Effect and molecular mechanism of Sulforaphane alleviates brain damage caused by acute carbon monoxide poisoning:Network pharmacology analysis, molecular docking, and experimental evidence.

Sulforaphane (SFN) has attracted much attention due to its ability on antioxidant, anti-inflammatory, and anti-apoptotic properties, while its functional targets and underlying mechanism of action on brain injury caused by acute carbon monoxide poisoning (ACOP) have not been fully elucidated. Herein, we used a systematic network pharmacology approach to explore the mechanism of SFN in the treatment of brain damage after ACOP. In this study, the results of network pharmacology demonstrated that there were a total of 81 effective target genes of SFN and 36 drug-disease targets, which were strongly in connection with autophagy-animal signaling pathway, drug metabolism, and transcription disorders in cancer. Upon the further biological function and KEGG signaling pathway enrichment analysis, a large number of them were involved in neuronal death, reactive oxygen metabolic processes and immune functions. Moreover, based on the results of bioinformatics prediction associated with multiple potential targets and pathways, the AMP-activated protein kinase (AMPK) signaling pathway was selected to elucidate the molecular mechanism of SFN in the treatment of brain injury caused by ACOP. The following molecular docking analysis also confirmed that SFN can bind to AMPKα well through chemical bonds. In addition, an animal model of ACOP was established by exposure to carbon monoxide in a hyperbaric oxygen chamber to verify the predicted results of network pharmacology. We found that the mitochondrial ultrastructure of neurons in rats with ACOP was seriously damaged, and apoptotic cells increased significantly. The histopathological changes were obviously alleviated, apoptosis of cortical neurons was inhibited, and the number of Nissl bodies was increased in the SFN group as compared with the ACOP group (p < .05). Besides, the administration of SFN could increase the expressions of phosphorylated P-AMPK and MFN2 proteins and decrease the levels of DRP1, Caspase3, and Casapase9 proteins in the brain tissue of ACOP rats. These findings suggest that network pharmacology is a useful tool for traditional Chinese medicine (TCM) research, SFN can effectively inhibit apoptosis, protect cortical neurons from the toxicity of carbon monoxide through activating the AMPK pathway and may become a potential therapeutic strategy for brain injury after ACOP.

[Effects of activated coke on soil properties and growth of two plant species in saline alkali soil in northern Shanxi Province, China.] / 活性焦对晋北盐碱地土壤性质和两种植物生长的影响.

Solid waste-based improver is one of the effective means to improve properties of saline-alkali soil. As a kind of porous waste, activated coke is expected to improve soil properties and alleviate salt-alkali stress. In order to understand the improvement effect of activated coke on saline alkali land in northern Shanxi Province, we examined the effects of different addition rates of activated coke (CK, 0 g·kg-1; A10, 10 g·kg-1; A20, 20 g·kg-1; A50, 50 g·kg-1) on the properties of saline alkali soil and the growth of two plant species. The results showed that activated coke addition could increase the content of water soluble soil aggregates, reduce soil salt content, soil pH, and the electrical conductivity (EC). Compared with CK, the mean weight diameters of the aggregates for the saline-alkali soils grown with Puccinellia distans and maize were increased by 5.1%-32.2%, soil pH was decreased by 0.4%-4.1%, sodium adsorption ratio (SAR) was decreased by 4.8%-18.7%, and the EC was decreased by 7.4%-8.2%. Applying appropriate amount of activated coke could promote plant growth through reducing the plasma membrane damage of plant cells, increasing plant chlorophyll and Ca2+ contents. The biomass of Puccinellia distans and maize both reached the maximum under the A20 treatment. It suggested that the application of 20 g·kg-1 activated coke (A20) in saline alkali soil could improve soil quality in the rhizosphere soil, increase plant selective Ca2+ absorption, thereby reducing salt damage to plant cells and promote plant growth in saline-alkali habitat.

Effects of human parathyroid hormone 1-34 on bone loss and lumbar intervertebral disc degeneration in ovariectomized rats.

