Polydatin ameliorates hepatic ischemia-reperfusion injury by modulating macrophage polarization

Background:Polydatin,a glucoside of resveratrol,has shown protective effects against various diseases.However,little is known about its effect on hepatic ischemia-reperfusion(I/R)injury.This study aimed to elucidate whether polydatin protects liver against I/R-induced injury and to explore the underlying mechanism.Methods:After gavage feeding polydatin once daily for a week,mice underwent a partial hepatic I/R procedure.Serum alanine aminotransferase(ALT)/aspartate aminotransferase(AST),hematoxylin-eosin(H&E)and TdT-mediated dUTP nick-end labeling(TUNEL)staining were used to evaluate liver injury.The severity related to the inflammatory response and reactive oxygen species(ROS)production was also investigated.Furthermore,immunofluorescence and Western blotting were used to detect macrophage polarization and the NF-κB signaling pathway in macrophages.Results:Compared with the I/R group,polydatin pretreatment significantly attenuated I/R-induced liver damage and apoptosis.The oxidative stress marker(dihydroethidium fluorescence,malondialdehyde,superoxide dismutase and glutathione peroxidase)and I/R related inflammatory cytokines(interleukin1β,interleukin-10 and tumor necrosis factor-α)were significantly suppressed after polydatin treatment.In addition,the result of immunofluorescence indicated that polydatin reduced the polarization of macrophages toward M1 macrophages both in vivo and in vitro.Western blotting showed that polydatin inhibited the pro-inflammatory function of RAW264.7 via down-regulating the NF-κB signaling pathway.Conclusions:Polydatin protects the liver from I/R injury by remodeling macrophage polarization via NFκB signaling.

Protective effects of polydatin against CCl_4-induced injury to primarily cultured rat hepatocytes

AIM To investigate the protective effects of polydatin (PD) against injury to primarily cultured rat hepatocytes induced by CCl 4. METHODS Rat hepatocytes were separated by methods of liver infusion in vivo and cultured medium (7 5×10 5 cells/mL). Two mL or 0 2mL was added into 24 well or 96 well plates respectively. Twenty four hours after cell preculture, PD at concentrations of 10 -7 mol/L-10 -4 mol/L was added into each plate. At the same time injury to hepatocytes was induced by adding 10mmol/L CCl 4. Then, 0 1mL or 1mL culture solution was removed from the 96 well or 24 well plates at 6h , 12h , 24h and 48h after CCl 14 intoxication respectively for the determination of GPT, GSH and MDA. At 48h , the survivability of rat hepatocytes was assayed by the MTT colormetric method. RESULTS After CCl 4 challenge, the release of GPT and the formation of MDA in rat hepatocytes markedly increased and maintained at a high level in 48h , whereas PD with different concentrations could markedly inhibit this elevation with 10 -5 mol/L PD having the strongest effects and inhibiting rate was over 50%. PD could also improve the decreased content of GSH caused by CCl 4 in accordance with the doses used. CCl 4 evidently decreased the hepatocyte survivability from 91 0%±7 9% to 35 4%±3 8%. On the other hand, PD at 10 -7 mol/L-10 -4 mol/L could reverse this change and improve the cell survival rates to 56 1%±5 2%, 65 8%±5 0%, 88 7%±6 8% and 75 2%±7 3%, respectively. CONCLUSION PD at 10 -7 mol/L-10 -4 mol/L could protect primarily cultured rat hepatocytes against CCl 4 induced injury.

Polydatin prevents the induction of secondary brain injury after traumatic brain injury by protecting neuronal mitochondria

Polydatin is thought to protect mitochondria in different cell types in various diseases.Mitochondrial dysfunction is a major contributing factor in secondary brain injury resulting from traumatic brain injury.To investigate the protective effect of polydatin after traumatic brain injury,a rat brain injury model of lateral fluid percussion was established to mimic traumatic brain injury insults.Rat models were intraperitoneally injected with polydatin(30 mg/kg)or the SIRT1 activator SRT1720(20 mg/kg,as a positive control to polydatin).At 6 hours post-traumatic brain injury insults,western blot assay was used to detect the expression of SIRT1,endoplasmic reticulum stress related proteins and p38 phosphorylation in cerebral cortex on the injured side.Flow cytometry was used to analyze neuronal mitochondrial superoxide,mitochondrial membrane potential and mitochondrial permeability transition pore opened.Ultrastructural damage in neuronal mitochondria was measured by transmission electron microscopy.Our results showed that after treatment with polydatin,release of reactive oxygen species in neuronal mitochondria was markedly reduced;swelling of mitochondria was alleviated;mitochondrial membrane potential was maintained;mitochondrial permeability transition pore opened.Also endoplasmic reticulum stress related proteins were inhibited,including the activation of p-PERK,spliced XBP-1 and cleaved ATF6.SIRT1 expression and activity were increased;p38 phosphorylation and cleaved caspase-9/3 activation were inhibited.Neurological scores of treated rats were increased and the mortality was reduced compared with the rats only subjected to traumatic brain injury.These results indicated that polydatin protectrd rats from the consequences of traumatic brain injury and exerted a protective effect on neuronal mitochondria.The mechanisms may be linked to increased SIRT1 expression and activity,which inhibits the p38 phosphorylation-mediated mitochondrial apoptotic pathway.This study was approved by the Animal Care and Use Committee of the Southern Medical University,China(approval number:L2016113)on January 1,2016.

