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Objective@#To investigate the protective effects and mechanism of keratinocyte growth factor (KGF) combined with hypoxia inducible factor-1α (HIF-1α) on intestinal crypt epithelial cells (IEC-6) of rats with hypoxia stress.@*Methods@#(1) The routinely cultured IEC-6 of rats were collected and divided into normoxia blank group, normoxia KGF group, normoxia HIF-1α group, and normoxia combine group, according to the random number table, and then the previous mediums were respectively replaced with dulbecco′s modified eagle medium (DMEM), medium with 0.5 ng/mL KGF, medium with 10.0 ng/mL HIF-1α, and medium with 0.5 ng/mL KGF and 30.0 ng/mL HIF-1α. And the cells were cultured in cell incubator with oxygen volume fraction of 21% for 24 hours. (2) Another batch of routinely cultured IEC-6 were collected and divided into normoxia control group, hypoxia control group, hypoxia KGF group, hypoxia HIF-1α group, and hypoxia combine group, according to the random number table. The previous mediums were replaced with DMEM, DMEM, medium with 0.5 ng/mL KGF, medium with 10.0 ng/mL HIF-1α, and medium with 0.5 ng/mL KGF and 30.0 ng/mL HIF-1α respectively. And then, the cells in normoxia control group were cultured routinely for 24 hours, and cells in the other 4 groups were cultured in cells incubator of 3 gases, with oxygen volume fraction of 5% for 24 hours. Cells cultured in normoxic and hypoxic incubators were collected, with 3 samples in each group, and morphological changes of cells were observed with optical microscope. Cells cultured in normoxic and hypoxic incubators were collected, with 3 samples in each group, and survival rates of cells were detected by cell count kit 8. Cells in normoxia control group and cells cultured in hypoxic incubator were collected, with 3 samples in each group. The cell cycle changes and apoptosis rates were detected by flow cytometer, the content of adenosine triphosphate (ATP) was detected by ultraviolet spectrophotometer, and protein expression of p53 was detected by Western blotting. Data were processed with one-way analysis of variance and least significant difference test.@*Results@#(1) After being cultured for 24 h, cells cultured in normoxic incubator grew well with oval or round shapes and clear cytoplasm, and cells cultured in hypoxic incubator showed irregular shapes such as fusiform or starlike shape, with black particle in cytoplasm. (2) After being cultured for 24 h, cell survival rates of normoxia blank group, normoxia KGF group, normoxia HIF-1α group, and normoxia combine group were (107.4±8.7)%, (109.8±2.9)%, (115.8±7.4)%, and (112.8±10.6)% respectively. There was no significantly statistical difference in general comparison of cell survival rates among the above groups (F=0.685, P=0.586). After being cultured for 24 h, cell survival rates of hypoxia control group, hypoxia KGF group, hypoxia HIF-1α group, and hypoxia combine group were (35.1±4.6)%, (52.9±6.8)%, (56.2±3.1)%, and (71.2±9.6)% respectively, which were significantly lower than (106.3±12.3)% of normoxia control group (P<0.001). Survival rates of cells in hypoxia KGF group, hypoxia HIF-1α group, and hypoxia combine group were significantly higher than the rate of cells in hypoxia control group (P=0.023, 0.009, <0.001). Survival rate of cells in hypoxia combine group was significantly higher than the rates of cells in hypoxia KGF group and hypoxia HIF-1α group (P=0.017, 0.045). (3) After being cultured for 24 h, percentage of cells in G1 phase in hypoxia control group was significantly higher than that of cells in normoxia control group (P=0.030), percentages of cells in S phase in hypoxia control group, hypoxia KGF group, and hypoxia HIF-1α group were obviously lower than the percentage of cells in normoxia control group (P=0.020, 0.031, 0.026), and percentages of cells in different phases in other groups were close to those of cells in normoxia control group (P=0.516, 0.107, 0.052, 0.985, 0.637, 0.465, 0.314, 0.591). After being cultured for 24 h, percentages of cells in G1 phase in hypoxia control group, hypoxia KGF group, and hypoxia HIF-1α group were obviously higher than the percentage of cells in hypoxia combine group (P=0.001, 0.030, 0.014), and percentages of cells in S phase in the above 3 groups were obviously lower than the percentage of cells in hypoxia combine group (P=0.001, 0.012, 0.010). (4) After being cultured for 24 h, compared with that of cells in normoxia control group, apoptosis rate of cells in hypoxia control group obviously increased (P=0.018), and apoptosis rate of cells in hypoxia combine group obviously decreased (P=0.008). After being cultured for 24 h, compared with that of cells in hypoxia control group, apoptosis rates of cells in hypoxia KGF group and hypoxia combine group obviously decreased (P=0.004, 0.001). Apoptosis rate of cells in hypoxia combine group was obviously lower than those of cells in hypoxia KGF group and hypoxia HIF-1α group (P=0.032, 0.002). (5) After being cultured for 24 h, compared with that of cells in normoxia control group, the content of ATP of cells in hypoxia combine group changed unobviously (P=0.209), and content of ATP of cells in the other groups obviously decreased (P= <0.001, 0.001, 0.002). Content of ATP of cells in hypoxia HIF-1α group and hypoxia combine group was obviously higher than that of cells in hypoxia control group (P=0.044, 0.001). Content of ATP of cells in hypoxia combine group was obviously higher than that of cells in hypoxia KGF group and hypoxia HIF-1α group (P=0.011, 0.020). (6) After being cultured for 24 h, protein expressions of p53 of cells in hypoxia control group, hypoxia KGF group, and hypoxia HIF-1α group were obviously higher than that of cells in normoxia control group (P<0.001), and protein expression of p53 of cells in hypoxia combine group was obviously lower than those of cells in hypoxia control group, hypoxia KGF group, and hypoxia HIF-1α group (P=0.001, 0.001, 0.002).@*Conclusions@#KGF combined with HIF-1α have significant protective effects on IEC-6 of rats with hypoxia stress, and can improve its survival in hypoxic environment by inhibiting cell cycle arrest, reducing the level of apoptosis, and increasing level of energy metabolism.
