Identification of G2/M phase transition by sequential nuclear and cytoplasmic changes and molecular markers in mice intestinal epithelial cells.

Although the regulatory network of G2/M phase transition has been intensively studied in mammalian cell lines, the identification of morphological and molecular markers to identify G2/M phase transition in vivo remains elusive. In this study, we found no obvious morphological changes between the S phase and G2 phase in mice intestinal epithelial cells. The G2 phase could be identified by Brdu incorporation resistance, marginal and scattered foci of histone H3 phosphorylated at Ser10 (pHH3), and relatively intact Golgi ribbon. Prophase starts with nuclear transformation in situ, which was identified by a series of prophase markers including nuclear translocation of cyclinB1, fragmentation of the Golgi complex, and a significant increase in pHH3. The nucleus started to move upwards in the late prophase and finally rounded up at the apical surface. Then, metaphase was initiated as the level of pHH3 peaked. During anaphase and telophase, pHH3 sharply decreased, while Ki67 was obviously bound to chromosomes, and PCNA was distributed throughout the whole cell. Based on the aforementioned markers and Brdu pulse labeling, it was estimated to take about one hour for most crypt cells to go through the G2 phase and about two hours to go through the G2-M phase. It took much longer for crypt base columnar (CBC) stem cells to undergo G2-prophase than rapid transit amplifying cells. In summary, a series of sequentially presenting markers could be used to indicate the progress of G2/M events in intestinal epithelial cells and other epithelial systems in vivo.

P68 RNA helicase as a molecular target for cancer therapy.

The DEAD-box family of RNA helicase is known to be required in virtually all cellular processes involving RNA, and p68 is a prototypic one of the family. Reports have indicated that in addition to ATPase and RNA helicase ability, p68 can also function as a co-activator for transcription factors such as estrogen receptor alpha, tumor suppressor p53 and beta-catenin. More than that, post-translational modification of p68 including phosphorylation, acetylation, sumoylation, and ubiquitylation can regulate the coactivation effect. Furthermore, aberrant expression of p68 in cancers highlights that p68 plays an important role for tumorgenesis and development. In this review, we briefly introduce the function and modulation of p68 in cancer cells, and put forward envisagement about future study about p68.

Experimental research on the management of combined radiation-burn injury in China.

Combined radiation-burn injury can occur in people exposed to nuclear explosions, nuclear accidents or radiological terrorist attacks. Using different combined radiation-burn injury animal models, the pathological mechanisms underlying combined radiation-burn injury and effective medical countermeasures have been explored for several years in China, mainly at our institute. Targeting key features of combined radiation-burn injury, several countermeasures have been developed. Fluid transfusion and the calcium antagonist verapamil can prevent early shock and improve myocardial function after combined radiation-burn injury. Recombinant human interleukin 4 (rhIL-4) is able to effectively reduce bacterial infection and increase intestinal immunological ability. Chitosan-wrapped human defensin 5 (HD5) and glucagon-like peptide 2 (GLP-2) nanoparticles can increase the average survival time of animals with severe combined radiation-burn injury. After treatment by cervical sympathetic ganglia block (SB), hematopoietic function is promoted and the release of inflammatory cytokines is suppressed. The optimal time for escharectomy and allo-skin grafting is 24 h after injury. Transfusion of irradiated (20 Gy) or stored (4°C, 7 days) blood improves the survival of allo-skin grafting and allo-bone marrow cells. In conclusion, as our understanding of the mechanisms of combined radiation-burn injury has progressed, new countermeasures have been developed for its treatment. Because of the complexity of its pathology and the difficulty in clinical management, further efforts are needed to improve the treatment of this kind of injury.

Protective effect of atorvastatin on radiation-induced vascular endothelial cell injury in vitro.