Polydatin attenuated food allergy via store-operated calcium channels in mast cell

AIM: To investigate the effect of polydatin (PD), a resveratrol glucoside, on mast cell degranulation and antiallergic activity. METHODS: After the rats were orally sensitized with ovalbumin (OVA) for 48 d and underwent PD treatment for 4 d, all the rats were stimulated by 100 mg/mL OVA for24 h and then sacrificed for the following experiments. The small intestines from all the groups were prepared for morphology examination by hematoxylin and eosin staining. We also used a smooth muscle organ bath to evaluate the motility of the small intestines. The OVA-specific immunoglobulin E (IgE) production and interleu-kin-4 (IL-4) levels in serum or supernatant of intestinal mucosa homogenates were analyzed by enzyme-linked immunosorbent assay (ELISA). Using toluidine blue stain, the activation and degranulation of isolated rat peritoneal mast cells (RPMCs) were analyzed. Release of histamine from RPMCs was measured by ELISA, and regulation of PD on intracellular Ca 2+ mobilization was investigated by probing intracellular Ca 2+ with fluo-4 fluo-rescent dye, with the signal recorded and analyzed. RESULTS: We found that intragastric treatment with PD significantly reduced loss of mucosal barrier integrity in the small intestine. However, OVA-sensitization caused significant hyperactivity in the small intestine of allergic rats, which was attenuated by PD administration by 42% (1.26 ± 0.13 g vs OVA 2.18 ± 0.21 g, P < 0.01). PD therapy also inhibited IgE production (3.95 ± 0.53 ng/mL vs OVA 4.53 ± 0.52 ng/mL, P < 0.05) by suppressing the secretion of Th2-type cytokine, IL-4, by 34% (38.58 ± 4.41 pg/mLvs OVA 58.15 ± 6.24 pg/mL, P < 0.01). The ratio of degranulated mast cells, as indicated by vehicles (at least five) around the cells, dramatically increased in the OVA group by 5.5 fold (63.50% ± 15.51% vs phosphate-buffered saline 11.15% ± 8.26%, P < 0.001) and fell by 65% after PD treatment (21.95% ± 4.37% vs OVA 63.50% ± 15.51%, P < 0.001). PD mediated attenuation of mast cell degranulation was further confirmed by decreased histamine levels in both serum (5.98 ± 0.17 vs OVA 6.67 ± 0.12, P < 0.05) and intestinal mucosa homogenates (5.83 ± 0.91 vs OVA 7.35 ± 0.97, P < 0.05). Furthermore, we demonstrated that administration with PD significantly decreased mast cell degranulation due to reduced Ca 2+ influx through store-operated calcium channels (SOCs) (2.35 ± 0.39vs OVA 3.51 ± 0.38,P < 0.01).CONCLUSION: Taken together, our data indicate that PD stabilizes mast cells by suppressing intracellular Ca 2+ mobilization, mainly through inhibiting Ca 2+ entry via SOCs, thus exerting a protective role against OVA-sensitized food allergy.

Effects of polydatin on the proliferation,migration,and invasion of ovarian cancer

To investigate the effects of polydatin on the proliferation,migration,and invasion of ovarian cancer,the change of proliferative ability,migration ability,and invasive ability of human ovarian cancer cell OVCAR-3,A2780,and HO-8910 was detected by using polydatin and up-regulating PI3K.The anticancer activity and mechanism of polydatin in ovarian cancer were analyzed.Polydatin could effectively inhibit the proliferation,migration,and invasion of OVCAR-3,A2780,and HO-8910,and inhibit the expression of PI3K protein.After the expression level of PI3K protein was up-regulated,the inhibitory effect of polydatin on the proliferative ability,migration ability,and invasive ability of OVCAR-3,A2780,and HO-8910 significantly decreased,suggesting that PI3K was the target of polydatin.Therefore,we concluded that polydatin could inhibit the proliferation,migration,and invasion of ovarian cancer cells by inhibiting the expression of PI3K protein,which provides an experimental basis for polydatin in the treatment of ovarian cancer.

DETERMINATION OF POLYDATIN IN POLYGONUM CUSPIDTUM SIEB. ET ZUCC. BY TLC-FLUORESCENCE SPECTROPHOTOMETRY

Objective A method of TLC-fluorescence spectrophotometry was established to assay the content of polydatin in polygonum cuspidatum sieb. et zucc. Methods: Polydatin was extracted by methanol and separated with chloroform-acetone-formic acid-water (4∶4∶0.5∶0.2) by thin layer chromatography. The excitation wavelength and emission wavelength were 284 nm and 384 nm, respectively. Results The linear regression equation of the calibration graph was y=7.02179x+4.5143, a linear regression correlative coefficient r=0.9936. Conclusion This method was proved simple, stable and sensitive. It can be used in quality control of herbs.

