Protective effects of panax notoginseng saponin on dextran sulfate sodium-induced colitis in rats through phosphoinositide-3-kinase protein kinase B signaling pathway inhibition

BACKGROUND Intestinal inflammation is a common digestive tract disease, which is usually treated with hormone medicines. Hormone medicines are effective to some extent, but long-term use of them may bring about many complications.AIM To explore the protective effects of panax notoginseng saponin(PNS) against dextran sulfate sodium(DSS)-induced intestinal inflammatory injury through phosphoinositide-3-kinase protein kinase B(PI3K/AKT) signaling pathway inhibition in rats.METHODS Colitis rat models were generated via DSS induction, and rats were divided into control(no modeling), DSS, DSS + PNS 50 mg/k, and DSS + PNS 100 mg/kg groups. Then, the intestinal injury, oxidative stress parameters, inflammatory indices, tight junction proteins, apoptosis, macrophage polarization, and TLR4/AKT signaling pathway in colon tissues from rats in each of the groups were detected. The PI3 K/AKT signaling pathway in the colon tissue of rats was blocked using the PI3K/AKT signaling pathway inhibitor, LY294002.RESULTS Compared with rats in the control group, rats in the DSS group showed significantly shortened colon lengths, and significantly increased disease activity indices, oxidative stress reactions and inflammatory indices, as well as significantly decreased expression of tight junction-associated proteins. In addition, the DSS group showed significantly increased apoptotic cell numbers,and showed significantly increased M1 macrophages in spleen and colon tissues.They also showed significantly decreased M2 macrophages in colon tissues, as well as activation of the PI3K/AKT signaling pathway(all P < 0.05). Compared with rats in the DSS group, rats in the DSS + PNS group showed significantly lengthened colon lengths, decreased disease activity indices, and significantly alleviated oxidative stress reactions and inflammatory responses. In addition, this group showed significantly increased expression of tight junction-associated proteins, significantly decreased apoptotic cell numbers, and significantly decreased M1 macrophages in spleen and colon tissues. This group further showed significantly increased M2 macrophages in colon tissues, and significantly suppressed activation of the PI3K/AKT signaling pathway, as well as a dose dependency(all P < 0.05). When the PI3K/AKT signaling pathway was inhibited, the apoptosis rate of colon tissue cells in the DSS + LY294002 group was significantly lower than that of the DSS group(P < 0.05).CONCLUSION PNS can protect rats against DSS-induced intestinal inflammatory injury by inhibiting the PI3K/AKT signaling pathway, and therefore may be potentially used in the future as a drug for colitis.

Effects of Panax notoginseng saponins on apoptosis induced by hydrogen peroxide in cultured rabbit bone marrow stromal cells via altering the oxidative stress level and down-regulating caspase-3

Objective： To investigate the effects of Panax notoginseng saponins （PNS） on hydrogen peroxide （H2O2）-induced apoptosis in cultured rabbit bone marrow stromal cells （BMSCs）. Methods： The effects of different concentrations of PNS on proliferation and early osteoblast differentiation of BMSCs were determined by the MTT assay and an alkaline phosphatase （ALP） assay. An optimal effective concentration of PNS was determined and used in subsequent experiments. The cultured BMSCs were divided into three groups： untreated control, H2O2 treated, and PNS pretreatment of H2O2 treated. The oxidative stress level was assessed by superoxide dismutase （SOD） and malondialdehyde （MDA） assays. Flow cytometry was used to determine BMSC apoptosis by staining with annexinV-FITC/propidium iodide （PI）. The activity of caspase-3 enzyme was measured by spectrofluorometry. Results： PNS （0.1g/L） significantly increased both BMSC proliferation rate and ALP activity, while it decreased the indicators of oxidative stress, caspase-3 activity, and the apoptosis rate of BMSCs induced by H2O2.. Conclusion： PNS, acting as a biological antioxidant, had a protective effect on H2O2-induced apoptosis in cultured rabbit BMSCs by decreasing oxidative stress and down-regulating caspase-3.

