| Objective:Radiationtherapy is the main treatment for Nasopharyngeal carcinoma and head and neck cancer. With conventional radiation way to nasopharyngeal carcinoma, the patients must bear external irradiation to both sides of the main salivary gland in radiation therapy and head and neck cancer, especially the parotidgland is located in the irrdiation target area. Radiation-induced salivary gland injury cause the gland dysfunction and reduce saliva secretion leading to xerostomia after radiotherapy in patients with serious impact on the quality of life.Lacking of effective prevention and control measures, must comprehensive symptomatic treatment is still used to radiation-induced xerostomia. Study of mechanisms against the radiation for salivary gland radiation-induced damage may find drugs to inhibit tumor and treat diseases, which has important clinical significance. To explore mechanism and effective doses of radiation-induced damage in this study, we observed firstly rat submandibular gland damage by different radiation-induced dose at early time(1-3 day), and observed secondly protective effect to the submandibular gland radiation damage with LJP drug intervention.To explore mechanism of LJP anti-human nasopharyngeal carcinoma cell, we observed lastly inhibition to human nasopharyngeal HONE1 and CNE2 cell proliferation and established up xenografts of human nasopharyngeal carcinoma HONE1 in nude mice model by detection technologies includining terminal deoxynuclotidyl transferase-mediated dTP nick end labeling(TUNEL), electronmicroscopy pathomorphology, immunohistochemistry, cell culture cytotoxicity test(MTT assay) and reverse transcriptase polymerase chain reaction(RT-PCR), which can provide experimental basis for salivary gland radiation and anti-nasopharyngeal carcinoma with LJP.Methods:The study is divided into three parts:Part one, the gene-related expression of apoptosis and morphology to different doses of 60Co y-ray irradiationon in rat submandibular gland. Part two,protective effect of LJP to destruction induced by 60Co y-rays in rat Submandibular gland.Parts three, inhibition to xenografts of human carcinoma Cell line in nude mice. The methods of part one,48 Wistarrats were randomly divided into:(1) normal control group(n=12); (2) radiotherapy 7.5Gy group(n=12); (3)radiotherapy 15Gy group (n=12); (4) radiation therapy 22.5Gy group (n=12). Radiotherapy group were irradiated by one-time total dose of y-ray irradiation according to group dose, while the control group were not irradiated.6 of each group in rat were sacrificed in 1 day and 3 days after irradiation All specimens were dehydrated, embeded in paraffin and serially cutted into 5μm slicesion.The expression of P53, Caspase-3 were detected by immunohistochemistry SP method and TUNEL assay cell apoptosis. All specimens taken randomly from a submandibular gland in each group were observed pathological changes of morphology by Transmission of electron microscope (TEM). The methods of part two,24 Wistar rats were randomly divided into:(1)normal control group(n=6); (2)LJP high-dose radiotherapy group(300mg/kg/one/day)(n=6); (3)the LJP low-dose radiotherapy group (30m/kg/one/day)(n=6); (4)radiotherapy control group(n=6). From 3 days before irradiation to 3 day after irradiation, each one was treated by intraperitoneal injection of LJP according to group dose.With one-time total dose of 15Gy of y-ray irradiation,each one was irradiated in radiotherapy group, while the control group were not irradiated.6 of each group in rat were sacrificed in 3 days after irradiation. All specimens were dehydrated, embeded in paraffin and serially cutted into 5μm slicesion. The expression of p53, Caspase-3 were detected by immunohistochemistry SP method and TUNEL assay cell apoptosis.15 Wistar rats were divided into three dose groups by 7.5Gy,15Gy,22.5 Gy for electron microscopy, and each group was divided into:(1)control group (n=1); (2)LJP high-dose radiotherapy group (n=1); (3) LJP low-dose radiotherapy group(n=1); (4) radiotherapy control group(n=1); (5)sucrose-negative