(HPLC UV和HPLC ELSD) ,并与原有的测定总胆酸含量的紫外分光光度法进行比较。方法 :高效液相色谱法测定 ,采取紫外与蒸发光散射 2种检测方式 ;结果 :同时测定了清开灵注射液中的 3种胆酸物质 ,方法回收率HPLC/ELSD为1 0 0 % ,HPLC/UV为 97% ,均高于原有的紫外方法 (95 %～ 96 % ) ;结论 :HPLC/ELSD测定清开灵中胆酸成分 ,方法简便准确 。

浙贝乙素胆汁酸盐的制备及其镇咳、祛痰和平喘活性的筛选

为了寻找具有优良镇咳、祛痰和平喘活性且毒性较低的药物,将贝母中的主要药效成分之一浙贝乙素和蛇胆中各主要药效成分进行酸碱反应,得到一系列浙贝乙素胆汁酸盐并对所得的盐进行镇咳、祛痰和平喘活性的筛选。活性筛选结果显示,浙贝乙素胆酸盐和浙贝乙素鹅去氧胆酸盐的镇咳、祛痰和平喘活性较强,尤其是二者显示出比磷酸可待因还强的镇咳活性值得关注。借鉴药物化学中的结构拼合思路有望在中药复方的研究开发中开辟出一条新的途径。

清开灵注射液及其有效成分诱导人急性早幼粒白血病细胞凋亡的研究

目的 :研究清开灵注射液及其有效成分治疗急性白血病的作用机理。方法 :采用体外培养技术、MTT法、细胞形态学、DNA凝胶电泳及流式细胞检测技术 ,对清开灵及其有效成分诱导的人急性早幼粒白血病 (HL 60 )细胞凋亡进行分析。结果 :清开灵及其有效成分黄芩甙、猪去氧胆酸对HL 60细胞均有很强的细胞毒作用 ,牛黄胆酸的作用次之 ;前三者在体外作用 6h即可诱导细胞凋亡 ,流式细胞仪检测出现典型凋亡峰 ;牛黄胆酸无诱导HL 60细胞凋亡作用。结论 :清开灵注射液及其有效成分可在体外诱导白血病细胞凋亡 ,可能是其治疗急性白血病的作用机理之一。