Panax notoginseng saponins promotes cerebral recovery from ischemic injury by downregulating LINGO-1 and activating the EGFR/PI3K/AKT signaling pathways in vivo

Objective:Panax notoginseng saponins (PNS),extracted from rhizome of the herb Radix et Rhizoma Notoginseng (Panax notoginseng (Burk.) F.H.Chen),was recently discovered to have beneficial effects against neurological damage.This study investigated the effects of PNS on cerebral ischemia and elucidated the molecular mechanisms underlying these effects.Methods:Middle cerebral artery occlusion rats were treated with PNS (3.6 mg/100 g or 7.2 mg/100 g per day) for 7 days,the gene of LINGO-1 was measured and the expression of protein synaptophysin,postsynaptic density protein 95,LINGO-1 and p-EGFR/p-PI3K/p-AKT were investigated.The weight and mNSS score of Sprague-Dawley rats in each group were recorded every day during the 7 days.Results:PNS promoted middle cerebral artery occlusion rats' weight and the recovery of neural function.PNS significantly decreased ischemia-induced LINGO-1 protein expression.PNS also elevated EGFR/PI3K/AKT phosphorylation levels.Conclusion:PNS promoted cerebral recovery from ischemic injury by accelerating synapse reconstruction and inhibiting the neuron growth negative regulatory protein LINGO-1 and activating the epidermal growth factor receptor (EGFR)/PI3K/AKT signaling pathway in vivo.

2～3年生三七花药愈伤组织诱导的比较研究

以盆栽2、3年生三七花药为外植体,在附加不同激素、浓度的MS培养基上进行愈伤组织的诱导试验。结果显示:在MS基本培养基中附加2,4-D 2.0 mg/L+BA 1.5 mg/L+IAA 1.0 mg/L+蔗糖6%+琼脂0.8%的条件下,2年生三七花药愈伤组织的诱导率可达43.0%,三年生三七花药愈伤组织的诱导率可达55.0%。2、3年生三七花药愈伤组织继代培养差别不大。

三七总皂甙预处理大鼠供肝对细胞凋亡及TNF-α、Caspase-3 表达的影响a(英文)

目的探讨三七总皂甙(PNS)预处理对大鼠供肝的保护作用及其对供肝细胞凋亡和TNF-α、Caspase-3mRNA表达的影响。方法雄性SD大鼠分别用作供、受体,采用Kamada's袖套法建立原位肝移植模型,根据供肝切取前1h是否静脉注射PNS(50mg/kg)将大鼠随机分为2组:PNS预处理组(P组)和NS对照组(N组);另设假手术作对照组(S组)。分别于供肝再灌注后2、6、24h处死各组动物,检测血清ALT、AST,HE切片作组织学检查,TUNEL法检测肝细胞凋亡,RT-PCR法检测TNF-α、Caspase-3mRNA的表达。结果供肝再灌注后2、6、24h各时点,P组大鼠血清ALT、AST水平及肝细胞凋亡指数(AI)均明显低于N组(P<0.05);供肝再灌注后2h、6h,P组大鼠肝组织TNF-α、Caspase-3mRNA的相对表达水平明显低于N组(P<0.05)。结论三七总皂甙(PNS)预处理大鼠供肝,可以有效地减轻移植肝的缺血/再灌注损伤和细胞凋亡,影响TNF-α、Caspase-3的表达,可能为PNS抗细胞凋亡的机制之一。

黄芪、三七及其配伍对MNNG诱导萎缩性胃炎癌前病变大鼠Gli1/2/3、SUFU及CyclinD1水平的影响

目的探讨黄芪、三七及其配伍对萎缩性胃炎癌前病变大鼠神经胶质瘤关联癌基因同源物(Gli1/2/3)、丝氨酸激酶抑制物(SUFU)及细胞周期蛋白Cyclin D1水平的影响。方法雄性Wistar大鼠随机分为空白组、模型组、黄芪组、三七组、黄芪三七组、Purmorphamine组、环巴明组及叶酸组,每组10只。采用MNNG负荷多因素法制备萎缩性胃炎癌前病变模型,并给予相应药物治疗8周。Western blot及RT-PCR检测大鼠Gli1/2/3蛋白及基因水平。量子点介导的免疫荧光化学法检测SUFU及Cyclin D1蛋白表达。结果与空白组相比,模型组大鼠Gli-1 mRNA含量降低明显(P<0.01),Gli-2及Gli-3蛋白含量均显著升高(P<0.01),SUFU蛋白表达明显增强(P<0.01),Cyclin D1蛋白表达明显减弱(P<0.01)。与模型组相比,三七组可明显升高Gli-1基因表达(P<0.05),降低Gli-2、Gli-3和SUFU的蛋白表达(P<0.05,P<0.01),黄芪三七组对Gli-3及SUFU的蛋白表达具有改善作用(P<0.05,P<0.01),而黄芪组仅能降低SUFU蛋白表达(P<0.01),对其余指标均未见明显改善。结论活血药三七可影响萎缩性胃炎癌前病变大鼠胃黏膜hedgehog信号通路中关键因子的表达,从而对萎缩性胃炎癌前病变起到防治作用。

