RNA interference experiments were performed as it was previously described [ 27 ]. For cell cycle analysis, all floating and adhering cells were trypsinized and then washed two times with cold PBS. Our goal was to introduce various levels of oxidative stress. By our definition mild oxidative stress does not induce cell death mechanism; while excessive level of oxidative stress turns on the self-killing mechanism.
A remarkable tendency to cell cycle arrest was already observed after 3 hours assuming a relatively quick cellular oxidative stress-response mechanism. The propidium iodide staining indicated that cell cycle machinery already answered to a minimal oxidative treatment, but full cell cycle block cannot be observed in this case.
Therefore we can distinguish an early stage of treatment when the cell response mechanism is just turned on in oxidative stress.
Hesperidin alleviates zinc oxide nanoparticle induced hepatotoxicity and oxidative stress
After fixation, cell cycle analysis was performed with propidium iodide staining. Curve fitting and calculating the different phases of cell cycle were measured by FlowJo Software. B Summary table of the cell cycle distribution in different samples. C Overlay histogram of the cell cycle analysis was also made by FlowJo Software. Although significant cell cycle arrest was not detected on population level upon short oxidative stress, we intended to check whether cell cycle machinery has already started to induce its response mechanism on molecular level.
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Since Pyo et al. GAPDH was used as a housekeeping gene. GAPDH was used as a loading control. For each of the experiments, three independent measurements were carried out. These results imply that diminish of Cyclin D1 is not required at early stage of cell cycle response upon mild oxidative stress.
Pharmacology of Free Radicals and the Impact of Reactive Oxygen Species on the Testis
Rather the control network requires some other mechanism to block cell cycle progressing. In this case Cyclin D1 level did not change after 1. However our data also indicate that Cyclin D1 level is not affected at early stage of mild oxidative stress. In our lab it got emerged that this key cyclin of cell cycle machinery is regulated with a much quicker process e.
It is well-known that Cyclin D1 is controlled by various inhibitors at the different phases of cell cycle progress. While stoichiometric inhibitors p15 and p16 have essential roles in G1 phase; both p21 and p27 inhibit Cyclin D1 in G2 phase [ 31 — 33 ]. Therefore we assume that Cyclin D1 activity is controlled by some of these inhibitors at early stage of oxidative stress. Cells growing 0. A Testing the effectiveness of RNA silencing. Next, we followed the Cyclin D1 inhibitors i. As shown in Fig 3B and 3C , the level of p15, p21 and 27 resulted in a time-dependent increase, suggesting their essential role with respect to oxidative stress.
These results assume that Cyclin D1 might be regulated by their stoichiometric inhibitors at mild level of TBHP and the induction of cell cycle block might be achieved by this posttranslational modification at early stage of oxidative stress. Besides that, PERK silencing did not have any effect on p16 protein level, suggesting that p16 was not part of the oxidative stress response mechanism. Collectively, these results suggest that PERK has an essential role in controlling cell cycle progression via inducing Cyclin D1 inhibitors at initial stage of permanent mild oxidative stress. Next, our goal was to verify the role of NRF2 in cell cycle progression upon oxidative stress.
Cullinan et al.
Besides the down-regulation of Cyclin D1 inhibitors i. However, it was not clear whether NRF2 is able to regulate the stoichiometric inhibitors of Cyclin D1 to induce cell cycle arrest upon oxidative stress. Recently, by a global mapping of binding sites for NRF2 Malhotra et al. They also claimed that NRF2 might have a positive effect on these stoichiometric inhibitors of Cyclin D1, although further studies were also suggested.
Therefore we investigated the direct role of NRF2 on Cyclin D1 inhibitors p15, p16, p21 and p27 upon oxidative stress. While the protein level of p15, p21 and p27 has significantly increased upon prolonged oxidative stress in a time-dependent manner, we observed the complete diminish of these stoichiometric inhibitors in NRF2-silenced cells Fig 4B and 4C. These results imply that NRF2 is required for initiation of early stage of cell cycle block to induce Cyclin D1 inhibitors upon mild oxidative stress.
Finally, we attempted to determine whether the activation profile of Cyclin D1 inhibitors shows any differences at early stage of mild and excessive oxidative stress levels. In contrast, p15, p21 and p27 levels resulted in a drastic increase at both mild and excessive oxidative stress levels, meanwhile p16 level was not affected at all. These results further confirm that although p16 is a well-known inhibitor of Cyclin D1, it does not have any role in controlling the early stage of cellular stress-response mechanism with respect to oxidative stress. C The hypothetic control network of cell cycle regulatory system with respect to oxidative stress.
Our results suggest that the posttranslational regulation of Cyclin D1 by its inhibitors always occurs at early stage of oxidative stress cell supposing that besides controlling Cyclin D1 level its down-regulation is also essential. Oxidative stress generated by internal metabolism or environmental toxicant exposure affects many cellular functions, including various signalling pathways.