control group (n=1), with above method of administration of radiotherapy, all rat were sacrificed in 3 days after irradiation and the morphology of rat submandibular gland were observed to understand pathological changes with drug intervention. The methods of part three, MTT assay was detected effect of LJP on inhibition to the proliferation of the human NPC cell line (HONE1 and of CNE2).30 male mice were randomly divided into groups:(1) normal control group(NS0.1ml/10g/d) (n=6); (2)LJP12.5 group (LJP12.5mg/kg/d) (n=6); (3)LJP25 group (LJP25mg/kg/d) (n=6); (4)LJP50 group (LJP50mg/kg/d)(n=6); (5) positive control group(DDP2mg/kg/d)(n=6). The drug intervention of LJP were used to detected effect on inhibition to human nasopharyngeal HONE1 and CNE2 cell proliferation, and the model of xenografts of human nasopharyngeal carcinoma HONE1 were established in nude mice, which were observed to detect the expression of Bax, Bcl-2,Caspase-3,8,9 messenger RNA (mRNA) with LJP drug intervention by RT-PCR.Results:Part I:①Gross specimen observation, there were differences on mental state, reaction of the outside and diet in rat after irradiation, the greater the radiation dose, the worse the mental status, ability to respond to outside and appetite.②p53 expression as follows:7.5 Gy Id,3d irradiation group compared with control group showed no statistically significant(p>0.05); 15 Gy 1d,3d irradiation group compared with control group increased statistically (p<0.01),22.5Gy 3d group compared with control group was statistically significant(p<0.05); 15 Gy group difference between Id and 3d was statistical significance(p<0.01), difference between 1d and 3d in 7.5Gy,22.5Gy groups were no significant differences(p>0.05).③Caspase-3 expression as follows: 1d,3d in 7.5Gy and 22.5Gy group,3d in 15Gy group compared with the control group was significant statistically significance(p<0.01); Id in 15Gy group compared with the control group was statistically significant (p<0.05). Difference between Id and 3d in 15Gy group was statistically significant (p<0.05); difference between 1d and 3d in 7.5Gy,22.5Gy group was not statistically significant(p>0.05).④TUNEL staining as follows:3d in 7.5Gy group compared with the normal group was statistically significant(p<0.05); Id in 15 Gy and 1d,3d in 22.5 Gy groups compared with the control group was statistically significant(p<0.05); 3d in 15 Gy group compared with the control group was statistically significant(p<0.01); difference between 1d and 3d in 7.5Gy,15Gy,22.5Gy groups was not statistically significant(p>0.05). Difference in 7.5Gy,15Gy,22.5Gy groups was not statistically significant(p>0.05).⑤Electron microscopy results:in 7.5Gy group, serous acinar cells showing as follows:the nuclear membrane integrity and nucleolus disappear, nuclear chromatin condensate integrated block and the set of edges in the nuclear membrane, the inside of the rough endoplasmic reticulum is slight expansion in the cytoplasm and the mitochondria is a little swelling, cristae blurred. Ductal cells showing that membrane shrinkage, the cytoplasm of mitochondrial swelling, cristae,reduced or disappear, the complete nucleus, nucleolus clear.15Gy group,serous acinar cells and ductal cells showing with cell shrinkage, increased electron density, the nuclear membrane integrity, the nucleolus disappears, the nuclear heterochromatin increased significantly, the condensate integrated block and the set of edges in the inside nuclear membrane; rough endoplasmic reticulum was significantly reduced and the expansion of the secretory granules also decreased significantly, and the particles inside the disorganized mitochondrial structure was mbiguous. 22.5Gy group, serous acinar cells and ductal cells showing that cell shrinkage, increased electron density, incomplete nuclear membrane, nucleus different chromatin coagulation integrated block and the edge set of the inside at the nuclear membrane, perinuclear gap widened; quality within the rough endoplasmic reticulum were significant reduction and dilated, the structural disorder, secretory granules disappeared, remnants of a small amount of mitochondria, nuclear heterochromatin began to increase. PartⅡ,①p53 expression as follows:radiotherapy group compared with control group was statistically significant(p<0.05); the difference in radiotherapy group was statistically significant(p<0.05), the apoptos of high-dose LJP group was the lowest value.