三七总皂苷对大鼠肝移植物细胞凋亡及Bcl-2和Caspase-3表达的影响

目的 探讨三七总皂苷(PNS)预处理对大鼠供肝缺血 再灌注损伤的保护作用及其对供肝细胞凋亡和Bcl 2及Caspase 3 mRNA表达的影响。方法 雄性SD大鼠分别用作供、受体,采用Kamada’s袖套法建立原位肝移植模型,根据供肝切取前1 h是否静脉注射 PNS(50 mg/kg)将大鼠随机分为 PNS预处理组(PNS组)和生理盐水对照组(NS组); 另设假手术作对照组(SO组)。分别于供肝再灌注后 2 h、6 h及 24 h处死各组动物,检测血清 ALT及AST,HE切片作病理组织学检查,TUNEL法检测肝细胞凋亡,RT PCR法检测Bcl 2及Caspase 3 mR NA的表达。结果 供肝再灌注后2 h、6 h及24 h各时点,PNS组大鼠血清 ALT和 AST水平及肝细胞凋亡指数(AI)均明显低于NS组(P<0.05,P<0.01); 6 h及24 h,PNS组大鼠肝组织Bcl 2 mRNA的表达水平明显高于NS组(P<0.05); 2 h及6 h,PNS组大鼠肝组织Caspase 3 mRNA的表达水平明显低于NS组(P<0.05)。结论 PNS预处理大鼠供肝,可以有效地减轻移植肝的缺血/再灌注损伤和细胞凋亡,影响细胞凋亡调控基因 Bcl 2 和Caspase 3的表达。这可能为PNS抗细胞凋亡的机理之一。

三七总皂苷诱导U937细胞凋亡及对Caspase-3表达的影响

目的观察三七总皂苷(PNS)对人急性单核细胞白血病细胞系U937细胞的增殖、凋亡及Caspase-3表达的影响。方法用不同浓度PNS作用于U937细胞,采用MTT法检测细胞增殖活性,用流式细胞术检测细胞周期和凋亡,Western-blot法检测Caspase-3蛋白表达。结果 PNS抑制U943细胞生长并呈时间和剂量依赖性;分别予100、200、400、800mg/L处理U937细胞48h,细胞凋亡率分别为(5.93±0.15)%、(7.43±0.70)%、(12.3±0.38)%和(20.4±0.91)%,对照组细胞凋亡率为(1.73±1.50)%(P<0.01),并诱导细胞G1期阻滞;Western-blot结果显示,PNS使Caspase-3蛋白表达上调。结论 PNS能抑制U937细胞增殖,并可能通过上调Caspase-3蛋白表达诱导细胞凋亡。

三七总皂苷抑制小鼠星形胶质细胞活化缓解完全弗氏佐剂所致炎性痛

目的探讨三七总皂苷(PNS)对完全弗氏佐剂(CFA)所致慢性炎性疼痛小鼠模型的镇痛作用及可能机制。方法48只C57BL/6J雄性小鼠随机分成生理盐水对照组(Ctrl)、CFA组(CFA)、CFA+PNS组、CFA+地塞米松(DEX)组(CFA+DEX)。采用Von Frey纤维丝检测小鼠机械疼痛;采用免疫组织化学法检测脊髓后角胶质纤维酸性蛋白(GFAP)阳性星形胶质细胞数目和形态结构变化;采用Western blotting检测各组小鼠相应节段脊髓GFAP、核苷酸结合寡聚化结构域(NOD)样受体热蛋白结构域相关蛋白3(NLRP3)、凋亡相关斑点样蛋白(ASC)、Caspase-1、白细胞介素(IL)-1β和IL-18的表达。结果与Ctrl组相比,CFA组小鼠机械痛阈值在第1、3、5、7、14天明显下降,小鼠脊髓NLRP3、ASC、Caspase-1、IL-1β和IL-18的表达均显著增加。PNS干预可缓解模型小鼠机械痛觉,下调小鼠脊髓NLRP3、ASC、Caspase-1、IL-1β和IL-18的表达,且与CFA+DEX组表达差异无显著性。免疫组织化学结果显示,与Ctrl组相比,CFA组小鼠脊髓后角GFAP阳性细胞数目显著增多;PNS干预后CFA+PNS组小鼠脊髓后角GFAP阳性细胞数目减少;DEX干预后对小鼠脊髓后角GFAP阳性细胞数目没有明显影响。Western blotting结果显示,与Ctrl组相比,CFA组小鼠脊髓GFAP表达明显增加;PNS干预后CFA+PNS组小鼠脊髓GFAP表达明显减少,DEX干预后对小鼠脊髓后角GFAP表达没有明显影响。结论PNS对炎性痛具有较好的缓解作用,其机制可能与抑制星形胶质细胞活化以及减少NLRP3炎性小体激活有关。