PURPOSE: Lumbar intervertebral disc degeneration is a common cause of lower back pain that affects the physical and mental health of patients and increases social burden. Parathyroid hormone has been reported to be effective at inhibiting disc degeneration; however, these effects have not been fully established in vivo in ovariectomized (OVX) rats. Thus, in this study, we aimed to address this issue and examine the effects of parathyroid hormone treatment in OVX rats. METHODS: Thirty female Sprague-Dawley rats, three months-old, were subjected to sham or ovariectomy surgery. Twelve weeks postsurgery, OVX rats were treated with either human parathyroid hormone [hPTH(1-34), 30 µg/kg/day] or vehicle (normal saline (NS)) treatment. The L3-6 spinal segments were harvested after 12 weeks treatment. Bone mineral density (BMD), micro-architectural parameters, and biomechanical assessment were measured at the lumbar vertebral bodies. Histology and immunohistochemistry were performed to analyze the characteristics of the lumbar intervertebral discs. RESULTS: OVX + PTH rats had significantly higher BMD, percentage bone volume density, trabecular thickness, and biomechanical strength compared with those in Sham and OVX + NS rats. Histology and immunostaining revealed that disc degeneration was not significantly different between the OVX + NS rats and the OVX + PTH rats, compared with the Sham group; the structure of nucleus pulposus was disordered, the expression of collagen I was increased, and collagen II and aggrecan were decreased. CONCLUSIONS: These findings confirmed that hPTH(1-34) treatment has substantial anabolic effects on bone mass and trabecular micro-architecture, while the excessively enhanced bone mass and strength were coupled with a non-significant effect on the disc degeneration in ovariectomized rats.

Orally administered simvastatin partially preserves lumbar vertebral bone mass but not integrity of intervertebral discs in ovariectomized rats.

The present study aimed to investigate the effect of orally administered simvastatin on lumbar vertebral bone mass and intervertebral disc (IVD) degeneration in ovariectomized (OVX) rats. A total of 30 female Sprague-Dawley (SD) rats were subjected to either bilateral ovariectomy (n=20) or sham surgery (n=10). After 12 weeks, the OVX rats were orally administered either saline vehicle (OVX + V group; n=10), or 5 mg/kg/day simvastatin (OVX + SIM group; n=10). Following 12 weeks of treatment, necropsy was conducted and bone mineral density (BMD) was determined in the L5-6 vertebrae. Furthermore, the microstructure and biomechanical properties of the L3 vertebrae were detected by micro-computed tomography and compression testing, respectively. The L5-6 vertebrae were analyzed by measurement of IVD height, observation of histological changes by van Gieson staining, and evaluation of collagen-II (col-II), aggrecan (AGG) and collagen I (col-I) expression by immunohistochemical analysis. Rats in the OVX+V group had lower BMD, bone volume/trabecular volume ratio, maximum load and elastic modulus than the sham group. Rats in the OVX + SIM group had higher BMD and biomechanical strength values than the rats in the OVX+V group. Histological analysis showed that the OVX + V and OVX + SIM groups exhibited significantly higher disc degeneration scores and significantly lower IVD height than the sham group. Immunohistochemical analysis revealed lower expression levels of col-II and AGG, but higher levels of col-I in the annulus fibrosis and endplate in OVX+V rats compared with the sham group. The OVX + SIM group exhibited levels of col-II, AGG and col-I expression comparable with those of OVX+V rats, with the exception of an upregulation of col-II expression in the annulus fibrosis. These data demonstrate that simvastatin treatment partially prevented bone loss and the deterioration of biomechanical properties of lumbar vertebrae, but not the progression of IVD degeneration in OVX rats.

Enhancement of Lumbar Fusion and Alleviation of Adjacent Segment Disc Degeneration by Intermittent PTH(1-34) in Ovariectomized Rats.

Osteoporosis, which is prevalent in postmenopausal or aged populations, is thought to be a contributing factor to adjacent segment disc degeneration (ASDD), and the incidence and extent of ASDD may be augmented by osteopenia. Parathyroid hormone (PTH) (1-34) has already been shown to be beneficial in osteoporosis, lumbar fusion and matrix homeostasis of intervertebral discs. However, whether PTH(1-34) has a reversing or retarding effect on ASDD in osteopenia has not been confirmed. In the present study, we evaluated the effects of intermittent PTH(1-34) on ASDD in an ovariectomized (OVX) rat model. One hundred 3-month-old female Sprague-Dawley rats underwent L4 -L5 posterolateral lumbar fusion (PLF) with spinous-process wire fixation 4 weeks after OVX surgery. Control groups were established accordingly. PTH(1-34) was intermittently administered immediately after PLF surgery and lasted for 8 weeks using the following groups (n = 20) (V = vehicle): Sham+V, OVX+V, Sham+PLF+V, OVX+PLF+V, OVX+PLF+PTH. The fused segments showed clear evidence of eliminated motion on the fusion-segment based on manual palpation. Greater new bone formation in histology was observed in PTH-treated animals compared to the control group. The extent of ASDD was significantly increased by ovariotomy. Intermittent PTH(1-34) significantly alleviated ASDD by preserving disc height, microvessel density, relative area of vascular buds, endplate thickness and the relative area of endplate calcification. Moreover, protein expression results showed that PTH(1-34) not only inhibited matrix degradation by decreasing MMP-13, ADAMTS-4 and Col-I, but also promote matrix synthesis by increasing Col-II and Aggrecan. In conclusion, PTH(1-34), which effectively improves lumbar fusion and alleviates ASDD in ovariectomized rats, may be a potential candidate to ameliorate the prognosis of lumbar fusion in osteopenia.

