| ObjectiveNowadays microwave technology is widely used for military, medical treatment,industry, communication and agriculture. It is inevitably that the organism is affectedby microwave radiation. It has been proved that microwave does harm tomulti-systems, and for example immune system which is also one of the focuses inthe field of bioelectromanetics. However, mechanism of injury induced by microwaveexposure on the immune system remains to be unclear and effective drugs withdefense to the injury has not been found. The aims of the study was to investigate theprecautionary effects of AduoLa Fuzhenglin (ADL) on rats' immune injury inducedby microwave exposure, the effects of NKG2D which is one of the important activereceptors of NK cell, and the role of NKG2D/ERK signal pathway in the procedure.Our study helps to expound the mechanism of injure induced by microwaveirradiation on the immune system and screen the defensive drugs.Materials and MethodsThis study included two parts: in vivo and in vitro experiments.(1)250male Wistar rats were randomized into2parts, one of which included125rats. Each part was randomized into5groups,3of which had been treated0.75,1.5and3g/(kg.d) ADL for two weeks or for four weeks before they were exposed to30mW/cm2microwave radiation for15min. The group without ADL treating orradiation was used as control, and that only with radiation as radiation group. Countsof leucocytes and lymphocytes, ratio of CD4+/CD8+, level of interleukin(IL)-2andIL-4in peripheral blood were detected by biochemical tests, flow cytometry andradioimmunoassay in7d and14d after microwave exposure. The changes in histologyand ultrastructure in rats' thymus and spleen were observed by light and transmissionelectron microscopy at6h,7d,14d after microwave exposure. The expression ofNKG2D in spleen was detected by Western Blot. (2) The cultured NK92cells were exposed to10,30,50mW/cm~2microwave for5minutes. Cell morphology was observed by inverted phase contrast microscope.Apoptosis, necrosis and cell cycles were detected by flow cytometry. Cytotoxicitywas tested by LDH ways. Real-time PCR, Western Blot and image analysis were usedto detect NKG2D and perforin mRNA and protein as well as ERK1/2, p-ERK1/2inNK92cells. The regulation of ERK signal pathway on perforin was investigated byintervention means of ERK inhibitor U0126.Results(1) The changes of immune function indexes in the rats' peripheral blood:When it cames to two weeks experiment, The ratio of CD4+/CD8+, counts ofleucocytes and lymphocytes decreased in14d after microwave exposure (p<0.05orp<0.01), which were lower than those in1.5g/(kg.d) group or in3g/(kg.d) group(p<0.05or p<0.01). Counts of leucocytes and lymphocytes in0.75g/(kg d) groupshowed no change when compared with the radiation group. The levels of IL-2orIL-4were of no significant difference among the5groups in7d or14d after exposure.Four weeks group had the same rule with two weeks one.(2) Histological and ultrastructural changes in the rats' thymus and spleen:When it came to two weeks experiment, obvious histological and ultrastructuraldamage such as apoptosis and necrosis of lymphocytes in rat spleen of radiation groupwere observed at6h and7d after microwave exposure. The pathological changes in1.5g/(kg.d) and3g/(kg.d) ADL groups were improved significantly at6h and7d afterstopping ADL. Structure injuries in0.75g/(kg d) group showed no change whencompared with the radiation group. The injuries recovered nearly at14d. Four weeksgroup had the same rule with two weeks one.(3) The expression changes of NKG2D in the rats' spleens: The expression ofNKG2D in the spleen was decreased significantly at6h (p<0.01) in the30mW/cm2group than normal control, and that was increased significantly in the1.5and3g/(kg·d) ADL prevention groups when compared to irradiation control (p<0.05orp<0.01).(4) The changes of morphology, rate of apoptosis and necrosis, and cellcycles of NK92cell: The outlines of cells became irregular and light refractiondecreased at1h in the10,30and50mW/cm2groups than control group, the ratio ofG0/G1increased significantly (p<0.01), and rate of S stage cells decreased significantly (p<0.05or p<0.01) and the rate of necroses increased (p<0.05or p<0.01).The ratio of G2/M and apoptosis increased at1h in the30and50mW/cm~2groupssignificantly (p<0.05or p<0.01). The rate of apoptosis increased at24h in the30and50mW/cm2groups than control group (p<0.05).(5) The changes of cytotoxicity of NK92cell: Cytotoxicity of NK92againstK562decreased at1h in the30and50mW/cm2groups than control group (p<0.01).(6) The changes of NKG2D expression in the NK92cell line in protein andmRNA levels: The expression of NKG2D decreased significantly at6h in bothmRNA and protein levels in the30mW/cm2group than the control group (p<0.05orp<0.01), and insignificantly at12h.(7) The changes of perforin expression in the NK92cell line in protein andmRNA level: The mRNA level of perforin decreased significantly at1h in the30mW/cm2group than the control group (p<0.05), and the protein level decreasedsignificantly at1h and6h respectively (p<0.05or p<0.01), and insignificantly at12h.(8) The changes of p-ERK1/2expression in NK92cell line: The expression ofp-ERK1/2decreased significantly at1h in the30mW/cm2group than the controlgroup (p<0.01).(9) The changes of p-ERK1/2and perforin after U0126Intervention: Theexpression of p-ERK1/2and perforin decreased significantly in U0126interventiongroup than the irradiation group (p<0.05or p<0.01).Conclusions(1) Being given1.5,3g/(kg d) ADL for two weeks or for four weeks is approvedto protect against immune injury caused by microwave exposure. The injuries can notbe protected obviously by being given0.75g/(kg d) ADL for two weeks or for fourweeks.(2) Being given ADL for two weeks has the same effect with for four weeks.1.5g/(kg.d) ADL is the effective and economical dosage. Two weeks is the bestdelivery cycle.(3)30or50mW/cm2microwave irradiation leads to damages of NK92cell line,which is displayed as cell morphology becoming irregular, rate of cell apoptosis andnecrosis increasing, and activity of proliferation and cytotoxicity decreasing in detail,but10mW/cm2alters apoptosis and killing activity of NK92cell line insignificantly.(4)30mW/cm2microwave irradiation leads to down-expression of NKG2D and perforin both in protein and mRNA levels.(5) Activation of ERK signal pathway up-regulates expression of perforin inNK92cell line after microwave exposure, while NKG2D/ERK/perforin pathway isinhibited by microwave irradiation.(6) The precautionary effect of ADL against microwave irradiation on immunedamage is supposed to attribute to promotion of NKG2D expression and activation ofNKG2D/ERK/perforin signal pathway.
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