Determination of Polydatin in Rat Serum and Its Changes by UPLC Method

[Objectives]To establish a UPLC-UV method for the determination of polydatin in the serum of Wistar rats.[Methods]Acquity UPLC BEH shield RP18 column(1.7μm,2.1 mm×100 mm,Waters Corporation,USA)was used as the analytical column,acetonitrile-water(55∶45)was used as the mobile phase,the flow rate was 0.5 mL/min,and the column temperature was 30℃and the detection wavelength was 306 nm.[Results]The linear range of the established serum sample analysis method was 1.0-20.0μg/mL,and the correlation coefficient was r=0.9994;the intraday and interday RSD of Wistar rat serum was less than 3.0%,and the accuracy was higher than 90%.[Conclusions]This method is sensitive,accurate,and rapid.It is suitable for monitoring the concentration of polydatin in serum after intragastric administration,and can also be used for pharmacokinetics and bioavailability studies.

虎杖苷通过抑制TGF-β/Smad信号通路改善动脉粥样硬化大鼠动脉粥样硬化斑块和内皮炎症反应

目的:探讨虎杖苷通过抑制转化生长因子β(TGF-β)/Smad信号通路对动脉粥样硬化(AS)大鼠AS斑块和内皮炎症反应的改善作用。方法:将60只大鼠随机分成空白对照组、模型对照组、虎杖苷低、中、高剂量组和阳性对照组,每组10只。正常对照组大鼠用普通饲料喂养,其余各组大鼠采用高脂饲料喂养与维生素D3腹腔注射联合的方法制备AS大鼠模型。虎杖苷低、中、高剂量组大鼠每天灌胃虎杖苷40、80、160 mg/kg,阳性对照组大鼠每天灌胃辛伐他汀5 mg/kg,模型对照组和空白对照组大鼠每天灌胃等量生理盐水。给药结束后,油红O染色观察大鼠主动脉AS斑块形成情况;全自动生化分析仪检测大鼠三酰甘油(TG)、总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)水平;酶联免疫吸附法检测大鼠血清中肿瘤坏死因子(TNF)-α、IL-6、IL-8、C反应蛋白(CRP)、单核细胞趋化蛋白1(MCP-1)、同型半胱氨酸(Hcy)、细胞间黏附分子1(ICAM-1)、内皮素-1(ET-1)含量;蛋白免疫印迹法检测大鼠TGF-β1、Smad2/3、p-Smad2/3蛋白表达水平。结果:与空白对照组相比,模型对照组大鼠主动脉内壁出现大量AS斑块,血清中TG、TC、LDL-C水平及TNF-α、IL-6、IL-8、CRP、MCP-1、Hcy、ICAM、ET-1含量明显升高,HDL-C水平明显降低,主动脉组织中TGF-β1蛋白表达、p-Smad2/3/Smad2/3显著升高(P<0.05)。虎杖苷干预后AS大鼠主动脉内壁斑块面积明显减小,血清中TG、TC、LDL-C水平及TNF-α、IL-6、IL-8、CRP、MCP-1、Hcy、ICAM、ET-1含量显著降低,HDL-C水平显著升高,主动脉组织中TGF-β1蛋白表达、p-Smad2/3/Smad2/3明显减小(P<0.05),以上指标变化均呈剂量依赖性(P<0.05)。阳性对照组TC、LDL-C、HDL-C水平与虎杖苷高剂量组无明显差异(P>0.05),其余指标均比虎杖苷高剂量组更低(P<0.05)。结论:虎杖苷通过调节血脂、降低细胞炎症因子的表达,从而减少AS斑块的形成,改善AS大鼠体内的炎症反应,其机制与抑制TGF-β/Smad信号通路激活有关。

虎杖苷对AML细胞增殖、迁移、侵袭及肿瘤生长的影响

目的探讨虎杖苷(PD)对急性髓系白血病(AML)细胞增殖、迁移、侵袭及肿瘤生长的影响。方法将人AML细胞KG-1分为正常组,低、中、高浓度PD组(10、30、60μmol/L的PD),SQ22536组[环磷酸腺苷(cAMP)抑制剂,100μmol/L],高浓度PD和SQ22536联用组(60μmol/L的PD+100μmol/L的SQ22536),考察PD对细胞活力、凋亡率、侵袭和迁移能力、cAMP水平和上皮-间充质转化(EMT)相关蛋白、蛋白激酶A(PKA)蛋白表达的影响。以BALB/c裸鼠为对象,通过皮下接种KG-1细胞悬液建立AML裸鼠移植瘤模型,并分为对照组、PD组、SQ22536组、PD+SQ22536组(每组6只),考察PD对其瘤体体积及质量的影响。结果与正常组细胞或对照组裸鼠比较,PD各浓度组的细胞活力、迁移细胞数、侵袭细胞数和波形蛋白、锌指转录因子Snail蛋白的相对表达量,以及PD组裸鼠的瘤体体积及质量均显著降低,而细胞凋亡率、cAMP水平、PKA和上皮钙黏素的相对表达量均显著升高,且呈浓度依赖性(P<0.05);SQ22536对细胞和裸鼠的作用效果与PD相反,并可显著逆转PD的抗肿瘤活性(P<0.05)。结论PD可能通过激活cAMP/PKA信号通路来抑制AML细胞的增殖、迁移、侵袭,诱导细胞凋亡,抑制EMT进程及肿瘤生长,从而发挥抗AML作用。