Age dependent changes in cartilage matrix, subchondral bone mass, and estradiol levels in blood serum, in naturally occurring osteoarthritis in Guinea pigs.

The Dunkin Hartley (DH) guinea pig is a widely used naturally occurring osteoarthritis model. The aim of this study was to provide detailed evidence of age-related changes in articular cartilage, subchondral bone mineral density, and estradiol levels. We studied the female Dunkin Hartley guinea pigs at 1, 3, 6, 9, and 12 months of age (eight animals in each group). Histological analysis were used to identify degenerative cartilage and electron microscopy was performed to further observe the ultrastructure. Estradiol expression levels in serum were assessed, and matrix metalloproteinase 3 and glycosaminoglycan expression in cartilage was performed by immunohistochemistry. Bone mineral density of the tibia subchondral bone was measured using dual X-ray absorptiometry. Histological analysis showed that the degeneration of articular cartilage grew more severe with increasing age starting at 3 months, coupled with the loss of normal cells and an increase in degenerated cells. Serum estradiol levels increased with age from 1 to 6 months and thereafter remained stable from 6 to 12 months. Matrix metalloproteinase 3 expression in cartilage increased with age, but no significant difference was found in glycosaminoglycan expression between 1- and 3-month old animals. The bone mineral density of the tibia subchondral bone increased with age before reaching a stable value at 9 months of age. Age-related articular cartilage degeneration occurred in Dunkin Hartley guinea pigs beginning at 3 months of age, while no directly positive or negative correlation between osteoarthritis progression and estradiol serum level or subchondral bone mineral density was discovered.

Alendronate retards the progression of lumbar intervertebral disc degeneration in ovariectomized rats.

OBJECTIVE: Increasing evidence has revealed a positive correlation between postmenopausal osteoporosis and intervertebral disc degeneration, the underlying mechanism of which might be associated with changes in the vertebral bone and endplate. Alendronate (ALN) can increase bone mass and improve the microstructure of osteoporotic vertebrae, which might be helpful in preserving disc morphology and mechanical properties. This study aims to investigate the effects of ALN on lumbar intervertebral disc degeneration related to osteoporosis using an ovariectomized (OVX) rat model. METHODS: Thirty female Sprague-Dawley rats aged 3 months were randomly divided into three groups (with 10 rats each) as follows: the Sham group underwent sham surgery; the OVX + ALN group had twice-a-week subcutaneous injections of ALN (15 µg/kg) for 6 months. The OVX + V group received an equivalent volume of saline solution as placebo post-OVX. After animals were sacrificed at 6 months post-OVX, the L3-6 spinal segments were harvested. Bone mineral density (BMD), micro-CT analysis and biomechanical testing were performed to evaluate the bone quality and microstructural changes in the lumbar vertebral bodies. Histological analysis with van Gieson stain and the histological score were used to identify the characteristics of the degenerative discs. The disc height and the thickness of the cartilage endplate were measured and compared. Immunohistochemistry and real-time PCR measurements for aggrecan, type I collagen, type II collagen, and matrix metalloprotease (MMP)-1, MMP-3 and MMP-13 expressions on the disc were performed to assess the underlying molecular signaling changes in matrix metabolism during intervertebral disc degeneration. RESULTS: The OVX + ALN group significantly maintained vertebrae BMD, percent bone volume and biomechanical strength, when compared with the OVX + V group. Histological evaluation suggests that there was no significant difference in disc height between the OVX + ALN and Sham groups, and ALN significantly prevented cartilage endplate thickening and the development of abnormal bony tissues within the cartilage endplate. The histological score in the OVX + ALN group was significantly lower than the OVX + V group, suggesting that ALN treatment was effective in delaying the process of the disc degeneration. The results of molecular analysis revealed a significant increase in aggrecan and type II collagen expressions, but marked reductions in MMP-1, MMP-3 and MMP-13 expressions at both the protein and mRNA levels in the OVX + ALN group. CONCLUSIONS: ALN can retard the progression of lumbar intervertebral disc degeneration in OVX rats. The underlying mechanisms might be related to preservation of the structural integrity and function of the adjacent structures, including the vertebrae and endplates, which further links with modulations in extracellular matrix metabolism to protect the disc from degeneration. These results suggest that ALN might be a promising drug agent for preventing lumbar intervertebral disc degeneration related to osteoporosis.

Effects of ovariectomy and 17beta-estradiol treatment on the renin-angiotensin system, blood pressure, and endothelial ultrastructure.

