Role of an endoplasmic reticulum Ca2+-independent phospholipase A2 in cisplatin-induced renal cell apoptosis

It has been demonstrated recently that rabbit renal proximal tubule cells (RPTC) express a novel Ca(2+)-independent phospholipase A(2) (iPLA(2)) whose activity localizes to the endoplasmic reticulum (ER-iPLA(2)) and is similar to group VIB PLA(2). In this study, the expression of group VIB PLA(2) was examined and the role of ER-iPLA(2) in cisplatin-induced apoptosis was determined. Cisplatin induced both time- and concentration-dependent RPTC apoptosis as determined by p53 nuclear localization, annexin V staining, caspase 3 activity, and chromatin condensation. Inhibition of ER-iPLA(2) with bromoenol lactone (5 microM) reduced cisplatin-induced annexin V binding 40%, chromatin condensation 55%, and caspase 3 activity 42%, but had no effect on p53 nuclear localization. Treatment of RPTC with the protein kinase C stimulator phorbol 12-myristate 13-acetate increased the activity of ER-iPLA(2) 2-fold and increased cisplatin-induced RPTC apoptosis. These studies demonstrate that group VIB PLA(2) is expressed in RPTC and suggest that RPTC ER-iPLA(2) is the rabbit homolog of group VIB PLA(2). These data also demonstrate that ER-iPLA(2) acts downstream of p53 and upstream of caspase 3 to mediate cisplatin-induced RPTC apoptosis. Finally, ER-iPLA(2) seems to be regulated by protein kinase C.     

Differential roles for cytosolic and microsomal Ca2+-independent phospholipase A2 in cell growth and maintenance of phospholipids

Physiological roles of microsomal (iPLA(2)gamma) and cytosolic (iPLA(2)beta)Ca(2+)-independent phospholipase A(2) were determined in two different epithelial cell models. R- and S-enantiomers of the iPLA(2) inhibitor bromoenol lactone (BEL) were isolated and shown to selectively inhibit iPLA(2gamma) and iPLA(2beta), respectively. The effect of these enantiomers on cell growth was assessed in human embryonic kidney 293 and Caki-1 cells using 3-(4-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT). S-BEL (0-5.0 microM) decreased MTT staining 35% after 24 h compared with control cells, whereas treatment with either R-BEL or R/S-BEL induced 15% decreases. Neither R-BEL nor S-BEL induced cell death as determined by annexin V and propidium iodide staining. Transfection of cells with iPLA(2)beta siRNA reduced MTT staining approximately 35%, whereas transfection of cells with iPLA(2)gamma siRNA only decreased MTT staining 10 to 15% compared with control cells. The effect of iPLA(2)beta and iPLA(2)gamma siRNA on cell number and protein was also determined, and iPLA(2)beta siRNA decreased cell number and protein 25% compared with control cells. In contrast, iPLA(2)gamma siRNA decreased cell number, but not cellular protein, compared with control cells. Selective inhibition of iPLA(2)beta, but not iPLA(2)gamma, decreased several arachidonic acid-containing phospholipids, including 16:1-20:4, 16:0-20:4, 18:1-20:4, and 18:0-20:4 phosphatidylcholine, showing that the ability of iPLA(2)beta inhibitors to decrease cell growth correlates with their ability to decrease arachidonic acid-containing phospholipids. These data show that iPLA(2)beta inhibition results in greater decreases in cell growth and proliferation than iPLA(2)gamma, identifies specific phospholipids whose expressions are differentially regulated by iPLA(2)beta and iPLA(2)gamma, and suggests novel roles for iPLA(2)beta in cell growth.     

Inhibition of Ca2+-independent phospholipase A2 decreases prostate cancer cell growth by p53-dependent and independent mechanisms