虎杖苷通过Hippo/YAP通路影响甲状腺癌8505C细胞的恶性生物学行为和顺铂敏感性

目的:探究虎杖苷通过Hippo/Yes相关蛋白(YAP)通路对人甲状腺癌8505C细胞的恶性生物学行为和顺铂(DDP)敏感性的影响。方法:体外培养8505C细胞,构建其DDP耐药细胞8505C/DDP,用CCK-8法检测0、25、50、75、100 nmol/L虎杖苷处理8505C和8505C/DDP细胞的增殖能力,以筛选虎杖苷的最佳作用浓度。将8505C细胞分为对照组、虎杖苷组、空载组、虎杖苷+YAP1过表达组;将8505C/DDP细胞分为对照组、DDP组、DDP+虎杖苷组、DDP+空载组、DDP+虎杖苷+YAP1过表达组。WB法检测各组8505C细胞中Hippo/YAP通路[YAP1、转录辅激活因子(TAZ)]和EMT(E-cadherin、N-cadherin)相关蛋白,8505C/DDP细胞中YAP1、TAZ、耐药相关蛋白[P-糖蛋白(P-gp)、多药耐药相关蛋白1(MRP1)]、凋亡相关蛋白(C-caspase-3、BAX、Bcl-2)的表达。Transwell小室和细胞划痕实验分别检测各组8505C、8505C/DDP细胞的侵袭、迁移能力。结果:虎杖苷可显著抑制8505C细胞的增殖活性(P<0.05)明显抑制8505C细胞中YAP1、TAZ蛋白、N-cadherin的表达(均P<0.05),提升E-caderin蛋白的表达(P<0.05),显著抑制8505C细胞的迁移和侵袭能力(均P<0.05),而8505C/DDP细胞对低浓度的虎杖苷具有耐药性(P<0.05);过表达YAP1则可逆转虎杖苷对8505C细胞的影响。50 nmol/L虎杖苷明显抑制DDP处理的8505C/DDP细胞中YAP1、TAZ、P-gp、MRP1、Bcl-2的蛋白的表达(均P<0.05),提升cleaved caspase-3、BAX蛋白的表达(均P<0.05)并诱导其细胞凋亡(P<0.05),过表达YAP1则可逆转虎杖苷对8505C/DDP细胞的影响。结论:虎杖苷抑制Hippo/YAP信号通路,从而抑制8505C细胞的恶性生物学行为和增强其对的DDP敏感性。

虎杖苷对间质性肺病模型小鼠免疫炎性代谢性损伤的影响

目的 探讨虎杖苷(Polydatin, PD)对博来霉素(Bleomycin, BLM)诱导肺间质病变小鼠的干预作用及机制。方法 采用SPF级雄性C57/BL6小鼠,体质量(20±2)g,经BLM气管内滴注诱导间质性肺病模型,将造模成功的30只小鼠按随机数表法分为模型组6只、阳性药组6只、PD高、中、低剂量组各6只,另选6只未造模小鼠作为正常对照组。正常对照组和模型组予蒸馏水10 ml/(kg·d),阳性药组给予泼尼松7.5 mg/(kg·d),PD高、中、低剂量组分别给予PD 200 mg/(kg·d)、100 mg/(kg·d)、50 mg/(kg·d)剂量灌胃给药,其中PD高剂量组2次/d灌胃给药。干预4周后处死小鼠,采集肺组织标本。以HE染色观察肺组织病理改变;RT-PCR技术检测肺组织AMPKα1、PPARγ、PGC-1α mRNA转录水平;Western Blot技术检测肺组织AMPKα1、pAMPKα1、PPARγ、PGC-1α、HMGB1、TGF-β1、NF-κB p65蛋白表达水平。结果 模型组小鼠较正常对照组有明显病理改变(P<0.01),主要表现在肺组织炎症细胞浸润、间质纤维化;阳性药组肺组织炎症病变较模型组有改善(P<0.05);PD各治疗组小鼠出现肺组织炎症细胞浸润、间质纤维化较模型组有改善(P<0.05)。RT-PCR结果显示模型组小鼠肺组织中AMPKα1、PPARγ、PGC-1α mRNA转录水平较正常对照组有下降趋势,阳性药及PD各治疗组小鼠肺组织中AMPKα1、PPARγ、PGC-1α mRNA转录水平较模型组均有升高趋势;阳性药组及PD各组间变化无明显差异。Western Blot结果显示,与正常组比较,模型组小鼠肺组织中HMGB1、NF-κB p65蛋白表达水平明显升高(P<0.01),AMPKα1、pAMPKα1、PPARγ、PGC-1α蛋白表达水平明显降低(P<0.05);与模型组比较,阳性药组及PD高剂量组AMPKα1蛋白表达水平明显升高(P<0.05),PD高、低剂量组pAMPKα1蛋白表达水平明显升高(P<0.05),PD中剂量组PPARγ蛋白表达水平明显升高(P<0.05);阳性药组HMGB1、PD低剂量组NF-κB p65蛋白表达水平明显下降(P<0.05),其余各治疗组HMGB1、TGF-β1、NF-κB p65较模型组均有下降趋势;阳性药组及PD各组间变化无明显差异。结论 PD能够改善CTD-ILD模型小鼠肺组织炎症病理损伤,改善肺间质纤维化病理改变,抑制免疫炎性损伤,其作用机制可能是通过激活AMPKα1/PPARγ/PGC-1α信号通路,抑制下游免疫炎性因子HMGB1、NF-κB的高表达,改善肺组织免疫炎性损伤;抑制纤维化细胞因子TGF-β1的释放,改善肺组织免疫炎性损伤及间质纤维化。