三七总皂苷抑制脑组织3-硝基酪氨酸形成的体外研究

目的研究三七总皂苷(PNS)对体外过氧化亚硝基离子(ONOO-)依赖途径和亚铁血红素-亚硝酸盐-过氧化氢(heme/NO_2^-/H_2O_2)依赖途径诱导的脑组织3-硝基酪氨酸(3-NT)形成的抑制作用。方法依据体内3-NT形成的主要途径,分别建立heme/NaNO_2/H_2O_2和ONOO^-诱导的3-NT形成体外模型;分别以牛血清白蛋白(BSA)/大鼠血浆蛋白、大鼠脑组织匀浆蛋白为底物,分为对照组、模型组及低、中、高浓度干预组(PNS终浓度分别为50、100及200 mg/L),Western blot法检测各组3-NT水平。结果与对照组相比,模型组BSA、血浆蛋白、脑组织匀浆蛋白中均有3-NT形成(均P<0.05)。与模型组相比,两种途径下,加入不同浓度PNS后,BSA、大鼠血浆蛋白中3-NT表达水平均降低(均P<0.05),而低浓度干预组脑组织匀浆蛋白中3-NT降低不明显(P>0.05),中、高浓度干预组脑组织匀浆蛋白3-NT水平降低明显,且高浓度干预组作用最大(均P<0.05)。结论中、高浓度的PNS对脑组织3-NT形成具有抑制作用,PNS可能对3-NT参与损伤机制的多种脑组织疾病(如颅脑外伤、脑卒中、神经系统变性疾病等)具有保护作用。

三七总皂苷对脊髓损伤大鼠神经元凋亡及Nrf2、caspase-3表达的影响

目的:探究三七总皂苷(TSPN)对脊髓损伤(SCI)大鼠神经元凋亡及Nrf2、Caspase-3表达的影响。方法:采用“自制击打器”建立SCI大鼠模型,将成功构建的100只SCI大鼠随机分为对照组、模型组、阳性对照组及实验组,每组25只。阳性对照组与实验组分别腹腔注射甲基强的松龙[30 mg/(kg·d)]与TSPN[100 mg/(kg·d)],对照组与模型组则腹腔注射等量生理盐水,连续干预14 d。采用HE染色与TUNEL法检测大鼠脊髓组织病理学变化与神经元凋亡;采用免疫组化法及Wb法检测脊髓组织Nrf2、Caspase-3蛋白表达。结果:与对照组比较,模型组大鼠脊髓组织Nrf2蛋白表达显著降低,且阳性对照组与实验组高于模型组,阳性对照组高于实验组;神经元凋亡率及Caspase-3蛋白表达升高,且阳性对照组与实验组低于模型组,阳性对照组低于实验组(均P<0.05)。结论:TSPN可减轻SCI大鼠脊髓组织病变程度,保护神经元,其作用机制可能与下调脊髓组织Caspase-3蛋白表达,上调Nrf2蛋白表达有关。