ABSTRACT
Objective To investigate the effect of 12-O-tetradecanoylophorbol-13-decanoate(TPD)on protection against acute intestinal radiation injury of mice and the possible mechanism.Methods Twenty female BALB/c mice aged 6-8 weeks were divided by random number table method into the control group and TPD groups(25,50,and 100 μg/kg). A radiation-damaged model of mice was irradiated by 10 Gy 60Co γ-rays,while the TPD groups were pretreated for 3 d with caudal vein injection before irradiation.The survival time of 20 days and the number of crypts at 3.5 days after irradiation were detected.Rat intestinal epithelial cells(IEC-6)were treated with 1 nmol/L TPD for 12 h before irradiation with 10 Gy 60Co γ-rays,and CCK-8 was used to detect the capability of cell proliferation at 0,1,2,3 and 4 d after irradiation. Results The mice in the control group survived for an average of 4.2 days,compared to 10 days in the optimal TPD group (100 μg/kg).The average number of crypts in the control group and the best TPD group was 11.0 ±1.3 and 35.1 ±1.9 respectively.The proliferation activity of IEC-6 was measured for four consecutive days.The average D value of the TPD groups was significantly higher than that of control.Conclusion TPD has a protective effect against acute intestinal radiation injury, and its protective mechanism may be achieved by promoting intestinal crypt cell proliferation and increasing the number of crypts in the intestine.
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Objective To study the protective function of resveratrol on radiation-induced small intestine injury and lethal effect in mice.Methods Mice were randomly divided into three groups:irradiation (IR) control,IR only,and IR + resveratrol.15 mice each group were irradiated on abdomen with 7.2 Gy γ-rays for cell lethal assay and 8 mice each group were irradiated with 6.5 Gy for small intestine injury assay.For the IR + resveratrol group,the mouse was given resveratrol by intragastric administration 24 h before irradiation and then was fed with resveratrol daily for 5 days.The control and IR alone groups were fed with placebo.After 30 days of IR,mouse survival rate was detected.For small intestine injury experiments,24 h after IR,the mice were terminated and the small intestines were treated with HE and immunohistochemical staining.Results Compared with the irradiation group,resveratrol increased mouse survival by 33.3％,decreased apoptosis in intestinal crypt cells (t =17.35,P ＜ 0.05),and increased Ki67 expression (t =13.62,P ＜ 0.05).Conclusion Resveratrol could protect small intestine injury from ionizing irradiation.
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PURPOSE: We examined the radioprotective effects of 5-androstendiol (5-AED), a natural hormone produced in the reticularis of the adrenal cortex, as a result of intestinal damage in gamma-irradiated C3H/HeN mice. MATERIALS AND METHODS: Thirty mice (C3H/HeN) were divided into three groups; 1) non-irradiated control group, 2) irradiated group, and 3) 5-AED-treated group prior to irradiation. Next, 5-AED (50 mg/kg per body weight) was subcutaneously injected 24 hours before irradiation. The mice were whole-body irradiated with 10 Gy for the histological examination of jejunal crypt survival and the determination of the villus morphology including crypt depth, crypt size, number of villi, villus height, and length of basal lamina, as well as 5 Gy for the detection of apoptosis. RESULTS: The 5-AED pre-treated group significantly increased the survival of the jejunal crypt, compared to irradiation controls (p<0.05 vs. irradiation controls at 3.5 days after 10 Gy). The evaluation of morphological changes revealed that the administration of 5-AED reduced the radiation-induced intestinal damages such as villus shortening and increased length of the basal lamina of enterocytes (p<0.05 vs irradiation controls on 3.5 day after 10 Gy, respectively). The administration of 5-AED decreased the radiation-induced apoptosis in the intestinal crypt, with no significant difference between the vehicle and 5-AED at 12 hours after 5 Gy. CONCLUSION: The results of this study suggest that the administration of 5-AED has a protective effect on intestinal damage induced by gamma-irradiation. In turn, these results suggest that 5-AED could be a useful candidate for radioprotection against intestinal mucosal injury following irradiation.
Subject(s)
Animals , Mice , Adrenal Cortex , Apoptosis , Basement Membrane , EnterocytesABSTRACT
Achievements of pharmacological studies on IEC-6 small intestinal crypt-like cells (IEC-6 cells) were reviewed. Based on the physiological research of IEC-6 cells, the pharmacological experiment model of IEC-6 cells was established and the pharmacological actions of spleen-strengthening and Qi-invigorating herbal medicine (SQHM) on intestinal epithelial stem cells were observed. Thiazolyl blue colorimetry (MTF), reverse transcription polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), high performance liquid chromatography (HPEC) and immune fluorescence labeling technology were applied in the experiments to observe the effect of SQHM on the proliferation, migration and differentiation of IEC-6. Its influences on the expression of villus protein, divalent mental transferrin, interleukin-6 (IL-6) and ornithine decarboxylase (ODC) and on ODC activity and putrescine content were also observed. The results showed that the extracted chemical components of Radix Codonopsis, Rhizoma Atractylodis Macrocephalae, Radix Astragali seu Hedysari and Radix Glycyrrhizae promoted or inhibited the proliferation, migration, differentiation and absorption function of IEC-6 cells by regulating ODC activity and putrescine production. It is indicated that IEC-6 cells are the pharmacological target of spleen-strengthening and Qi-invigorating herbal medicine.