The mechanisms by which Ca(2+)-independent phospholipase A(2) (iPLA(2)) mediates cell growth in p53-positive LNCaP and p53-negative PC-3 prostate cancer cell lines were studied. Exposure of cells to the iPLA(2) selective inhibitor bromoenol lactone (BEL; 0-20 microM) induced concentration- and time-dependent decreases in cell growth based on 3-(4, dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide staining and cell number. Decreased cell growth was not caused by cell death as BEL exposure did not alter nuclear morphology or increase annexin V (apoptotic cell marker) or propidium iodide (necrotic cell marker) staining after 48 h. Decreased growth correlated to a G(1)/G(0) arrest in LNCaP cells and aG(2)/M arrest in PC-3 cells. In LNCaP cells, G(1) arrest was preceded by time- (0-48 h) and concentration-dependent (0-10 microM) increases in the expression of the tumor suppressor protein p53 and the cyclin-dependent kinase inhibitor p21. Increases in p53 expression preceded increases in p21 expression by 8 h. In LNCaP cells, BEL treatment decreased the expression of the p53 antagonist Mdm2, while increasing Akt phosphorylation. BEL treatment also increased Akt phosphorylation in PC-3 cells, but Mdm2 was not detected. The ability of BEL to increase Akt phosphorylation was inhibited by the phosphoinositide 3-kinase inhibitor LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one]. BEL treatment also decreased agonist-induced activation of the epidermal growth factor receptor. These data suggest that inhibition of iPLA(2) decreases prostate cancer cell growth by p53-dependent and independent mechanisms. Furthermore, alterations in Mdm2 and epidermal growth factor receptor activation following BEL exposure suggest novel roles for iPLA(2) in prostate cancer cell signaling.     

Reversibility of gelsolin/actin interaction in macrophages. Evidence of Ca2+-dependent and Ca2+-independent pathways

We have developed an immunoadsorption technique for quantitating EGTA-resistant gelsolin/actin complexes in macrophages extracted with Triton X-100. We report here that the proportion of gelsolin complexed irreversibly to actin is low in freshly harvested macrophages. The amount of the EGTA-resistant complex increases spontaneously during incubation of the cells in suspension at 37 degrees C, or after exposure to the Ca2+ ionophore ionomycin. On the other hand, exposure of suspended cells to the chemotactic oligopeptide, FMLP, or plating of the cells onto tissue culture dishes causes the EGTA-resistant complex to dissociate rapidly. Plating even prevents Ca2+ ionomycin-treated cells with elevated intracellular Ca2+ from inducing this complex. Therefore, our results suggest that macrophages possess a mechanism, not directly involving Ca2+, for dissociating actin/gelsolin EGTA-resistant complexes. This mechanism may be a Ca2+-independent signal for leukocyte activation.     

Inhibition of Ca2+-induced relaxation by oxidized tungsten wires and paratungstate

Recent studies of rat mesenteric arteries using a wire myograph detected decreased Ca2+ and acetylcholine-induced relaxation responses. Preliminary experiments indicated the reduced responses were associated with the tungsten wire used in the myograph system. Compared with earlier observations, arteries mounted on aged 28-microm tungsten wire showed decreased maximal Ca2+-induced relaxation responses of arteries precontracted with phenylephrine (91.9 +/- 1.5 versus 54.8 +/- 4.5%, p < 0.001) and reduced sensitivity to Ca2+ (ED50 = 1.65 +/- 0.07 versus 4.58 +/- 0.16 mM, p < 0.001). Similar shifts were seen for acetylcholine. When the surface of the wire was cleaned by abrasion with fine sandpaper, both the ED50 for Ca2+ and maximal relaxation significantly improved. An enhanced sensitivity to Ca2+ was also seen when arteries were mounted on newly purchased 14-microm tungsten or 14-microm 24K gold wire with the rank order: 14-microm gold > 14-microm tungsten > 28-microm aged tungsten wire. Laser Raman spectral analysis of the aged 28-microm tungsten wire showed that the surface was in an oxidized state that shared spectral characteristics with the paratungstate [W12O42](-12) anion. The effect of the paratungstate anion on arterial relaxation was therefore tested. Paratungstate, but not the structurally dissimilar tungstate and metatungstate anions, significantly reduced the sensitivity and magnitude of relaxation induced by Ca2+ and to a lesser extent, relaxation induced by acetylcholine. To learn whether paratungstate inhibits relaxation through the generation of oxygen radicals, the effect of the superoxide dismutase mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (1 mM) was assessed and found to have no effect. Since Ca2+-induced relaxation is inhibited by iberiotoxin, the effect of paratungstate on K+ channel activity was assessed. Paratungstate had no effect on currents through large conductance, Ca2+-activated K+ channels in whole-cell recordings from vascular smooth muscle cells, ruling out an action at the BK(Ca) channel. We conclude that: 1) surface oxidation of tungsten wire commonly used in wire myography significantly and adversely affects vascular responses to vasodilator compounds, 2) the effect is likely mediated by the paratungstate anion, and 3) the effects of the anion are not associated with free radical generation or K+ channel inhibition.     