Vascular endothelial cells are very sensitive to ionizing radiation, and it is important to develop effective prevent agents and measures in radiation exposure protection. In the present study, the protective effects of atorvastatin on irradiated human umbilical vein endothelial cells (HUVEC) and the possible mechanisms were explored. Cultured HUVEC were treated by atorvastatin at a final concentration of 10 µ mol/ml for 10 minutes, and then irradiated at a dose of 2 Gy or 25 Gy. Twenty-four hours after irradiation, apoptosis of HUVEC was monitored by flow cytometry, and the expression of thrombomodulin (TM) and protein C activation in HUVEC was respectively assessed by flow cytometry and spectrophotometry. After treatment with atorvastatin for 24 h, the rate of cell apoptosis decreased by 6% and 16% in cells irradiated with 2 Gy and 25 Gy, respectively. TM expression increased by 77%, 59%, and 61% in untreated cells, 2 Gy irradiation-treated cells, and 25 Gy irradiation-treated cells, respectively. The protein C levels in 2 Gy and 25 Gy irradiation-treated cells were reduced by 23% and 34% when compared with untreated cells, but up-regulated by 79% and 76% when compared with cells which were irradiated and treated with atorvastatin. In conclusion, these data indicate that atorvastatin exerts protective effects on irradiated HUVEC by reducing apoptosis by up-regulating TM expression and enhancing protein C activation in irradiated HUVEC.

CXCR4 gene transfer enhances the distribution of dermal multipotent stem cells to bone marrow in sublethally irradiated rats.

Our previous study indicated that systemically transplanted dermal multipotent cells (DMCs) were recruited more frequently to bone morrow (BM) of rats with sublethal irradiation than that of normal rats, and the interactions between stromal-derived factor (SDF-1) and its receptor (CXC chemokine receptor 4, CXCR4) played an important role in this process. In the present study, we aimed to investigate whether CXCR4 gene transfer could promote the distribution of DMCs into irradiated BM and accelerate its function recovery. Firstly, adenovirus vector of CXCR4 (Adv-CXCR4) and green fluorescent protein (Adv-GFP) were constructed. Then male DMCs infected by Adv-CXCR4 (group A), or infected by Adv-GFP (group B), and non-infected DMCs (group C) were transplanted into irradiated female rats, and real-time polymerase chain reaction for the sex-determining region of Y chromosome was employed to determined the amount of DMCs in BM. The functional recovery of BM was examined by hematopoietic progenitor colonies assay. The results showed that the amount of DMCs in BM of group A was greater than that in group B and group C from day 5 after injury (P < 0.05), and the amount of CFU-F, CFU-E and CFU-GM were greater than that in group B and group C from day 14 after injury (P < 0.05). These findings suggest that DMCs infected by Adv-CXCR4 distributed more frequently to the bone marrow of sublethally irradiated rats and could accelerate hematopoiesis function recovery.

[Studies on the treatment of combined radiation-burn injury].

Combined radiation-burn injuries mainly occur under the circumstances of nuclear explosion, nuclear accident, nuclear terrorism, depleted uranium attack, as well as secondary injuries following attack on nuclear installation. Combination of burn and radiation injuries bring along more serious whole body damage, more complicated pathological mechanism and much more difficult management. Research progress on the pathological mechanism and medical management of several key links of combined injury were discussed in this paper. (1) Enhancement of early first aid and prevention of early death of wounded. (2) Damage and restoration of hemopoietic function. (3) Disturbance of immune function and prevention and treatment of infection (mainly on the intestinal mucosa immunity and enterological infection). (4) Management of burn wound. (5) The role of several important measures in the comprehensive treatment.

Recruitment of transplanted dermal multipotent stem cells to sites of injury in rats with combined radiation and wound injury by interaction of SDF-1 and CXCR4.