虎杖苷通过AMPK/Nrf2途径调控糖尿病大鼠伤口愈合和血管生成的机制研究

目的:基于AMPK/Nrf2途径探讨虎杖苷(PD)对糖尿病(DM)大鼠伤口愈合和血管生成的作用机制。方法:建立DM大鼠模型,将45只DM大鼠随机分为DM组、DM+PD组和DM+PD+ML385组(n=15),另取15只健康大鼠作为对照组,建立伤口愈合受损的模型,DM+PD组大鼠予PD灌胃(20mg/kg),DM+PD+ML385组大鼠在PD的基础上腹腔注射ML385抑制AMPK/Nrf2通路(30mg/kg)。比较各组大鼠伤口愈合情况,通过免疫组化染色检测分析微血管密度,Westernblot及RT-qPCR分析转化生长因子β1(TGF-β1)、血管内皮生长因子(VEGF)以及AMPK/Nrf2通路转录和翻译水平。结果:四组小鼠的上述指标比较差异显著(P<0.05)。DM组的伤口愈合百分比、生成血管数目、TGF-β1和VEGF蛋白水平、AMPK和Nrf2 mRNA和蛋白表达水平显著低于对照组(P<0.05)。DM+PD组的伤口愈合百分比、生成血管数目、TGF-β1和VEGF蛋白水平、AMPK和Nrf2mRNA和蛋白表达水平显著高于DM组(P<0.05)。DM+PD+ML385组的伤口愈合百分比、生成血管数目、TGF-β1和VEGF蛋白水平、AMPK和Nrf2 mRNA和蛋白表达水平显著低于DM+PD组而显著高于DM组(P<0.05)。结论:PD可能通过调控AMPK/Nrf2通路诱导血管生成,进而促进DM模型大鼠的伤口愈合。

虎杖苷调节Akt/MDM2/p53信号通路对胆囊癌细胞增殖、迁移和细胞周期的影响

目的探讨虎杖苷(PD)调节蛋白激酶B/原癌基因MDM2/抑癌基因p53信号通路对胆囊癌细胞增殖、迁移和细胞周期的影响。方法以人胆囊癌细胞株(GBC-SD)为研究对象,体外培养人胆囊癌细胞株(GBC-SD),使用浓度为10~160 mmol/L的虎杖苷处理细胞24、48、72 h,采用CCK-8法检测细胞的增殖能力,确定最佳实验浓度。将GBC-SD细胞分为对照组(Control组)、虎杖苷低、中、高浓度组(PD-L组、PD-M组、PD-H组)、虎杖苷+Akt激活剂组(PD+SC79组),Transwell小室法评价细胞的迁移能力,Hoechst染色观察细胞的凋亡,流式细胞术检测细胞周期与细胞凋亡,Western blot检测Akt、MDM2、p53磷酸化水平,建立荷瘤小鼠模型评价虎杖苷对胆囊癌肿瘤生长的影响。结果浓度为10~160 mmol/L的虎杖苷处理细胞24 h,可显著抑制GBC-SD细胞的增殖活性,选择10、20、40 mmol/L的虎杖苷进行后续实验;与Control组比较,PD-L组、PD-M组、PD-H组GBC-SD细胞的迁移数、细胞凋亡率、G2/M期细胞比例及S期细胞比例、P-Akt、P-MDM2蛋白表达显著降低,G0/G1期细胞比例、P-p53蛋白表达显著升高,且呈浓度依赖性(P<0.05);与PD-H组比较,PD+SC79组GBC-SD细胞的迁移数、细胞凋亡率、G2/M期细胞比例及S期细胞比例、P-Akt、P-MDM2蛋白表达显著升高,G0/G1期细胞比例、P-p53蛋白表达显著降低(P<0.05);虎杖苷干预治疗后,小鼠移植瘤的生长速度显著降低(P<0.05)。结论虎杖苷可以通过调节Akt/MDM2/p53信号通路使细胞周期阻滞,抑制胆囊癌细胞增殖、迁移。