Diminished inotropic response to amrinone in ventricular myocytes from myopathic hamsters is linked to depression of high-gain Ca2+-induced Ca2+ release

This study investigates whether amrinone (100-1000 microM), a phosphodiesterase-III inhibitor, can alleviate depression of contractions in ventricular myocytes from prefailure cardiomyopathic (CM) hamsters (80-100 days). Cell shortening and ion currents were measured in voltage-clamped cells at 37 degrees C. Normal myocytes exhibited low-gain Ca(2+)-induced Ca(2+) release (CICR) initiated by test steps from -40 mV and high-gain CICR initiated from more negative potentials. In normal myocytes, amrinone selectively increased contractions initiated by high-gain CICR (fractional shortening increased from 3.6 +/- 0.5% to 5.3 +/- 0.6%, 300 microM amrinone) but had no effect on low-gain CICR. Amrinone decreased L-type Ca(2+) current (I(Ca-L); -5.5 +/- 0.8 to -3.7 +/- 0.5 picoAmp/picoFarad, 300 microM amrinone). In contrast, in CM myocytes, high-gain CICR was virtually absent, and amrinone had no inotropic effect. Amrinone inhibited I(Ca-L) less in CM than normal myocytes. Sarcoplasmic reticulum (SR) Ca(2+) stores, assessed by caffeine, were significantly increased by amrinone in normal but not CM myocytes. Thus, the positive inotropic effect of amrinone in normal hamster myocytes was mediated by selective enhancement of high-gain CICR. This effect was not mediated by stimulation of I(Ca-L) because I(Ca-L) is inhibited by this drug in hamster. High-gain CICR, which is depressed in CM myocytes, cannot be restored by amrinone. However, minimal stimulation of adenylyl cyclase with forskolin restored the positive inotropic effect of amrinone in CM cells. This positive inotropic effect of amrinone may reflect increased SR Ca(2+) stores because increased stores accompanied the positive inotropic effect in normal myocytes but were absent in CM myocytes.     

The mitochondrial permeability transition pore in cell death: A promising drug binding bioarchitecture

Bioenergetic failure often features programmed cell death involved in some severe pathologies. When the cell is fated to die, the inner mitochondrial membrane becomes permeable to ions and solutes, due to the formation and opening of a channel known as mitochondrial permeability transition pore (mPTP). Up to now, the still-elusive mPTP structure and mechanism prevented any attempt to identify/design drugs to rule its formation and limit cell death. Latest advances, which strongly suggest that the F₁F_O-ATPase can coincide with the mPTP, open new perspectives in therapy. Compounds targeting and inhibiting cyclophilin D, a known mPTP promoter, could be exploited to block mPTP formation. Moreover, if the mPTP-F₁F_O-ATPase connection will be consolidated, selected F₁F_O-ATPase inhibitors could represent novel therapeutic options to attenuate mPTP-related diseases by directly acting on mPTP molecular mechanism. This intriguing perspective, which raises new hopes to counteract mPTP-related diseases, stimulates further studies to clarify the mPTP architecture and mechanism.     

Role of phospholipase C-beta in the modulation of epithelial tight junction permeability

The results presented in this study establish an association between phospholipase C-beta (PLC-beta) and tight junction permeability across Madin-Darby canine kidney (MDCK) cell monolayers, an in vitro model for epithelial tissue. These results further show that PLC-beta modulates tight junction permeability by affecting actin filament organization. Hexadecylphosphocholine (HPC) inhibited PLC-beta and increased tight junction permeability in MDCK cells. Interestingly, the analogs of HPC, a series of alkylphosphocholines containing various lengths of linear alkyl chains, inhibited PLC-beta and increased tight junction permeability with a wide range of potency. The potency of alkylphosphocholines as enhancers of tight junction permeability significantly correlated (p < 0.05) with their potency as PLC-beta inhibitors. U73122, a steroid derivative that is structurally unrelated to alkylphosphocholines, inhibited PLC-beta and increased tight junction permeability with potencies that fit into the correlation observed for the alkylphosphocholine series. U73122 and HPC induced disorganization of actin filaments in MDCK cell monolayers. The potencies to cause disorganization of actin filaments were consistent with the potencies of these agents as inhibitors of PLC-beta and enhancers of tight junction permeability. Furthermore, ATP, an activator of PLC-beta, attenuated U73122-induced increase in tight junction permeability as well as disorganization of actin filaments. These results provide strong evidence that PLC-beta inhibition leads to increased tight junction permeability across MDCK cell monolayers through disorganization of actin filaments.     