Systemic transplantation of dermal multipotent stem cells has been shown to accelerate both hematopoietic recovery and wound healing in rats with combined radiation and wound injury. In the present study, we explored the mechanisms governing the recruitment of dermal multipotent stem cells to the sites of injury in rats with combined injury. Male dermal multipotent stem cells were transplanted into female rats, and using quantitative real-time PCR for the sex-determining region of Y chromosome, it was found that the amounts of dermal multipotent stem cells in irradiated bone marrow and wounded skin were far greater than those in normal bone marrow and skin (P < 0.01). However, incubation of dermal multipotent stem cells with AMD3100 before transplantation, which specifically blocks binding of stromal cell-derived factor 1 (SDF-1) to its receptor CXCR4, diminished the recruitment of dermal multipotent stem cells to the irradiated bone marrow and wounded skin by 58 +/- 4% and 60 +/- 4%, respectively (P < 0.05). In addition, it was confirmed that the expression of SDF-1 in irradiated bone marrow and wounded skin was up-regulated compared to that in their normal counterparts, and in vitro analysis revealed that irradiated bone marrow and wounded skin extracts had a strong chemotactic effect on dermal multipotent stem cells but that the effect decreased significantly when dermal multipotent stem cells were preincubated with AMD3100 (P < 0.05). These data suggest that transplanted dermal multipotent stem cells were recruited more frequently to the irradiated bone marrow and wounded skin than normal bone marrow and skin and that the interactions of SDF-1 and CXCR4 played a crucial role in this process.

Effects of peritoneal lavage fluid from radiation or/and burn injured rats on the growth of hematopoietic progenitor cells.

PURPOSE: To evaluate the effects of peritoneal lavage fluids from radiation injury, burn injury and combined radiation-burn injury on the growth of hematopoietic progenitor cells (HPC). MATERIALS AND METHODS: Rats were divided into four groups: A radiation group (RG), a burn group (BG), a combined radiation-burn group (CRBG) and normal control group (NG). RG and CRBG rats were irradiated with 12 Gy, and burns of 30% total body surface area were generated in group BG and group CRBG. Peritoneal lavage fluids were collected and tested for their effects on the growth of erythrocyte progenitor cells or granulocyte-macrophage progenitor cells of BALB/c mice in vitro. RESULTS: The numbers of colony forming units-erythroid (CFU-E), burst forming units-erythroid (BFU-E) and colony-forming units-granulocyte-macrophage (CFU-GM) formed after treatment with lavage fluids from BG or CRBG were significantly higher than those from NG. However, fewer CFU-E, BFU-E or CFU-GM colonies were found after treatment with lavage fluid from the RG. In lavage fluid from BG and CRBG, the concentration of interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor alpha (TNFalpha) was increased in comparison to NG and RG. Treatment with these cytokines had similar promoting effects on the growth of hematopoietic colonies and neutralizing antibodies inhibited these effects significantly. CONCLUSIONS: Burns increase the responsiveness of the system and help the proliferation of hematipoietic progenitor cells, while radiation decreases all these responses relative to both the controls and the burn plus radiation group.

Effects of serum from rats with combined radiation-burn injury on the growth of hematopoietic progenitor cells.

BACKGROUND: This study aims to observe the effects of blood serum from rats with radiation injury, burn injury, and combined radiation-burn injury on the growth of hematopoietic progenitor cells and to explore the possible mechanisms. METHODS: Serum from rats with radiation injury, burn injury, and combined radiation-burn injury were collected at 3 hours, 12 hours, 24 hours, 48 hours, 72 hours, and 96 hours after injury and then was added to the culture medium to see its effect on the growth of hematopoietic progenitor cells (HPCs) at a final protein concentration of 10 microg/mL. Radioimmunoassay and enzyme-linked immunosorbent assay were employed to measure the level of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 in each group, and the effect of TNF-alpha and IL-6 on the growth of HPC was also observed. RESULTS: The number of HPCs colonies formed after addition of the serum from rats with burn or combined radiation-burn injuries was significantly higher than that from normal rats at 3 hours, 12 hours, 24 hours, 48 hours, 72 hours, and 96 hours after injury and reached its peak value at 24 hours after injury. However, fewer HPCs colonies were found after the addition of the serum from irradiated rats. At the same time, the levels of TNF-alpha and IL-6 in the serum of burn group and combined radiation-burn injury group were significantly higher than that of normal group, and much higher than that of the irradiation injury group (p < 0.01). Also, TNF-alpha and IL-6 demonstrated promoting effect on the growth of HPC. CONCLUSION: Serum from rats with burn injury and combined radiation-burn injury stimulates the growth of HPCs, while serum from irradiated rats shows inhibitory effects on the growth of HPCs. These effects may lie in the different level of TNF-alpha and IL-6 in the serum of each group.