虎杖苷调节YAP1/TAZ信号通路对缺氧性肺动脉高压新生大鼠肺动脉平滑肌细胞增殖与凋亡的影响

目的 探究虎杖苷(PD)对缺氧性肺动脉高压(HPH)新生大鼠肺动脉平滑肌细胞(PASMCs)增殖与凋亡的影响及其作用机制。方法 将新生大鼠随机分为6组:对照组、模型组、PD低剂量组、PD中剂量组、PD高剂量组、PD高剂量+Hippo通路抑制剂(PD高剂量+XMU-MP-1)组,每组10只。缺氧2周后,检测各组大鼠右心室收缩压(RVSP)和右心室肥厚指数(RVHI),苏木精-伊红(HE)染色观察肺组织病理学变化,并计算肺动脉管壁厚度占总厚度的百分比(WT%)和管壁面积占总面积的百分比(WA%)。分离各组新生大鼠PASMCs,分为NC组、低氧(Hypoxia)组、PD低剂量组、PD中剂量组、PD高剂量组、PD高剂量+XMU-MP-1组。CCK-8和EdU检测细胞增殖,流式细胞术检测细胞凋亡,免疫印迹(Western blot)检测肺组织和PASMCs中Yes相关蛋白1(YAP1)、PDZ结合域的转录共刺激因子信号通路(TAZ)、哺乳动物不育系20样激酶1(MST1)、B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关蛋白(Bax)蛋白表达。结果 与对照组相比,模型组大鼠肺动脉管壁明显增厚,管腔变窄,RVSP、RVHI、WT%、WA%、YAP1、MST1、TAZ蛋白表达显著升高(均P<0.05);与模型组相比,PD低、中、高剂量组大鼠肺动脉增厚减少,管腔变大,RVSP、RVHI、WT%、WA%、YAP1、MST1、TAZ蛋白表达显著降低,且PD各组间差异存在剂量依赖性(均P<0.05);XMU-MP-1可逆转这一结果。与NC组相比,Hypoxia组细胞A_(450nm)值、EdU阳性率、YAP1、MST1、TAZ、Bcl-2蛋白表达显著升高,凋亡率、Bax蛋白表达显著降低(均P<0.05);与Hypoxia组相比,PD低、中、高剂量组细胞A_(450nm)值、EdU阳性率、YAP1、MST1、TAZ、Bcl-2蛋白表达显著降低,凋亡率、Bax表达显著升高,且PD各组间有剂量依赖性(均P<0.05);XMU-MP-1可逆转这一结果。结论 PD可能通过抑制YAP1/TAZ信号通路抑制HPH新生大鼠PASMCs增殖,促进凋亡。

虎杖苷调节Sirt1-FoxO1信号通路对高糖诱导的心肌细胞焦亡的影响

目的 基于沉默信息调节因子1(silence information regulator1,Sirt1)-叉头状转录因子O1(forkhead transcription factor O1,FoxO1)信号通路研究虎杖苷(polydatin,PD)抑制高糖诱导的心肌细胞焦亡的作用机制。方法 以心肌细胞H9c2为研究对象,使用不同浓度PD处理筛选PD浓度。将细胞分为模型组、PD低、中、高剂量组(PD-L、M、H,20、40、80μmol/L)、空白对照(control)组、Sirt1抑制剂[PD-H+sirtinol (10μmol/L)]组。细胞计数试剂盒(cell counting Kit-8,CCK8)法检测细胞活性;酶联免疫吸附试验(enzyme-linked immunosorbent assay,Elisa)法检测细胞内乳酸脱氢酶(lactate dehydrogenase,LDH)释放量、白细胞介素1β(Interleukin-1β,IL-1β)水平;二氯荧光素双醋酸盐(dichlorodihydrofluorescein diacetate,DCFH-DA)荧光探针检测细胞活性氧(reactive oxygen species,ROS)水平;流式细胞术检测细胞焦亡情况;实时荧光定量PCR检测细胞中Nod样受体蛋白3(Nod-like receptor protein 3,NLRP3) mRNA、消皮素D(gasdermin D,GSDMD) mRNA表达水平;Western blot检测蛋白表达。结果 与control组比较,模型组细胞活性下降(P<0.01),经过PD不同浓度处理后,细胞活性逐渐上升(P<0.01);与control组比较,模型组LDH释放量、IL-1β水平、ROS水平、细胞焦亡率、NLRP3 mRNA、GSDMD mRNA表达、焦亡蛋白NLRP3、裂解的半胱氨酸天冬氨酸酶(cleaved-caspase-1,c-caspase-1)、GSDMD、GSDMDN表达均上升,Sirt1-Foxo1信号通路蛋白表达下降(P<0.01);与模型组比较,PD各组及PD-H+sirtinol组LDH释放量、IL-1β水平、ROS水平、细胞焦亡率、NLRP3 mRNA、GSDMD mRNA表达、焦亡蛋白NLRP3、c-caspase-1、GSDMD、GSDMD-N表达均下降,Sirt1-FoxO1信号通路蛋白表达上升(P<0.05)。sirtinol可以逆转PD对于高糖诱导的心肌细胞焦亡的保护作用。结论 PD可以通过上调Sirt1-FoxO1信号通路蛋白表达,改善细胞炎症损伤,抑制高糖诱导的心肌细胞焦亡。