非钙依赖性调节肌球蛋白活性对维持平滑肌张力的影响

目的:揭示肌球蛋白轻链激酶(myosinlightchainkinase,MLCK)的一个活性片段对肌球蛋白功能的非钙依赖性调节作用,进一步完善平滑肌收缩的非钙依赖性调节机制研究,并为平滑肌系统疾病的治疗以及新药开发提供理论基础。方法:实验于2004-01/05在大连医科大学生化药理实验室完成。用胰蛋白酶(trypsin)水解MLCK,再经离子交换层析分离纯化而获得MLCK的活性片段(myosinlightchainkinasefragment,MLCKF,61ku);用west-ernblot检测MLCKF与完整MLCK的同源性;用SDS-PAGE及ScoinImage扫描软件检测肌球蛋白轻链的磷酸化程度;用分光光度法测定肌球蛋白Mg2+-ATP酶的活性。结果:在无Ca2+/CaM条件下,61kuMLCKF能轻度磷酸化20ku肌球蛋白轻链,并提高肌球蛋白Mg2+-ATP酶活性;其磷酸化效力强于无Ca2+/CaM时MLCK的磷酸化效力,但弱于有Ca2+/CaM时MLCK的磷酸化效力,且持续时间长,较稳定,不易被MLCK抑制剂ML-9抑制。结论:MLCKF能以非钙依赖性方式轻度磷酸化肌球蛋白轻链,并提高肌球蛋白Mg2+-ATP酶活性,对低钙时平滑肌张力的维持可能起一定作用。     

小剂量环孢霉素A抑制大鼠缺血性急性肾衰竭中肾小管上皮细胞线粒体通透性转运

目的研究小剂最环孢霉素A(CsA)对大鼠缺血性急性肾衰竭诱导的肾小管上皮细胞线粒体通透性转运的影响,探讨其抑制肾小管上皮细胞凋亡的作用机制。方法Wistar大鼠双侧肾动脉夹闭30min诱导缺血性急性肾衰竭。再灌注18h后留取肾组织及血液标本。比色法测定血清肌酐(SCr)浓度,原位末端标记(TUNEL)法检测肾小管上皮细胞凋亡情况,Westernblot测定肾组织中胞浆细胞色素C、caspase-3、9蛋白质表达。结果肾脏缺血30min／再灌注18h后大鼠SCr明显升高[(106．9±19．4)μmol／Lvs(56．5±7．1)μmol／L,P<0．05],肾小管上皮细胞凋亡数目显著增加[(20．14±3．70)％vs(0．99±0．17)％,P<0．05],肾组织胞浆细胞色素c、caspase-3、9表达增加(P<0．05);CsA能显著降低SCr[(87．5±18．5)μmol／L],减轻肾小管上皮细胞凋亡[(14．61±3．39)％],下调胞浆细胞色素C、caspase-3、9表达(P<0．05)。结论小剂量CsA能抑制大鼠缺血性急性肾衰竭引起的线粒体通透性转运,从而减轻肾小管上皮细胞凋亡,保护肾功能。     

Ginsenoside-Rh2-induced mitochondrial depolarization and apoptosis are associated with reactive oxygen species- and Ca2+-mediated c-Jun NH2-terminal kinase 1 activation in HeLa cells