Crucial role of SDF-1/CXCR4 interaction in the recruitment of transplanted dermal multipotent cells to sublethally irradiated bone marrow.

Our previous study indicated that dermal multipotent cells (DMCs) could engraft into bone morrow (BM) of rats with sublethal irradiation and promote hematopoietic recovery after being transplanted systemically, but the mechanisms determining the recruitment of DMCs to the irradiation injured BM remain unclear. In the present study, we investigated the role of stromal cellderived factor-1 (SDF-1)/CXCR4 interaction in this process. Male DMCs were isolated and transplanted into female rats systemically, and by employing quantitative real-time TaqMan polymerase chain reaction for the sex-determining region of Y chromosome, it was found that the amount of DMCs in BM of rats with sublethal irradiation was about 3 times more than that of normal rats (P < 0.01). Incubation of DMCs with AMD3100 before transplantation, which specifically blocks binding of SDF-1 to its endogenous receptor CXCR4, diminished recruitment of DMCs to the injured BM by 57.2 +/- 5.5% (P < 0.05). In addition, it was confirmed that the expression of SDF-1 in injured BM was up-regulated when compared with that in normal BM, and in vitro analysis revealed that BM extracts from irradiated rats had a strong chemotactic effect on DMCs, which decreased significantly when DMCs were pre-incubated with AMD3100 (P < 0.05). These data suggest that transplanted DMCs were recruited more frequently to irradiation-injured BM than normal BM and the interactions of SDF-1/CXCR4 played an important role in this process.

[Effects of blood serum from rats with combined radiation-thermal injury on the bone marrow hematopoietic progenitor cells growth].

To observe the effects of blood serum from rats with radiation injury, thermal injury and combined radiation-thermal lesions on growth of hematopoietic progenitor cells and the change of their serum cytokine levels, total body irradiation of rats was performed with 12 Gy gamma ray from a (60)Co source, and 30% total body surface area III degree thermal lesion on the back was inflicted with a 5 kW bromotungsten lamp. The blood serum from these animals was collected at 3, 12, 24, 48, 72 and 96 hours after injury. Then the blood serum was added to the culture medium of erythrocyte progenitor cells (CFU-E, BFU-E) or granulocyte-macrophage progenitor cells (CFU-GM) at final concentration of 10 microg/ml. The results showed that the colony number of CFU-E, BFU-E and CFU-GM formed after addition of the blood serum from rats with thermal or combined radiation-thermal injury was significantly higher than that from normal rats at 3, 12, 24, 48, 72 and 96 hours after injury and reached its peak value at 24 hours after injury (342.8, 261.6 and 228.4% respectively from burned rats, 252.4, 205.1 and 174.2% respectively from rats with combined radiation-thermal injury as compared with that of normal rats). However, a few CFU-E, BFU-E or CFU-GM formation was found after addition of the blood serum from irradiated rats. At the same time, the level of TNF alpha and IL-6 in serum of burn group and combined radiation-thermal injury group was markedly higher than that of normal group, even more higher than that of irradiation injury group (P < 0.01). It is concluded that the blood serum from rats with thermal lesion or combined radiation-thermal injury improves the growth of erythrocyte and granulocyte progenitor cells. On the contrary, the blood serum from the irradiated rats shows the inhibiting effects, definitely related to their serum cytokines changes.

[Effects of exogenerous nucleotides on the apoptosis of intestinal epithelial cells IEC-6].

OBJECTIVE: To investigate the effects of exogenous nucleotides on apoptosis of a normal rat small intestinal epithelial cell line, IEC-6. METHODS: Cultured IEC-6 cells were treated by four kinds of monophosphate nucleotides and their mixture prepared according to their composition in human milk, then the cell apoptosis was determined by flow cytometry measurement, morphologic characterization, and electron-microscope observation. RESULTS: IEC-6 cells treated with AMP or GMP showed a apotosis peak in flow cytometry measurement, but only AMP produce typical apoptosis characteristics in electron-microscope observation. Pyrimidine nucleotides (UMP and CMP)and nucleotides mixture could not induce apoptosis. However, UMP could significantly eliminate the apoptosis-inducing effects of AMP or GMP. CONCLUSION: Purine nucleotides induce apoptosis of IEC-6, inducing effects of purine nucleotides. pyrimidine nucleotides UMP could abolish the apoptosis-inducing effects of purine nucleotides.