三七总皂苷预处理通过激活HIF-1α/BNIP3线粒体自噬信号通路减少心肌细胞H/R损伤的机制研究

目的探索三七总皂苷(panax notoginseng saponins,PNS)预处理心肌细胞减少缺氧/复氧(hypoxiareoxygenation,H/R)损伤的作用机制。方法设置对照组(常规培养H9c2细胞)、模型组(H/R损伤的H9c2细胞)、PNS组(加入PNS处理的H/R损伤的H9c2细胞)、二甲氧基雌二醇(2-methoxyestradiol,2-ME2)组(加入2-ME2处理的H/R损伤的H9c2细胞)和PNS+2-ME2组心肌细胞(同时加入PNS和2-ME2处理的H/R损伤的H9c2细胞),采用酶联免疫吸附法检测心肌细胞损伤,蛋白质印迹法检测线粒体自噬信号通路表达,免疫荧光法检测心肌细胞自噬活性。结果模型组高于对照组的指标有肌酸激酶(creatine kinase,CK)(0.37±0.05 vs.2.41±0.66)、乳酸脱氢酶(lactate dehydrogenase,LDH)(2.20±0.69 vs.4.05±1.08)、天门冬氨酸氨基转移酶(aspartate amino transferase,AST)(38.17±9.02 vs.107.83±20.89)、细胞自噬标志物LC3(0.17±0.05 vs.0.63±0.66)、低氧诱导因子-1α(hypoxic inducible factor-1α,HIF-1α)(0.22±0.09 vs.0.66±0.08)和B淋巴细胞瘤-2/腺病毒E1B 19kDa相互作用蛋白3(B lymphocytoma-2/adenovirus E1B 19kDa interacting protein 3,BNIP3)(0.15±0.02 vs.0.59±0.13),差异均有统计学意义(P<0.05)。PNS组低于模型组的指标有CK(2.41±0.66 vs.1.55±0.39)、LDH(4.05±1.08 vs.3.19±0.88)、AST(107.83±20.89 vs.66.52±11.43),差异均有统计学意义(P<0.05);PNS组高于模型组的指标有LC3(0.63±0.66 vs.0.85±0.23)、HIF-1α(0.66±0.08 vs.0.91±0.22)和BNIP3(0.59±0.13 vs.0.82±0.17),差异均有统计学意义(P<0.05)。2-ME2+PNS组低于模型组的指标有CK(2.41±0.66 vs.2.08±0.54)、LDH(4.05±1.08 vs.3.51±0.92)、AST(107.83±20.89 vs.83.49±19.53);2-ME2+PNS组高于模型组的指标有LC3(0.63±0.66 vs.0.70±0.54)、HIF-1α(0.66±0.08 vs.0.72±0.10)和BNIP3(0.59±0.13 vs.0.64±0.12),差异均有统计学意义(P<0.05)。2-ME2组高于模型组的指标有CK(2.41±0.66 vs.3.62±0.98)、LDH(4.05±1.08 vs.5.67±1.39)、AST(107.83±20.89 vs.192.13±28.97),差异均有统计学意义(P<0.05);2-ME2组低于模型组的指标有HIF-1α(0.66±0.08 vs.0.27±0.03)和BNIP3(0.59±0.13 vs.0.25±0.07),差异均有统计学意义(P<0.05)。与对照组相比,模型组心肌细胞LC3荧光斑点数明显增加(P<0.05);与模型组相比,PNS组和2-ME2+PNS组心肌细胞LC3荧光斑点数明显增加,2-ME2组LC3荧光斑点数明显减少(P<0.05)。结论PNS能够通过激活HIF-1α/BNIP3线粒体自噬信号通路,减少心肌细胞H/R损伤。

Panax Notoginseng Saponin Attenuates Gastric Mucosal Epithelial Cell Injury Induced by Dual Antiplatelet Drugs through COX and PI3K/Akt/ VEGF-GSK-3β-RhoA Network Pathway

Objective To elucidate the underlying mechanism of Panax notoginseng saponin(PNS)on gastric epithelial cell injury and barrier dysfunction induced by dual antiplatelet(DA).Methods Human gastric mucosal epithelial cell(GES-1)was cultured and divided into 4 groups:a control,a DA,a PNS+DA and a LY294002+PNS+DA group.GES-1 apoptosis was detected by flow cytometry,cell permeability were detected using Transwell,level of prostaglandins E2(PGE2),6-keto-prostaglandin F1α(6-keto-PGF1α)and vascular endothelial growth factor(VEGF)in supernatant were measured by enzyme linked immunosorbent assay(ELISA),expression of phosphatidylinositide 3-kinase(PI3K),phosphorylated-PI3K(p-PI3K),Akt,phosphorylated-Akt(p-Akt),cyclooxygenase-1(COX-1),cyclooxygenase-2(COX-2),glycogen synthase kinase-3β(GSK-3β)and Ras homolog gene family member A(RhoA)were measured by Western-blot.Results DA induced apoptosis and hyper-permeability in GES-1,reduced supernatant level of PGE2,6-keto-PGF1αand VEGF(P<0.05).Addition of PNS reduced the apoptosis of GES-1 caused by DA,restored the concentration of PGE2,6-keto-PGF1αand VEGF(P<0.05).In addition,PNS attenuated the alteration of COX-1 and COX-2 expression induced by DA,up-regulated p-PI3K/p-Akt,down-regulated RhoA and GSK-3β.LY294002 mitigated the effects of PNS on cell apoptosis,cell permeability,VEGF concentration,and expression of RhoA and GSK-3βsignificantly.Conclusions PNS attenuates the suppression on COX/PG pathway from DA,alleviates DA-induced GES-1 apoptosis and barrier dysfunction through PI3K/Akt/VEGF-GSK-3β-RhoA network pathway.