②Caspase-3 expression results as follows:radiotherapy group compared with control group was statistically significant(p<0.05); difference between LJP30 and LJP300 group was not statistically significant(p>0.05), LJP30 and LJP300 group compared with radiotherapy group reduced statistically significant(p<0.05).③TUNEL staining results:LJP30 and LJP300 group compared with radiotherapy group was statistically significant (p<0.05); there were no statistically significant difference between the LJP30 and LJP300 group.④Electron microscopy results:the nucleus, mitochondria, endoplasmic reticulum and secretory vesicles structure of cells in 7.5 Gy,15Gy,22.5 Gy groups showing the injuries characteristics aggravated with increasing dose. Compared with radiation and sucrose control group, damage of cell structure in high and low LJP concentrations became lighter, which mean a protective effect on cell structure with LJP.PartⅢ:①MTT results, LJP IC10 of HONE 1 cells 76.85μg/ml, IC20 115.31μg/ml, IC30153.77μg/ml, IC50 240μg/ml; LJP CNE2 cells IC10 18.92μg/ml, IC20 57.38μg/ml, IC30 98.85μg/ml, IC50 180μg/ml.②Intervention results of the xenografts in nude mice:DDP group average tumor inhibition rate was 63.5%(compared with NS group, p<0.01), the LJP 12.5 group of transplanted tumors average tumor inhibition rate was 16.4% (with NS group, p>0.05) and inhibitory effect was not obvious, and the LJP 25 and LJP 50 group of transplanted tumor inhibition rates were 33.7%(compared with the NS group, p<0.05) and 47.0%(compared with NS group, p<0.01).③RT-PCR, Bax mRNA expression of LJP12.5, LJP25, LJP50, DDP2 group compared with the control group(NS) was statistically significant(p<0.05); difference of Bcl-2 mRNA expression in LJP12.5, LJP25, LJP 50, DDP2 group was statistically significant (p<0.05); the ratio of Bax/Bcl-2 in LJP50, LJP12.5, LJP25, DDP2 group compared with the control group was statistically significant (p<0.05); expression of Bax mRNA as follows:LJP25/LJP50> LJP12.5>DDP2, which difference was statistically significant(P<0.05); expression of Bcl-2 mRNA as follows:LJP25/LJP12.5>LJP50>DDP2, the difference was statistically significant (P<0.05). Expression of Caspase-3,9 mRNA in LJP12.5, LJP25, LJP50, DDP2 group compared with control group difference was statistically significant (p<0.05); LJP25, LJP50, DDP2 group of Caspase-8 mRNA expression and the control group the difference was statistically significance (p<0.05). Difference of Caspase-3 mRNA expression were statistically significant as follows:DDP2>LJP50>LJP25>LJP12.5; Caspase-8 mRNA expression between the NS and LJP12.5 group was not different (p>0.05) and no differences in LJP25 and LJP50 as well as LJP25 and DDP2; expression of Caspase-9 mRNA in LJP12.5, LJP25, LJP50 group was no difference, and were lower than expression of DDP2 treatment group.Conclusions:1.60Co y-ray irradiation could lead to apoptosis of rat submandibular gland in the early stage (1-3 days).2.60Co y-ray irradiation induced apoptosis of rat submandibular gland cells in a dose-effect relationship.3. Damage of submandibular gland cells could be induced by 15 Gy dose irradiation in the early stage (1-3 days),which has changes in the repair and apoptosis.4. Radiation-induced apoptosis of rat submandibular gland is inhibited by LJP.5. LJP can protect cell injury of rat submandibular gland induced by radiation.6. LJP inhibit human nasopharyngeal HONE1 and CNE2 cells proliferation. 7.LJP inhibit nasopharyngeal HONE1 and CNE2 cells of human in a concentration-dependent effect.8. One of the inhibition mechanism to xenografts of human nasopharyngeal carcinoma HONE1 is to promote apoptosis of cancer cells.
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