[Effect of lipopolysacharide on the biological features and growth factor secretion power of U937 cell line].

OBJECTIVE: To investigate the effect of lipopolysacharide (LPS) in different concentrations on the biological features and growth factor secretion power of U937 cell line. METHODS: In vitro cultured U937 cells were stimulated by 0 (as control), 0.1, 1.0, 10.0, 50.0 and 100 micro g/ml LPS respectively for 24 hours. Thereafter, the cell proliferation ability was determined by MTT method. The cell apoptosis rate was determined by flow cytometry. The changes in the contents of transforming growth factor beta(1) (TGFbeta(1)) and vascular endothelial growth factor (VEGF) of the supernatant of the cell culture were assessed by ELISA. RESULTS: Apoptosis and TGFbeta(1) secretion could be induced by LPS in dose of 0.1 to 100 micro g/ml when compared with that without LPS challenge (P < 0.05 - 0.01). In detail, LPS in lower dose (0.1, 1.0 and 10.0 micro g/ml) could promote the proliferation of U937 (P < 0.05 - 0.01) but exerted no effect on VEGF secretion. In contrary, LPS in high dose (50 and 100 micro g/ml) could promote VEGF secretion (P < 0.01) but exerted no effects on the proliferation of U937 cells. CONCLUSION: U937 cells could be activated to increase the secretion of TGFbeta(1) by LPS in optimal dose of 0.1 - 10.0 micro g/ml, but the secretion of VEGF could only be promoted by LPS in higher concentration.

Transplantation of dermal multipotent cells promotes the hematopoietic recovery in sublethally irradiated rats.

Our previous study indicated that dermal multipotent cells with the differentiation capacity to form cells with the phenotypic properties of osteocytes, adipocytes, chondrocytes, and neurons in specific inducing media could be isolated from the enzymatically dissociated dermal cells of newborn rats by their adherence to culture dish plastic. We have also observed that the systemic transplantation of dermal multipotent cells could not repopulate the hematopoietic system in lethally irradiated rats. In this paper, we found that a transplantation of plastic-adherent dermal multipotent cells into sublethally irradiated rats led to a significant increase of white blood cells in peripheral blood, nucleated cells, CFU-GM, and CFU-F colonies in bone marrow. FISH analysis, using a Y-chromosome specific probe, showed that dermal multipotent cells could engraft into bone marrow in recipients. Flow cytometry (FACS) analysis also showed that the proportion of CD2 and CD25 positive lymphocytes in peripheral blood did not change significantly in two weeks after transplantation. By these results, we infer that dermal multipotent cells may represent an alternative origin of mesenchymal stem cells to restore marrow microenvironment and promote the survival, engraftment, and proliferation of hematopoietic cells.

[The role of p38 on the differentiation of MSCs to myoblasts].

AIM: Inducing mesenchymal stem cells (MSCs) differentiate to myoblasts with 5-azacytidine(5-Aza-CR), investigating the expression of Myf5 and the role of the signal transduction case of p38 in all the course of differentiation. METHODS: Separating and purifying bone marrow-derived MSCs, inducing MSCs differentiation to myoblasts with 10 micromol/L 5-Aza-CR, assaying the gene expression time of Myf5 with RT-PCR method, the antigen expression of myosin with immunohistochemistry method and observing the changes of the activity of phosphorylation p38 before and after inhibited by SB203580 with Western-blot method. RESULTS: MSCs begin to express Myf5 delayed to the 9th day after inhibited by SB203580. Some of MSCs express myosin at the 7th day after induced; The phosphorylation p38 activity of MSCs enhanced after induced by 5-Aza-CR but obviously decreased after inhibited by SB203580. CONCLUSION: MSCs can express myogenic regulator factors and orientation differentiate to myoblasts after induced by 5-Aza-CR, p38 really have a positive signal transduction affection in this course.

[The role of homeobox B2 gene in vascular endothelial proliferation and the protective effects of VEGF on the endothelia against radiation injury].

