| Objectives: Radiation therapy is an essential treatment for head and neck cancer,but salivary glands are damaged inevitably in the meantime.DNA is the principal intracellular target of radiation.DNA double-strand breaks may compromise the genome’s stability and even lead to cell death.Actin-related protein 2/3 complex(ARP2/3 complex)drives DNA breaks for repair through nucleating actin structures,but its role in the irradiated submandibular gland(SMG)has not been reported.Our previous study revealed that phenylephrine(PE)has the property of alleviating cellular oxidative stress and maintaining mitochondrial homeostasis in irradiated SMG.However,the role of PE in DNA damage is still unclear.Therefore,we investigated the effects of PE and ARP3(the essential subunit of ARP2/3 complex)on DNA damage in irradiated SMG cells.Materials and Methods:(1)In vitro: Rat SMG epithelial polarized cell line SMG C6 was used and divided into four groups: Control group,PE group(PE 10 μM),IR group(X-ray 2.5,5,10,and 15 Gy),and PE pretreatment group(PE 10 μM was given 0.5 h before radiation).The level of cellular reactive oxygen species(ROS)was detected with the ROS fluorescent probe;immunoblotting was performed to detect the expression ofγH2AX and ARP3 after radiation;the localization of ARP3 protein in SMG cells was detected by immunocytochemistry.(2)In vivo,24 Wistar rats were randomly divided into four groups(six in each group): Control group,PE group(intraperitoneal injection,PE 5 mg/kg),IR group(X-ray 20 Gy),and PE pretreatment group(PE 5 mg/kg was injected intraperitoneally 0.5 h before irradiation).The SMG tissue was removed on day seven after radiation.Total protein from SMGs was extracted for proteomics analysis;immunohistochemistry was performed to detect the distribution of ARP3 protein in SMG tissue.Results:(1)In vitro: Cellular ROS detection showed that compared with the control group,the cellular ROS level in IR group increased by 168.66%(P < 0.05)at 1 h after X-ray(10 Gy).Pretreatment with PE reduced cellular ROS level by 108.44%(P < 0.05)compared with the IR group.The results of γH2AX from western blot showed that compared with the control group,the expression of γH2AX protein in IR group increased by 185.44%,552.51%,590.49% and 972.71%(P < 0.05)respectively at 1 h after X-ray(2.5,5,10,and 15 Gy);with PE pretreatment,the expression of γH2AX were significantly reduced by 146.67%,389.68%,247.25% and 419.79%(P < 0.05)respectively.The results from immunocytochemistry showed that ARP3 protein is located at both cytoplasm and nucleus in rat SMG cells.The results of ARP3 from western blot showed that compared with the control group,the expression level of nuclear ARP3 in IR group decreased by 9.83%(P < 0.05)at 4 h after X-ray(10 Gy),which were increased by 12.85%(P <0.05)with PE pretreatment.(2)In vivo,the results from proteomics showed that compared with the control group,the expression of ARP3 protein decreased by 31.71%(P < 0.05)after irradiation,which increased by 9.42%(P< 0.05)with PE pretreatment.The results of immunohistochemistry showed that ARP3 protein was distributed in rat submandibular gland tissues and the expression level of ARP3 in the IR group was 48.9% lower than that in the control group(P < 0.05),while pretreatment with phenylephrine increased the radiation-reduced ARP3 expression level by 31.7%(P < 0.05)Conclusions: PE could prevent radiation-induced DNA damage in SMG cells.The mechanisms may be related to activation of ARP3 and alleviation of cellular oxidative stress.This study will provide a new target as well as experimental and theoretical basis for PE in preventing radiation sialadenitis. |
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