The transcription factor NRF2 is a key element of the main signalling pathway responsible to cell defence against oxidative stress. NRF2 is an emerging regulator of cellular resistance to oxidative attacks by controlling the transcription of hundreds of mainly cytoprotective genes. The role of NRF2 in maintaining the cellular redox balance is deeply explored, although its effect in cell cycle progression upon oxidative stress is not well-studied yet. Recently Pyo et al. Since it has been also shown that NRF2 activity is regulated by PERK [ 24 ], in this report we explored whether this cell cycle regulation is NRF2-dependent or not with response to oxidative stress.
Using HEKT cell line we successfully set up a protocol when the initiation stage of cell cycle arrest was detected during oxidative stress. With TBHP we could find proper treatment when cells seemed to be already committed to cell cycle arrest, but has not blocked yet in G2 phase Fig 1. Pyo et al have already shown that Cyclin D1 quickly got decreased at oxidative stress resulting in a cell cycle block in G2 phase [ 12 ].
We observed similar depletion of Cyclin D1 when the cells were treated with high level of TBHP; however no Cyclin D1 degradation was detected upon mild level of oxidative stress Fig 2. This suggests that Cyclin D1 might get accumulate at the early stage of mild oxidative stress; therefore the control system requires a completely different mechanism to induce cell cycle block. Interestingly, the level of stoichiometric inhibitors of Cyclin D1 i.
This is the first study we have shown that cell cycle arrest got achieved via posttranslational modification of Cyclin D1 rather protein degradation upon mild oxidative stress. Our results revealed that the absence of either of these proteins has a dramatic effect on oxidative stress response mechanism suggesting the abolition of cell cycle block. Namely, stoichiometric inhibitors of Cyclin D1 i.
Cyclin D1 depletion was clearly observed by generating a high level of oxidative stress with H 2 O 2 addition [ 12 ]; however they did not check the effect of a much weaker oxidative exposure. In this study we reveal that PERK pathway by inducing its other downstream target NRF2 controls cell cycle progression both at permanent mild and short but excessive level of oxidative stress. Cyclin D1 degradation becomes significant even at early stage of excessive level of oxidative stress i. Here we assume that PERK controls cell cycle progression via both substrates i. At early stage of mild oxidative stress the control system quickly enhances the initiation of cell cycle arrest via enhancing Cyclin D1 inhibitors in an NRF2-dependent manner.
Since Cyclin D1 inhibitors got activated even at high level of oxidative stress Fig 5A and 5B , we suggest that NRF2-dependent stress response mechanism always plays an essential role in early stage of oxidative stress.
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Edited by Alexander Kokorin. Published: April 25th DOI: Introductory Chapter By Volodymyr I. Semchyshyn Open access peer-reviewed 2. Lushchak Open access peer-reviewed 3. Mohamed Open access peer-reviewed 5.
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Lemanski and Herbert Weissbach Open access peer-reviewed Valenzuela-Soto Open access peer-reviewed Petkova and Nuria Pujol-Carrion Open access peer-reviewed Teixeira Open access peer-reviewed The extract was administered with an intragastric tube daily at am. Since there was no reference compound or positive control available, two groups received only physiological saline solution, which was also the solution used to dissolve the extract, and were used as controls.
At the end of the 3-month study period, the animals were subdivided into acutely exercised and non-exercised groups. The rats were divided into 8 animals per group as follows:. Acute exercise. Under anesthesia with ip urethane 1. The time elapsed from the end of treadmill exercise to anesthesia and muscle excision was min. Soleus and red gastrocnemius are considered to be oxidative muscles since they are composed of type I and IIa fibers, whereas white gastrocnemius is a glycolytic muscle mainly composed of type IIb fibers, and plantaris is considered to be a mixed muscle composed of type I, IIa and IIb fibers.
Strenuous or exhaustive physical exercise increases free radical production; however, because of the difficulties in measuring free radicals by direct and reliable methods, it is necessary to use indirect methods such as MDA levels, GSH status and some enzymatic activities. Muscle from the right hind limb was weighed and homogenized in 0. Spectrophotometric assays were performed on a Hitachi U spectrophotometer Tokyo, Japan in 1-ml cuvettes, with a 1-cm light path. Reaction rates were proportional to protein concentrations. CS EC 4. Muscle samples 0.
After homogenization, the samples were incubated at room temperature and 2,4 dinitrofluorobenzene was added to attach a chromophore to the amino groups. Peroxidative damage to cell lipid constituents was determined by measuring MDA concentration in muscle homogenates by the method of Buege and Aust After centrifugation at g for 10 min, the absorbance of the supernatant was measured at nm. Two-way analysis of variance was performed on all variables. The significance of the difference between the means obtained was determined by the Newman-Keuls test.