OBJECTIVE: To explore the role of HOXB2 gene in the proliferation of primary human umbilical vein endothelial cells (HUVECs) and the protective effects of VEGF on the endothelia against radiation injury. METHODS: HUVECs were isolated, cultured, subcultured and identified. (1) Liposome coated oligodeoxynucleotide (odn) and homeoboxB2 antisense oligodeoxyncleotide (HOXB2asodn) were prepared prepared in the concentrations of 0.25, 0.5, 1.0 and 2.5 mg/L for the stimulation of HUVEC. (3)H-TdR incorporation test and MTT method were employed to determine the proliferation activity of HUVECs after activation. The cell cycle analysis of HUVECs was determined by flow cytometry. The expression level of HOXB2mRNA within HUVECs was detected by RT-PCR (reverse transcription polymerase chain reaction). (2) HUVECs were separately treated with the addition of VEGF in concentration of 50 microg/L, by radiation in the dose of 6 Gy or 12 Gy (60)Co gamma gamma ray, or radiation with 12 Gy (60)Co gamma gamma ray followed by the addition of VEGF in dose of 50 microg/L. The cellular morphology was observed and the cellular proliferation activity was determined by MTT method. RESULTS: (1) The proliferation activity of HUVECs could be markedly inhibited by liposome coated HOXB2asodn in comparison to liposome-odn (P < 0.05 or 0.001), and the inhibition effect was positively correlated with the increase in asodn concentration. The cell ratio in S phase and the expression level of the HOXB2mRNA could be lowered by asodn in dose of 2.5 mg/L (P < 0.05 or 0.001). (2) Radiation by (60)Co gamma ray could lead to the nuclear enlargement, vacuolation in the cytoplasm, multiplicity of nucleus and nuclear swelling. The proliferative activity of HUVECs was increased from 0.365 +/- 0.047 and 0.487 +/- 0.022 without radiation to 0.557 +/- 0.042 and 0.648 +/- 0.021 24 and 48 hours after 6 Gy radiation However it was decreased to 0.263 +/- 0.038 and 0.306 +/- 0.024 (P < 0.01) after 12 Gy (60)Co gamma ray radiation. Nevertheless, the cell morphology was obviously improved and the proliferation was enhanced by the addition of VEGF after 12 Gy radiation. CONCLUSION: HOXB2 gene played important roles in the biological activities of HUVECs. Small dose (6 Gy) gamma-radiation could promote, but large dose (12 Gy) could decrease the mRNA expression of HOXB2 gene in HUVECs. In addition, VEGF could protect HUVECs against radiation injury.

Downregulation of glucocorticoid receptors of liver cytosols and the role of the inflammatory cytokines in pathological stress in scalded rats.

Preliminary experiments indicated that target cells were resistant to glucocorticoid (GC) after pathological stress. This study was designed to investigate the alterations in plasma corticosterone level and GC receptor (GR) of liver cytosols, to assess the relative inflammatory cytokines contribution to GC resistant, and to observe the action of alpha-melanocyte-stimulating hormone (alpha-MSH) on the potential implications of glucocorticord regulatory effects in burned rats. Male Wistar rats (weight range, 180-200g) received a 35% total body surface area immersion scald and were randomly divided to receive either tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), polyclonal antibody (pAb), alpha-MSH, Ac-D-Lys-L-Pro-D-Val (KPV peptide), or saline (control). The binding capacity (Rt) of the steroid-binding sites was measured by radioligand binding assay, using [3H]dexamethasone as the ligand. We examined plasma levels of IL-1beta, TNFalpha, IL-10, and corticosterone following scald challenge in rats. The Rt of GR (208.45+/-30.78fmol/mg of protein) in hepatic cytosol in rats, 12h later the scald was significantly lower than that (306.71+/-27.96fmol/mg of protein) of the control group (P<0.01). The injections of anti-rat TNFalpha (257.80+/-12.82fmol/mg of protein), IL-1beta antibody (254.46+/-21.21fmol/mg of protein), alpha-melanocyte-stimulating hormone (278.32+/-7.76fmol/mg of protein) and KPV peptide (263.46+/-17.46fmol/mg of protein) might prevent the Rt of GR from decreasing in hepatic cytosols of rats with scald, respectively (all of P<0.05) in vivo. Scald-induced robust increases in plasma IL-1beta (214.08+/-27.25pg/ml), TNFalpha (111.18+/-23.97pg/ml), IL-10 (177.50+/-15.79pg/ml) and corticosterone (2680+/-443.23ng/ml) levels after 12h. The administration of TNFalpha, IL-1beta pAb, alpha-MSH and KPV might attenuate these increases. These studies suggest that pro-inflammatory cytokines are involved in downregulation of GRs and thus alpha-MSH and KPV might increase the level of GR in rats with immersion scald.