三七茎叶总皂苷抑制七氟醚诱导的海马神经元凋亡机制研究

目的探讨三七茎叶总皂苷(PNSLS)调节七氟醚诱导海马神经元凋亡的作用机制。方法以不同剂量七氟醚(0,4.1%,8%)干预小鼠海马神经元细胞系HT22,使用不同质量浓度的PNSLS(1.5,3.0,6.0,12.0μg/mL)预处理神经元。将HT22细胞分为对照组、暴露于4.1%七氟醚的细胞组(4.1%Sev组)、使用6μg/mL PNSLS对细胞进行预处理1 h后将细胞暴露于4.1%七氟醚组(4.1%Sev+PNSLS组)、在PNSLS处理前1 h加入丝裂原激活蛋白激酶(MAPK)激活剂anisomycin(An)再暴露于七氟醚和PNSLS的共培养组(4.1%Sev+PNSLS+An组)。通过CCK-8法检测神经元活力,流式细胞术及免疫印迹(Western blot)法检测海马神经元凋亡,比色活性测定试剂盒测定胱天蛋白酶-3(caspase-3)活性,Western blot法测定内质网应激相关蛋白及p38 MAPK/核转录因子-κB p65(p38 MAPK/NF-κB p65)信号通路相关蛋白的表达水平。结果4.1%七氟醚显著抑制海马神经元活力(P<0.05),6μg/mL PNSLS显著升高神经元活力并抑制海马神经元凋亡(P<0.05)。与Sev组比较,Sev+PNSLS组细胞中凋亡蛋白Bax表达水平显著降低(P<0.05),B细胞淋巴瘤2家族蛋白(Bcl-2)表达水平显著升高(P<0.05),caspase-3活性显著降低(P<0.05);Sev+PNSLS组内质网应激诱导的凋亡信号通路相关蛋白78000葡糖调节蛋白(GRP78)、内质网应激相关蛋白(CHOP)表达水平均显著降低(P<0.01),p38和IκBα的磷酸化水平均显著降低(P<0.01),细胞质中NF-κB p65水平显著升高(P<0.01),细胞核中NF-κB p65水平显著降低(P<0.01)。与Sev+PNSLS组比较,Sev+PNSLS+An组p38和IκBα的磷酸化水平均显著升高(P<0.01),细胞质中NF-κB p65水平显著降低(P<0.01),细胞核中NF-κB p65水平显著升高(P<0.01),海马神经元活力显著降低(P<0.01),凋亡率显著升高(P<0.01)。结论PNSLS通过激活p38 MAPK/NF-κB p65信号通路抑制七氟醚诱导的神经元凋亡。

Effects of Panax Notoginseng Saponins on Proliferation and Differentiation in NIH3T3 Cells

Objective： To investigate the effects of Panax notoginseng saponins （PNS） on the proliferation and differentiation in NIH3T3 cells. Methods： NIH3T3 cells were treated by various concentrations of PNS 0, 0.05, 0.10, 0.20, and 0.40 g/L. The vitality and proliferation potential of cells were detected by 3-（4,5-dimethylthiazol- 2-yl）-2, 5-diphenyltetrazolium bromide （MTT） assay, the alkaline phosphatase （ALP） activity was measured by p-nitrophenyl phosphate （pNPP） assay, and the mineralization formation ability was tested for the cellular differentiation toward osteoblast, as well as the expression level of phosphorylated extrecellular signal-regulated kinasel/2（P-ERK1/2）, extracellular signal-regulated kinasel/2 （ERK1/2） protein kinase was analyzed by Western blot with total cell lysate of NIH3T3 cells treated by PNS. Results： Both MTT and pNPP assay showed that optical density （OD） values were increased in response to PNS treatment at a dose-dependent pattern. The mineralization formation ability was enhanced in PNS-treated NIH3T3 cells compared with untreated cells. Meanwhile, the expression level of P-ERK1/2 protein kinase was up-regulated in PNS-treated NIH3T3 cells, while, the expression level of ERK1/2 protein kinase revealed no obvious difference with or without PNS treated cells. Conclusion： PNS could pay a role to promote the proliferation and differentiation in NIH3T3 cells by means of up-regulation of P-ERK1/2 protein kinase.