GSSG levels varied, without any clear tendency to a change caused by the effect of treatment and exercise. Lipid peroxidation measured on the basis of MDA levels was higher in all muscles after acute exercise; however, ginseng extract had an important antioxidant effect after exhaustive exercise, as shown by the lower MDA levels Figure 1. Figure 1. Table 1. Oxidative enzyme activities of citrate synthase and 3-hydroxyacyl-CoA dehydrogenase in skeletal muscle of rats treated with ginseng extract. Table 2. Glutathione content of rat soleus, gastrocnemius red and white and plantaris skeletal muscles.
The purpose of this research was to study the effects of oxidative stress induced by exhaustive exercise on muscle mitochondrial function and antioxidant response after long-term administration of the standardized ginseng extract. The most important finding of this in vivo study was that ginseng administration decreased the free radical generation in skeletal muscles after exhaustive exercise. In vitro studies 7,8,14 have long reported that ginseng is a scavenger of free radicals and the present study showed this effect in rat tissues obtained in vivo.
Free radical damage in muscle is associated with multiple pathophysiological processes occurring in muscle diseases and with aging. Different types of skeletal muscles possess distinct morphological, physiological and biochemical properties. Mixed muscles rich in IIa fibers seem to be more susceptible to exercise-induced oxidative stress 1, Acute pre-treatment of mice with crude extract increased their endurance in the swimming and running tests, but the reason for this increase was linked to the availability of muscle substrates and their utilization Lee et al.
Long-term administration of ginseng extract inhibited decomposition of unsaturated fatty acid caused by iron- and hydrogen peroxide-induced lipid peroxidation. The antioxidant effect of ginseng extract may occur not only at the initiation of the radical chain reaction but also at the propagation stage 7.
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Ginseng administration significantly increased CS and HADH activities in oxidative muscles such as soleus and red gastrocnemius Table 1. These activities are focused on mitochondria as the major site of free radical generation. Ferrando et al. On the other hand, down-regulation in CS activity has been reported in muscle and liver measured by GSH depletion and mitochondrial oxidative damage Fu and Ji 34 showed that a high dose 2.
GSH is an important cytosolic component that protects against damage by oxidative stress. GSH levels have been reported to decrease during physical exercise, especially in the liver In agreement with Lee et al. However, after exhaustive exercise the animals treated with ginseng extract showed lower GSH levels. Both the workload involved and the duration of exercise may be important in eliciting disturbances in muscle GSH levels 25, A concomitant increase in GSSG was not observed.
This has been demonstrated in skeletal muscle, liver, heart, and erythrocytes Exhaustive exercises increase the concentration of lipid peroxides in skeletal muscle and plasma The effect of acute exercise on MDA levels seems to be similar in both human and rat models and in the present study MDA levels were significantly increased in all muscles studied after exhaustive exercise, in agreement with other reports 17, Concerning membrane integrity and damage induced by free radicals, ginseng may stabilize the lipid structure of the membrane perturbed by the attack of free radicals.
The muscles showing the highest protection through decreases in MDA levels were the soleus and the red gastrocnemius. These muscles are mainly composed of type I and IIa fibers and show the highest antioxidant capacity Long-term administration of ginseng extract exhibited antioxidant capacity. Since free radicals have a very short half-life, we measured antioxidant capacity indirectly by determining MDA levels.
According to Jiang et al. In contrast, in a study of Vietnamese ginseng, Huong et al. A possible mechanism that could be invoked to explain the suppression of lipid peroxidation is through the inhibition of enzymes such as lipoxygenase involved in the metabolism of unsaturated fatty acids such as arachidonic acid to prostaglandins. Ginseng has well-known anti-inflammatory actions 12,38 , an effect probably caused by a decrease in phospholipase A 2 activity 39 and by the reinforcement of muscle fiber membranes, as also observed for prednisone, another anti-inflammatory agent Attele et al.
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D. Allan Butterfield | Chemistry
The long-term administration of ginseng extract was able to protect muscle from exercise-induced oxidative stress independent of fiber type but the mechanism of the antioxidant activity of ginseng extract is still unclear. In the present investigation, we studied the overall effects of a standardized ginseng extract.
The antioxidant action of ginseng is complex since ginsenosides and other constituents of ginseng may show many kinds of biological activity. In fact, ginseng extract contains several groups of compounds other than ginsenosides, such as phenolic substances maltol, salicylic acid, vanillic acid , with direct and indirect consequences in terms of antioxidant activity 6, Therefore, the exact mechanism of action of ginseng remains unclear because of the complex composition of the extract. Actually it is important to study the role of compounds of nutritional or pharmacological relevance by focusing on the potentiation of cellular antioxidant defense against toxins and various diseases.
The model established in the present study to induce oxidative stress and muscle tissue damage was exhaustive exercise.