Protective Effect Of Tubuloside B On TNFα-induced Apoptosis in Neuronal Cells

Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com

Min DENG, Jin-yuan ZHAO, Xiao-dong JU, Peng-fei TU, Yong JIANG, Zheng-bin LI

Apoptosis plays an important homeostatic role in several cellular processes as well as in the development of the nervous systems[1]. Programmed cell death may also contribute to various pathological conditions, such as cerebral ischemia[2,3], neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, Huntington disease, and amyotrophic lateral sclerosis[4-7]; as well as delayed encephalopathy after acute carbon monoxide poisoning[8]. Several lines of evidence have strongly suggested that oxidative stress, a cellular imbalance between the production and elimination of reactive oxygen species (ROS), led to neuronal apoptosis andnecrosis[9-11]. Therefore, it is valuable to identify compounds that can antagonize the deleterious action of ROSand act as an antioxidant to protect neurons from apoptosis.

Tubuloside B is one of the phenylethanoids isolated from the stems of Cistanche salsa, a Chinese herbal medicine, which is an important crude drug used as both anti-selenium and anti-fatigue agent[12]. Severalphenylethanoids have been shown to possess free radical scavenging properties and protect oxidative stress-induced toxic injuries[13-15]. Tumor necrosis factor-alpha (TNFα) is a toxic-interfering agent because of its important role in neurodegenerative diseases. Death by either necrosis or apoptosis has been seen in response to TNFα[16]. Many studies suggest that oxidative stress plays a critical role in the mechanism of TNFα-mediated cell apoptosis. Increased levels of ROS have been documented in cells following treatment with TNFα[17,18] andTNFα-mediated death can be inhibited by free radicalscavengers[19]. Thus, we studied whether tubuloside B can protect against TNFα-induced apoptosis and oxidative stress in cultured SH-SY5Y neuronal cells.

Cistanches salsa (CA Mey) G Beck, one species of Cistanches that belongs to the Orobanchaceae family, is a parasitic plant native in the northwest of China. The stem of this plant is an important traditional Chinese medicine and is used for kidney deficiency, female infertility, morbid leucorrhea, neurasthenia, and senile constipation due to colonic inertia. The major active constituents of this herb are phenylethanoid glycoside[28].In recent years, the results of several studies highlighted the function of tubuloside B in promoting various pharmacological and biological activities[13-15]. However, the cellular and molecular mechanisms that underlie the actions are not fully understood. The present study demonstrated that tubuloside B had significant neuroprotective effects on TNFα-induced apoptosis in SH-SY5Yneuronal cells by maintaining the mitochondrial function, decreasing the generation of ROS, reducing the level of intracellular calcium, and inhibiting caspase-3 activity through an antioxidation mechanism. These mechanisms may be through the individual neuroprotective effect of tubuloside B or its interaction with other factors and then may lead to a decreased ratio of apoptosis in cells.

Previous studies have shown that oxidants or pro-oxidants are important regulators of apoptosis and can induce apoptosis[29,30]. Oxidative stress is a common element of apoptosis induced by various stimuli such as TNFα and environmental toxin exposure, which usually do not exert a direct oxidant action. The central role of oxidative stress in apoptosis is strongly supported by the ability of various cellular antioxidants to block apoptosis induced by diverse agents[31]. Compounds with antioxidant properties could have a protective effect in different situations of cellular dysfunction through the scavenging of free radicals[32]. Moreover, several phenylpropanoid glycosides were reported to possess free radical scavenging properties and protect oxidative stress-induced toxic injuries. In line with this idea, we found that tubuloside B decreased the level of ROS induced by TNFα, it might protect neurons against apoptosis by directly scavenging intracellular reactive oxygen species.

Much evidence suggested that major alterations in mitochondrial function were critically involved in the apoptotic process[33]. Disorders of calcium homeostasis and alterations of mitochondrial membrane potential were found to promote the opening of the mitochondrial permeability transition pore (MPTP) or induce cytochrome c release through MPTP-independent mechanisms, being directly responsible for the activation of the apoptotic cascade and may precede nuclear signs of apoptosis[34]. These biochemical changes may result from alternations in the function of mitochondria[35]. The facts that tubuloside B inhibited the reduction of mitochondrial membrane potential and reduced the rise in intracellular calcium induced by TNFα suggest that tubuloside B may have the capacity to counteract the toxicity of TNFα by inhibiting the opening of MPTPand suppressing the dysfunction of mitochondria.

Outer stimuli can initiate apoptosis through the above mechanisms and may converge on the caspase pathway to execute the final phase of the apoptotic process[36]. The caspase family of proteases consists of at least 14 mammalian members that are constitutively expressed in almost all cell types as inactive proenzymes(zymogens) that become processed and activated in response to a variety of pro-apoptotic stimuli[37].Caspase-3 is a downstream member of the caspase cascade and acts as a central effector in the execution phase. When caspase-3 precursor protein CPP32 is activated by upstream signals such as the release of mitochondrial cytochrome c, the active caspase-3cleaves specific aspartate residues in proteins with various structural, housekeeping, and regulatory functions[38-40]. These proteolytic events can lead to cell apoptosis and contribute to DNA fragmentation and nuclear morphologic changes. Thus, substances that can inhibit the activity of caspase-3 might protect cells from apoptosis[2,31]. Since tubuloside B markedly inhibited the caspase-3 activity in the TNFα-treated cells, it has neuroprotective capacity.

In conclusion, tubuloside B had a multifunctional protective effect on damaged neurons. Because of its powerful anti-apoptosis and antioxidative stress activities, it might be for clinical use in neurodegenerative and neurologic disabilities involving neuron apoptosis.