Changes in glucocorticoid and mineralocorticoid receptors of liver and kidney cytosols after pathologic stress and its regulation in rats.

OBJECTIVE: As effectors, glucocorticoid and mineralocorticoid receptors play an important role in pathologic stress. This study was designed to observe the changes in glucocorticoid receptor of liver cytosols and mineralocorticoid receptor of kidney cytosols after pathologic stress in rats. DESIGN: Controlled laboratory study. SETTING: Medical university. SUBJECTS: Male Wistar rats (weight range, 180-200 g). INTERVENTIONS: Rats received a low-degree or heavy-degree immersion scald that covered 10% or 35% total body surface area and were randomly divided to receive either tumor necrosis factor-alpha, interleukin-1beta polyclonal neutralizing antibody, alpha-melanocyte-stimulating hormone, KPV peptide (Ac-D-Lys-L-Pro-D-Val), or saline (control). The binding capacity and the apparent dissociation constant of the steroid-binding sites of normal, low-degree, and heavy-degree scalded rats were measured by radioligand-binding assay, with [3H]dexamethasone and aldosterone as the ligand, respectively. MEASUREMENTS AND MAIN RESULTS: The binding capacity of glucocorticoid receptor in hepatic cytosols in rats 12 hrs after heavy-degree scald (208.45 +/- 30.78 fmol/mg of protein) was lower than that of the control group (306.71 +/- 27.96 fmol/mg of protein; p < .01). The binding capacity of glucocorticoid receptor in hepatic cytosols in rats 12 hrs after low-degree scald (296.64 +/- 16.06 fmol/mg of protein) was not significantly different compared with the control group (p > .05). There were two types of mineralocorticoid receptor in kidney cytosols in rats, and their binding capacity and apparent dissociation constant were not identical. The binding capacity of mineralocorticoid receptor in rats 12 hrs after heavy-degree scald (binding capacity 1, 22.40 +/- 5.40 fmol/mg of protein; binding capacity 2, 196.30 +/- 32.50 fmol/mg of protein) was lower than that of the control group (binding capacity 1, 41.60 +/- 7.20 fmol/mg of protein; binding capacity 2, 317.60 +/- 70.00 fmol/mg of protein; p < .01). The binding capacity of mineralocorticoid receptor in kidney cytosols in rats 12 hrs after low-degree scald (binding capacity 1, 41.40 +/- 5.00 fmol/mg of protein; binding capacity 2, 314.80 +/- 45.70 fmol/mg of protein) was not significantly different compared with the control group (p > .05). The injections of anti-rat tumor necrosis factor-alpha, interleukin-1beta polyclonal neutralizing antibody, alpha-melanocyte-stimulating hormone, and KPV peptide (Ac-D-Lys-L-Pro-D-Val) might prevent a reduction in the binding capacity of glucocorticoid receptor in hepatic cytosols and mineralocorticoid receptor in kidney cytosols in rats with heavy-degree scald in vivo. CONCLUSIONS: These studies suggest that the glucocorticoid receptor of hepatic cytosols and the mineralocorticoid receptor of renal cytosols decreased in rats with heavy-degree immersion scald and that the injections of anti-rat tumor necrosis factor-alpha, interleukin-1beta polyclonal neutralizing antibody, alpha-melanocyte-stimulating hormone, and KPV peptide might increase the level of glucocorticoid receptor and mineralocorticoid receptor in vivo.