基于Nrf2/HO-1通路探究虎杖苷对妊娠期糖尿病大鼠的治疗效果及作用机制

[目的]探究虎杖苷(PD)对妊娠期糖尿病(GDM)大鼠的治疗效果及作用机制。[方法]高脂高糖喂养雌性SD大鼠8周,雌雄同笼制备孕鼠84只,随机分为7组(12只/组):空白组、模型组、PD低、中、高剂量组(30、75、150 mg/kg的PD)、阳性对照组(200 mg/kg盐酸二甲双胍)、抑制剂组[150 mg/kg的PD+30 mg/kg的核转录因子E2相关因子2(Nrf2)/血红素加氧酶-1(HO-1)通路抑制剂ML385],除空白组外,其余各组在妊娠5 d后,腹腔注射链脲佐菌素(35 mg/kg)复制GDM大鼠模型。测量各组孕鼠体质量及空腹血糖(FBG);酶联免疫吸附(ELISA)法测定大鼠空腹胰岛素(FINS)、白细胞介素(IL)-6、IL-1β、肿瘤坏死因子(TNF)-α水平,计算胰岛素抵抗指数(HOMA-IR)、胰岛β细胞功能指数(HOMA-β)、胰岛素敏感指数(ISI);全自动生化分析仪分析大鼠血清中血脂水平;苏木精-伊红(HE)染色观察大鼠胰腺组织损伤;蛋白免疫印迹(Western Blot)法检测各组大鼠胰腺组织中Nrf2/HO-1信号通路蛋白表达。[结果]与空白组比较,模型组大鼠体质量、FBG、FINS、HOMA-IR、IL-6、IL-1β、TNF-α水平、总胆固醇(TC)、三酰甘油(TG)、低密度脂蛋白胆固醇(LDL-C)、游离脂肪酸(FFA)含量升高,ISI、HOMA-β、高密度脂蛋白胆固醇(HDL-C)含量、胰岛数量、Nrf2、HO-1蛋白表达水平降低(P<0.05);与模型组比较,PD低、中、高剂量组大鼠体质量、FBG、FINS、HOMA-IR、IL-6、IL-1β、TNF-α水平、TC、TG、LDL-C、FFA含量降低,ISI、HOMA-β、HDL-C含量、胰岛数量、Nrf2、HO-1蛋白表达水平升高(P<0.05);与阳性对照组比较,PD高剂量组大鼠上述指标差异均无统计学意义(P>0.05),提示与阳性对照药物疗效相当;Nrf2/HO-1通路抑制剂ML385可以逆转高剂量PD对GDM大鼠的保护作用(P<0.05)。[结论]PD通过降低血糖、血脂和炎症反应来改善GDM,其作用机制可能与激活Nrf2/HO-1信号通路有关。

虎杖苷对β淀粉样蛋白诱导体外皮质神经元线粒体氧化应激和功能障碍的保护作用

目的探讨虎杖苷对β淀粉样蛋白25~35(Aβ_(25~35))诱导的原代培养大鼠皮质神经元损伤的保护作用及可能的作用机制。方法原代培养SD大鼠皮质神经元,分为对照组、虎杖苷组、Aβ_(25~35)组和联合组(Aβ_(25~35)+虎杖苷)。采用四甲基偶氮唑盐检测皮质神经元活力,2,7-二氯荧光素二乙酸酯探针或红色线粒体超氧化物荧光探针染色检测细胞内和线粒体活性氧水平,线粒体膜通透性转换孔(MPTP)试剂盒检测MPTP开放程度,蛋白免疫印记法检测细胞色素C及线粒体转录因子A(TFAM)表达。另外,测定细胞内电子传递链复合物(包括复合物Ⅰ、Ⅱ、Ⅲ、Ⅳ)活性和三磷酸腺苷(ATP)水平。采用高效液相色谱法测定线粒体中8-羟基脱氧鸟苷(8-OHdG)。结果与对照组比较,Aβ_(25~35)组皮质神经元细胞活力、线粒体荧光强度、线粒体呼吸链酶活性(复合物Ⅰ、Ⅱ、Ⅲ、Ⅳ)、细胞内ATP、线粒体内TFAM表达明显降低,差异有统计学意义(P<0.05,P<0.01);与Aβ_(25~35)组比较,虎杖苷组线粒体荧光强度、线粒体呼吸链酶活性(复合物Ⅰ、Ⅱ、Ⅲ、Ⅳ)、细胞内ATP、线粒体内TFAM表达明显增高,皮质神经元暴露3、6、12、24h细胞内和线粒体内活性氧明显降低(P<0.05,P<0.01),联合组细胞色素C细胞质/线粒体比值、线粒体内8-OHdG水平明显低于Aβ_(25~35)组[3.02±0.28 vs 5.73±0.45,P<0.05;(8.07±1.45)×10^(6)dG vs(16.07±2.29)×10^(6)dG,P<0.05]。结论虎杖苷可有效地保护皮质神经元免受Aβ_(25~35)诱导的损伤,至少部分作用是通过抑制线粒体氧化应激和改善线粒体功能实现。