Panax Notoginseng Saponins Increases the Blood Concentration of Nifedipine by Inhibiting CYP3A4 Enzyme through PXR-and CAR-Mediated Pathway

Objective:To explore the mechanism underlying the effect of Panax notoginseng saponins(PNS)on the pharmacokinetics of nifedipine(NF)in rats.Materials and Methods:Twenty-four rats were randomly divided into blank(BL)group,PNS group,NF group,and PNS+NF group,with six rats in each group.Noncompartmental analysis and t-test were carried out to determine the difference between the pharmacokinetic parameters of NF in different groups.CYP3 A4 enzyme activity was calculated using the probe drug method.The mRNA and protein contents of CYP3 A4,nuclear receptor CAR,and PXR in rat liver were quantitatively analyzed by qRT-PCR and Western blot.Results:After the rats were treated with the combination of PNS and NF,the plasma concentration,half-life,peak time,and area under the concentration-time curve of NF increased,whereas the clearance rate decreased.The inhibitory effect on CYP3 A4 enzyme activity was in the following order:PNS+NF group(strongest)>PNS group>NF group,and BL group(weakest).Similar changes were observed for the inhibitory effect on CYP3 A4,CAR,and PXR mRNA and protein content,and the order was as follows:PNS+NF group(weakest)<PNS group<NF group,and BL group(strongest).Conclusion:In combination with NF,PNS may inhibit the mRNA and protein expression of nuclear receptor CAR and PXR and the activity of CYP3 A4 enzyme,slowing down the pharmacokinetics of NF in rats,increasing its blood concentration,and enhancing the therapeutic effect of NF.

三七总皂苷对大鼠脑缺血再灌注后Caspase表达的影响

目的研究大鼠局灶性脑缺血再灌注后脑组织中天冬氨酸特异性半胱氨酸蛋白酶(Caspase-1、Caspase-3、Caspase-8)蛋白表达的变化,以及三七总皂苷(PNS)对其表达的影响。方法采用线栓法建立大鼠大脑中动脉栓塞局灶性脑缺血再灌注模型。实验动物随机分为假手术组、模型组、三七总皂苷组和尼莫地平对照组,于缺血前5 m in、缺血后12、24、36 h给药,共给药4次。缺血2 h再灌注46 h后,采用免疫组织化学方法检测脑组织中Caspase-1、Caspase-3、Caspase-8蛋白表达的变化。结果缺血2 h再灌注46 h后,可诱导脑组织Caspase-1、Caspase-3表达增强,PNS能下调Caspase-1、Caspase-3蛋白的表达;但对Caspase-8的表达无影响。结论PNS能抑制脑缺血再灌注后Caspase-1、Caspase-3蛋白的表达,这可能是其促进脑缺血后神经元存活及损伤后修复的机制之一。

三七皂苷对激素性股骨头坏死早期细胞凋亡的影响及其机制

目的探讨三七皂苷(panax notoginseng saponins,PNS)对激素性股骨头坏死(steonecrosis of the femoral head,SONFH)早期细胞凋亡的影响及其作用机制。方法选取健康成年新西兰兔24只,分为正常对照组、模型组及PNS组3组。模型组采用马血清(10mL/kg)加激素(40mg/kg)的方法建立兔SONFH模型;PNS组在模型组基础上同时给予PNS(50mg/kg)干预;正常对照组常规饲养。2周后处死动物取双侧股骨头进行脱钙包埋,TUNEL染色观察细胞内凋亡变化,BCA法检测股骨头内caspase-3活性变化,免疫组化及Western blot检测各组股骨头内Bax及Bcl-2含量的变化。结果 TUNEL染色结果发现,模型组骨组织内可见大量凋亡细胞,PNS组骨组织内凋亡细胞明显减少(P<0.05);与模型组相比,经PNS预处理后股骨头组织内的caspase-3活性明显下降(P<0.05);免疫组化染色及Western blot检测结果证实,模型组骨组织内Bax蛋白表达量明显增加,而Bcl-2蛋白表达量明显减少,而与之相反的是PNS组骨组织内检测到的Bax蛋白表达量明显下降(P<0.05),而Bcl-2蛋白表达量明显增加(P<0.05)。结论PNS可能通过增强Bcl-2表达,降低Bax表达,抑制caspase-3活性,减少早期SONFH股骨头组织内的细胞凋亡。