We show here that Ca(2+) and reactive oxygen species (ROS) are involved in the up-regulation of c-Jun NH(2)-terminal kinase 1 (JNK1) activity during apoptosis induced by ginsenoside Rh2 (G-Rh2) in HeLa, MCF10A-ras, and MCF7 cells. Addition of antioxidants such as N-acetyl-l-cysteine or catalase attenuates G-Rh2-induced ROS generation, JNK1 activation, and apoptosis. The overexpression of catalase down-regulates caspase-3 and JNK1 activities. G-Rh2 treatment of cells results in mitochondrial depolarization, second mitochondrial activator of caspase release, and translocation of Bax into the mitochondria, and these events are inhibited by antioxidants. Ca(2+) is also involved in mitochondrial depolarization during G-Rh2-induced apoptosis. These results suggest that ROS and Ca(2+) are important signaling intermediates leading to stress-activated protein kinase/extracellular signal-regulated kinase kinase 1/JNK1 activation and depolarization of the mitochondrial membrane potential in G-Rh2-induced apoptosis.     

大鼠心肺复苏后神经细胞线粒体通透性转换孔的变化

目的 研究大鼠心肺复苏后神经细胞线粒体通透性转换孔(MPTP)的变化规律.方法 建立窒息联合冰氯化钾致大鼠心搏骤停、心肺复苏(CA/CPR)的动物模型,断头后提纯大脑皮层组织内线粒体,采用分光光度法测定线粒体MPTP在不同时相的开放程度.结果 心肺复苏后大鼠神经细胞线粒体功能明显受损,恢复自主循环(ROSC)后神经细胞MPTP持续处于开放状态,开放程度具有时间依赖性,具体表现为在ROSC后6 h以内开放程度保持低水平,6 h以后开始迅速大量开放,直至12 h开放程度达到最大,但并没有一直维持这一水平,而是在ROSC后24 h开放程度略有缩小,表明线粒体开始收缩,至48 h开放程度再次加大,72 h MPTP明显缩小,但未达到正常水平.结论 CA/CPR后大鼠线粒体MPTP出现明显规律性改变,复苏后12 h以内可能是改善复苏后脑功能的最佳治疗窗.     

Reactive oxygen species (ROS)-induced ROS release: a new phenomenon accompanying induction of the mitochondrial permeability transition in cardiac myocytes

We sought to understand the relationship between reactive oxygen species (ROS) and the mitochondrial permeability transition (MPT) in cardiac myocytes based on the observation of increased ROS production at sites of spontaneously deenergized mitochondria. We devised a new model enabling incremental ROS accumulation in individual mitochondria in isolated cardiac myocytes via photoactivation of tetramethylrhodamine derivatives, which also served to report the mitochondrial transmembrane potential, DeltaPsi. This ROS accumulation reproducibly triggered abrupt (and sometimes reversible) mitochondrial depolarization. This phenomenon was ascribed to MPT induction because (a) bongkrekic acid prevented it and (b) mitochondria became permeable for calcein ( approximately 620 daltons) concurrently with depolarization. These photodynamically produced "triggering" ROS caused the MPT induction, as the ROS scavenger Trolox prevented it. The time required for triggering ROS to induce the MPT was dependent on intrinsic cellular ROS-scavenging redox mechanisms, particularly glutathione. MPT induction caused by triggering ROS coincided with a burst of mitochondrial ROS generation, as measured by dichlorofluorescein fluorescence, which we have termed mitochondrial "ROS-induced ROS release" (RIRR). This MPT induction/RIRR phenomenon in cardiac myocytes often occurred synchronously and reversibly among long chains of adjacent mitochondria demonstrating apparent cooperativity. The observed link between MPT and RIRR could be a fundamental phenomenon in mitochondrial and cell biology.     

A role for N-arachidonylethanolamine (anandamide) as the mediator of sensory nerve-dependent Ca2+-induced relaxation