光甘草定-白藜芦醇-虎杖苷组合物的协同美白抗氧抗炎活性

围绕体外美白相关功效,对10种美白成分进行了功效对比,并从中优选出强功效成分进行组合筛选,发现了由虎杖苷、白藜芦醇、光甘草定构成的组合物具备较强美白效果,即使在较低浓度0.0001μmol/L,该组合物可显著抑制细胞内黑色素生成(抑制率19.47%)及酪氨酸酶活性(抑制率23.7%),同时表现出较强自由基清除能力,DPPH的IC50为0.3445 mmol/L,羟自由基的IC50为19.82 mmol/L。另外,该组合物具备一定抗炎活性,可抑制一氧化氮(NO)和肿瘤坏死因子(TNFα)的生成,并显著下调小眼畸形相关转录因子(MITF)通路相关基因表达。为其在化妆品类中的应用提供了理论依据。

虎杖苷调节Hippo/YAP信号通路对冠心病大鼠心肌损伤的影响

目的探讨虎杖苷(PD)对冠心病(CHD)大鼠心肌损伤及Hippo-Yes相关蛋白(YAP)信号通路的影响。方法构建CHD大鼠模型,将造模成功大鼠随机分为模型组(CHD组)、虎杖苷低、高剂量组(PD-L、PD-H组)、虎杖苷高剂量+Hippo/YAP信号通路激活剂PY-60组(PD-H+PY-60组),每组18只,另取18只正常大鼠作为对照组(Control组),超声心动图检测大鼠心功能。每组随机选择6只大鼠处死取心脏组织,HE染色观察大鼠心肌组织的病理形态变化,Masson染色观察大鼠心肌纤维化程度及胶原纤维化面积;TUNEL染色观察心肌组织中细胞凋亡情况;Western blot法检测B淋巴细胞瘤-2相关X蛋白(Bax)、半胱氨酸蛋白酶-3(Caspase-3)、p-YAP/YAP、具有PDZ结合模体的转录激活因子(TAZ)表达。结果CHD组较Control组心肌细胞异常,心肌纤维结构紊乱,炎性细胞浸润及组织水肿明显,蓝色胶原纤维沉积增多,心肌胶原纤维化面积占比增多,细胞凋亡率及Bax、Caspase-3、p-YAP/YAP、TAZ表达升高,左室射血分数(LVEF)、左室缩短分数(LVFS)、舒张早期运动速度/舒张晚期运动速度(Em/Am)水平降低(均P<0.05);PD-L组、PD-H组较CHD组心肌细胞形态相对正常,细胞坏死减少,炎性细胞浸润及组织水肿明显减轻,蓝色胶原纤维沉积减少,心肌胶原纤维化面积占比减少,细胞凋亡率及Bax、Caspase-3、p-YAP/YAP、TAZ表达降低,LVEF、LVFS、Em/Am水平升高(均P<0.05);PD-H+PY-60组较PD-H组心肌组织损伤加重,蓝色胶原纤维沉积增多,心肌胶原纤维化面积占比增多,细胞凋亡率及Bax、Caspase-3、p-YAP/YAP、TAZ表达升高,LVEF、LVFS、Em/Am水平降低(均P<0.05)。结论虎杖苷可改善CHD大鼠心肌损伤,其作用机制与抑制Hippo/YAP通路有关。

虎杖苷激活MEK/ERK信号通路改善东莨菪碱诱导的干眼症大鼠眼表功能障碍和病变

目的:探究虎杖苷对干眼症(DED)大鼠眼表功能障碍和病变的影响及其保护机制。方法:90只SD大鼠随机选取18只为对照组,其余皮下注射氢溴酸东莨菪碱建立DED模型,建模后分为模型组、0.05%虎杖苷组、0.5%虎杖苷组、0.5%虎杖苷+U0126[丝裂原活化蛋白激酶激酶(MEK)抑制剂]组,每组18只;各组给予相应剂量药物进行干预,对照组和模型组大鼠给予等量生理盐水。干预第7、14、21、28天,进行Schirmer测试检测各组大鼠泪液分泌量,荧光素染色检测各组大鼠泪膜破裂时间(BUT)和角膜损伤;RT-qPCR检测角膜和结膜组织促炎因子TNF-α、IFN-γ和IL-1βmRNA表达;HE染色观察角膜组织病理学变化;PAS染色评估结膜杯状细胞数量;Western blot检测角膜和结膜组织MEK/细胞外调节蛋白激酶(ERK)通路相关蛋白表达。结果:与对照组相比,模型组大鼠泪液分泌量、BUT、结膜杯状细胞数量显著减少(P<0.05),角膜损伤评分、TNF-α、IFN-γ和IL-1βmRNA水平显著升高(P<0.05),角膜上皮层变薄,可见大量炎症细胞浸润和新生毛细血管;与模型组相比,0.05%虎杖苷组和0.5%虎杖苷组泪液分泌量、BUT、结膜杯状细胞数量、p-MEK1/2/MEK1/2和p-ERK1/2/ERK1/2显著升高(P<0.05),角膜损伤评分、TNF-α、IFN-γ和IL-1βmRNA水平显著降低(P<0.05),角膜组织病理损伤明显缓解;U0126可显著减弱虎杖苷对DED大鼠眼表功能障碍和病变的保护作用。结论:虎杖苷可能通过激活MEK/ERK通路改善东莨菪碱诱导的DED大鼠眼表功能障碍和病变。