| Objective:Through the detection of reactive oxygen species,inflammatory factors,apoptosis and proliferation factors in the parotid gland tissues of rats after X-ray irradiation,the protective effect of Sarcandra glabra on the parotid gland radiation damage in rats and its possible mechanism were studied.Methods :(1)Establishment of experimental animal model :120 male rats were randomly divided into 5 groups: blank control group(control group),single irradiation group(single irradiation group),low,medium and high dose Sarcandra glabra plus irradiation group(low,medium and high dose drug group),24 rats in each group.The rats in the low-dose,medium-dose and high-dose drug groups(corresponding to 6.7,13.4 and 26.8g Sarcandra glabra /kg/d,respectively)began to be fed with the corresponding dose of nine-dose tea one week before the irradiation,until the end of observation.Single 15-gy X-ray irradiation of parotid gland tissues was given under general anesthesia.The single irradiation group was given the same amount of normal saline within the corresponding time of administration of the drug group,and the irradiation method was the same as that of the drug group.The control group was only fed with the same amount of normal saline without other intervention.8 rats in each group were randomly captured on day 10 d,day 40 d and day 70 d after irradiation.After abdominal anesthesia,blood was collected from the abdominal aorta and tissues of the parotid gland were removed for further detection.(2)Enzyme-linked immunosorbent assay(ELISA)was used to detect the amount of reactive oxygen species(ROS)in the serum of each group of rats.Pathological changes and ultrastructural changes of parotid gland cells were observed by hematoxylin-eosin(HE)staining and transmission electron microscopy.The expression levels of TNF-α,PCNA,bcl-2 and Bax in parotid gland tissues were detected by immunohistochemical method,and the proliferation and apoptosis of parotid gland cells were detected by TUNEL.Results:(1)The experimental animal model of parotid gland radiation injury under the intervention of Sarcandra glabra was successfully established.(2)At the same time point,ROS content and TNF-expression in the single irradiation group were significantly increased compared with that in the control group(P < 0.05),while the above observation indexes in each drug group were in between.The values of the high-dose drug group were lower than those of the low-dose drug group(P < 0.05).The structure of the parotid gland in the control group was intact.Hyperemia,edema and infiltration of inflammatory cells were observed in the parotid gland tissues of the single irradiation group for 10 days after exposure,and tissue fibrosis was aggravated at 40 days after exposure.However,compared with the single irradiation group,the inflammatory response of the parotid gland tissues of each drug group in the same period was significantly reduced and negatively correlated with drug dose.The ultrastructure of parotid gland cells in the blank group was normal.There were only a few secretory granules in the single irradiation group,a large number of lysosomes appeared,and the structure of rough endoplasmic reticulum was destroyed,showing irregular arrangement.The chromatin in the nucleus is compact and scattered on the nuclear membrane,and the nucleus is very irregular,and the nucleoli are squeezed to the edge of the nuclear membrane.The secreted granules in the parotid gland cells in the high-dose drug group were significantly higher than that in the single irradiation dose group,but the quantity was not up to the normal level,and there was a very small amount of lysosomes.The chromatin in the nucleus is evenly dispersed on the nuclear membrane,and the nucleus is basically round.Mitochondria are basically normal.The level of cell damage in the low and medium dose drug groups was between that of the single irradiation group and that of the high dose drug group.Immunohistochemical results showed that,after irradiation,PCNA expression levels were significantly increased in all groups exposed to radiation compared with the control group,with statistically significant differences(P < 0.05).At 10 and 40 days after the irradiation,PCNA expression level in the intervention group with medium and high doses of Sarcandra glabra was significantly increased,with statistically significant differences(P < 0.05).Moreover,at 40 days after the irradiation,the effect was more obvious in the high-dose drug group than in the low-dose drug group,with statistically significant differences(P < 0.05).Compared with the control group,the expression level of bcl-2 in the single irradiation group was significantly reduced,and the difference was statistically significant(P < 0.05).After 10 days and 70 days of exposure,the expression level of bcl-2 in the treatment group with Sarcandra glabra was significantly higher than that in the single irradiation group,with statistically significant differences(P < 0.05).In the single irradiation group,the expression level of bcl-2 showed a trend of decreasing with the extension of time.Compared with the control group,Bax expression level in the single irradiation group was significantly increased,and the difference was statistically significant(P < 0.05).After 10 and 70 days of exposure,the Bax expression level in the treatment group with Sarcandra glabra was significantly lower than that in the single irradiation group,with statistically significant differences(P < 0.05).In the single irradiation group,Bax expression level showed an increasing trend with the extension of time.At 10 d and 40 d after irradiation,TUNEL staining results showed that the average absorbance(A)of positive cells indicating apoptosis in the parotid gland of rats after radiation was significantly increased compared with that of the control group,and the difference was statistically significant(P < 0.05).However,the mean absorbance(A)of positive cells was significantly reduced in the treatment group with high-dose Sarcandra glabra on day 10,and the difference was statistically significant(P < 0.05).Moreover,the effect was more significant in the treatment group with low-dose Sarcandra glabra on day 40,and the difference was statistically significant(P < 0.05).Conclusions:(1)The experimental animal model of parotid gland radiation damage can be successfully established by single 15 Gy vertical irradiation of parotid gland tissues with single anterior field on the abdominal surface.(2)Sarcandra glabra can effectively slow down the radiation damage of parotid gland by removing the ROS produced by the radiation of parotid gland in rats and reducing the inflammatory reaction.(3)The inhibitory effect of Sarcandra glabra on the apoptosis of parotid gland cells after radiotherapy may be realized by inhibiting the expression of Bax and upregulating the expression of bcl-2.The increased apoptosis of adenocytes induced by X-rays may be one of the mechanisms of parotid gland radiation damage.Sarcandra glabra can better protect parotid gland damage caused by radiation,and its possible protective mechanism is to inhibit excessive apoptosis of gland cells and promote cell proliferation and repair. |
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