We tested the hypothesis that an endogenous cannabinoid (CB) receptor agonist, such as N-arachidonylethanolamine (anandamide), is the transmitter that mediates perivascular sensory nerve-dependent Ca2+-induced relaxation. Rat mesenteric branch arteries were studied using wire myography; relaxation was determined after inducing contraction with norepinephrine. Cumulative addition of Ca2+ caused dose-dependent relaxation (ED50 = 2.2 +/- 0.09 mM). The relaxation was inhibited by 10 mM TEA and 100 nM iberiotoxin, a blocker of large conductance Ca2+-activated K+ channels, but not by 5 microM glibenclamide, 1 mM 4-aminopyridine, or 30 nM apamin. Ca2+-induced relaxation was also blocked by the selective CB receptor antagonist SR141716A and was enhanced by pretreatment with 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (pefabloc; 30 microM), an inhibitor of anandamide metabolism. Anandamide also caused dose-dependent relaxation (ED50 =.72 +/- 0.3 microM). The relaxation was not inhibited by endothelial denudation, 10 microM indomethacin, or 1 microM miconazole, but was blocked by 3 microM SR141716A, 10 mM TEA, precontraction with 100 mM K+, and 100 nM iberiotoxin, and was enhanced by treatment with 30 microM pefabloc. Mesenteric branch arteries were 200-fold more sensitive to the relaxing action of anandamide than arachidonic acid (ED50 = 160 +/- 7 microM). These data show that: 1) Ca2+ and anandamide cause hyperpolarization-mediated relaxation of mesenteric branch arteries, which is dependent on an iberiotoxin-sensitive Ca2+-activated K+ channel, 2) relaxation induced by both Ca2+ and anandamide is inhibited by CB receptor blockade, and 3) relaxation induced by anandamide is not dependent on its breakdown to arachidonic acid and subsequent metabolism. These findings support the hypothesis that anandamide, or a similar cannabinoid receptor agonist, mediates nerve-dependent Ca2+-induced relaxation in the rat.     

磷脂酶A2在小儿急性肺损伤中的作用及意义

目的探讨磷脂酶A2在急性肺损伤(ALI)恶化进程中的作用及意义.方法结合危重病例评分,采用ELISA检测28例ALI患儿血清PLA2含量,并监测患儿脏器功能及动脉血气.结果 ALI患儿血清PLA2含量明显高于正常组(P＜0.001),低于多脏器功能衰竭(MOF)组(P＜0.01).结论 PLA2参与ALI的病理生理过程,与ALI的病情有关,并可作为早期警示ALI及MOF的实验室参数.     

Role in host cell invasion of Trypanosoma cruzi-induced cytosolic-free Ca2+ transients

Trypanosoma cruzi enters cells by a unique mechanism, distinct from phagocytosis. Invasion is facilitated by disruption of host cell actin microfilaments, and involves recruitment and fusion of host lysosomes at the site of parasite entry. These findings implied the existence of transmembrane signaling mechanisms triggered by the parasites in the host cells before invasion. Here we show that infective trypomastigotes or their isolated membranes, but not the noninfective epimastigotes, induce repetitive cytosolic-free Ca2+ transients in individual normal rat kidney fibroblasts, in a pertussis toxin-sensitive manner. Parasite entry is inhibited by buffering or depleting host cell cytosolic-free Ca2+, or by pretreatment with Ca2+ channel blockers or pertussis toxin. In contrast, invasion is enhanced by brief exposure of the host cells to cytochalasin D. These results indicate that a trypomastigote membrane factor triggers cytosolic-free Ca2+ transients in host cells through a G-protein-coupled pathway. This signaling event may promote invasion through modulation of the host cell actin cytoskeleton.     

有氧运动对大鼠骨骼肌线粒体Na+,K+-ATP酶和Ca2+-ATP酶及线粒体肿胀的影响

背景:研究表明有氧运动可提高线粒体功能,但在不同时期的作用特点还不明确.目的:观察不同周期有氧运动对大鼠骨骼肌线粒体Na+,K+-ATP酶和Ca2+-ATP酶以及线粒体肿胀的影响.方法:将SD大鼠随机分为正常对照组,有氧运动2,4和6周组.正常对照组不进行有氧运动,其余3组则参照BedfordTG标准,采用跑台运动方式,建立有氧运动模型进行相应的运动周期锻炼.测定各组大鼠骨骼肌线粒体Na+,K+-ATP酶和Ca2+-ATP酶的活性以及线粒体肿胀程度.结果与结论:有氧运动2周组各指标与对照组比较无差异.有氧运动4和6周组线粒体Na+,K+-ATP酶、Ca2+-ATP酶活性均增高(P<0.05),线粒体肿胀程度降低(P<0.05).实验结果表明,有氧运动可保护线粒体Na+,K+-ATP酶、Ca2+-ATP酶的活性,提高线粒体功能,但需要一定的时间积累.