三七总皂甙降低兔肺缺血再灌注损伤和抑制细胞凋亡

目的探讨细胞凋亡与肺缺血再灌注损伤的关系以及三七总皂甙的作用及机制。方法健康日本大耳白兔84只,随机分为对照组、肺缺血再灌注1、3、5 h组和相应三七总皂甙干预组。复制肺缺血再灌注损伤模型。用原位缺口末端标记(TUNEL)法、聚丙烯酰胺凝胶电泳观测肺组织细胞凋亡,原位杂交技术检测肺组织细胞Fas/FasL系统和caspase-3基因表达。结果肺缺血再灌注组肺组织细胞凋亡指数(肺缺血再灌注5 h组:22.08±1.93;对照组:2.04±0.67)、Fas/FasL和caspase-3基因表达(肺缺血再灌注5 h组:0.241±0.029;对照组:0.121±0.015)均显著高于对照组(P<0.01),并出现电泳梯形条带结构;三七总皂甙干预组Fas/FasL mRNA及其caspase-3的表达(三七总皂甙干预5 h组:0.199±0.020;肺缺血再灌注5 h组:0.241±0.029)显著低于缺血再灌注组(P<0.01),肺组织细胞凋亡指数(三七总皂甙干预5 h组:12.58±1.82;肺缺血再灌注5 h组:22.08±1.93)也显著低于缺血再灌注组(P<0.01),梯形条带结构基本消失。肺组织细胞凋亡指数分别与caspase-3 mRNA及Fas/FasL mRNA之间均呈显著正相关(P<0.01)。结论三七总皂甙可能通过抑制Fas/FasL系统的激活,阻遏肺组织细胞凋亡,从而减轻肺缺血再灌注损伤。

丹皮酚联合三七总皂苷对AngⅡ诱导的心脏成纤维细胞增殖及TGF-β/Smads信号通路的影响

目的探讨丹皮酚联合三七总皂苷应用对血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)诱导新生大鼠的心脏成纤维细胞(cardiac fibroblasts,CFs)增殖及TGF-β/Smads信号通路的影响。方法采用胰酶消化法分离培养的CFs随机分为6组:正常对照组,模型组,三七总皂苷组,丹皮酚组,丹皮酚与三七总皂苷联合用药大、小剂量组。采用MTT比色法测定各组培养液中CFs增殖情况,免疫细胞化学法检测α-平滑肌肌动蛋白(α-SMA)的蛋白表达水平,RTPCR法检测转化生长因子-β1(transforming growth factor-β1,Tgfb1)的mRNA表达水平,Western blot法检测TGF-β1、磷酸化的Smad2/3(p-Smad2/3)的蛋白表达水平。结果与正常对照组比较,模型组大鼠CFs增殖明显增高,Tgfb1的mRNA表达显著增加,α-SMA及TGF-β1、p-Smad2/3的蛋白表达显著增加;与模型组比较,丹皮酚组、三七总皂苷组、联合用药的大剂量和小剂量组均可抑制由AngⅡ诱导的CFs的增殖,Tgfb1的mRNA表达显著减少,α-SMA及TGF-β1、p-Smad2/3的蛋白表达显著减少;与丹皮酚组、三七总皂苷组比较,联合用药大、小剂量组在抑制CFs增殖的作用上显著增强,抑制Tgfb1的mRNA表达效果显著增强,抑制α-SMA及TGF-β1、p-Smad2/3的蛋白表达效果显著增强;与联合用药小剂量组比较,联合用药大剂量组对以上指标的抑制效果更佳,显示一定的剂量依赖性。结论丹皮酚与三七总皂苷联合应用对抑制CFs增殖,呈现明显的协同作用。其作用机制与抑制TGF-β/Smads信号通路的传导有关。