The purpose of this study was to determine whether the renin-angiotensin system (RAS), nitric oxide (NO), atrial natriuretic peptide (ANP), blood pressure (BP), ultrastructural characteristics, and endothelium-dependent relaxation of thoracic aorta were modulated by the estrogen level. Rats were divided into 3 groups: ovariectomized (OVX); not ovariectomized (sham); and ovariectomized and treated with subcutaneous 17beta-estradiol (15 microg/kg/day, OVX+E(2)) (n=15-17 per group). For 13 weeks after surgery, blood pressure, serum estrogen, NO, plasma angiotensin II (Ang II), ANP, and renin activity levels were monitored. Thirteen weeks after surgery, the vasodilator responses of the aortic rings to acetylcholine and the ultrastructural characteristics of the thoracic aorta were determined. In the 9th and 13th week, OVX rats had a significantly higher blood pressure than the other two groups (p<0.05). Ovariectomy led to a significant decrease in plasma Ang II level and a significant increase in renin activity in OVX rats compared to sham rats; this effect could be reversed by estrogen treatment. In the 5th, 9th, and 13th weeks, the serum NO level was significantly lower in the OVX group than in the sham group (p<0.05); this effect could be reversed by estrogen treatment. Plasma ANP levels in the 9th and 13th weeks were significantly lower in the OVX group (p<0.05), and plasma ANP levels could be completely restored by estrogen treatment. Ovariectomy markedly reduced endothelium-dependent relaxation in response to acetylcholine in isolated rat thoracic aortic rings; chronic estrogen treatment significantly restored endothelium-dependent relaxation in response to acetylcholine. Under electron microscopy, the endothelial cells in OVX rats were swollen, even necrosed; estrogen treatment inhibited these changes. These results strongly suggest that estradiol protects rats from the development of hypertension and has a protective effect on the endothelium by increasing NO and ANP levels while decreasing renin activity. However, there was a discordance between the effects that estradiol had on angiotensin II and on blood pressure. This might be the result of negative feedback that ultimately results in the overall suppression of the RAS.

17Beta-oestradiol regulates the expression of Na+/K+-ATPase beta1-subunit, sarcoplasmic reticulum Ca2+-ATPase and carbonic anhydrase iv in H9C2 cells.

1. It is necessary to improve our understanding of the effect of 17beta-oestradiol (E2) on the heart at a molecular and cellular level. In the present study, the effects of E2 on Na(+)/K(+)-ATPase, sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA) and carbonic anhydrase IV (CAIV) in H9C2 cells were investigated. To identify the mechanism of action of E2 on these proteins, the oestrogen receptor (ER) antagonist tamoxifen was used. 2. The results indicated that 1 and 100 nmol/L E2 can enhance the activity of Na(+)/K(+)-ATPase and SERCA and upregulate the expression of the Na(+)/K(+)-ATPase beta1-subunit, SERCA2a and CAIV at both the mRNA and protein level compared with 0 and 0.01 nmol/L E2. 17beta-Oestradiol had the greatest effect at 100 nmol/L; 1 micromol/L E2 did not further protein expression compared with 100 nmol/L E2. 3. Tamoxifen (10 nmol/L) significantly decreased the activity of SERCA, as well as the expression of the Na(+)/K(+)-ATPase beta1-subunit and SERCA at the mRNA and protein level, in H9C2 cells cultured with 1 nmol/L E2. Tamoxifen alone had no significant effect on these proteins in H9C2 cells. 4. It may be hypothesized that a suitable E2 concentration has a protective effect on the heart and that the actual dose of E2 used in hormone-replacement therapy is important in menopausal women.

[Application of allogeneic bone in surgical treatment of benign bone neoplasm].

OBJECTIVE: To evaluate the clinical outcomes of allogeneic bone grafting for bone defect resulting from benign neoplasm resection and discuss the clinical application and bone defect repair mechanisms of allogeneic bone. METHODS: A retrospective review was conducted of 135 patients with benign neoplasm resection who received bone defect filling with the allogeneic bone graft. RESULTS: In the 104 patients with complete clinical follow-up data, 96 achieved bone union, 7 experienced relapses to require surgical intervention and 1 had severe infection to lead to failure of the operation. The mean time for bone union was 9.7 months, and during the follow-up, no viral disease in relation to the graft was found after surgery. CONCLUSION: Bone defect filling with allogeneic bone graft can be simple and safe in comparison with that with autograft or other biomaterials, and the bone healing time, infection rate and local tumor recurrence can be comparable with the autograft.

Adult islets cultured in collagen gel transdifferentiate into duct-like cells.

AIM: To establish a model of islet-ductal cell transdifferentiation to identify the transdifferentiated cells. METHODS: Collagen was extracted from rat tail at first. Purified rat islets were divided into three groups, embedded in collagen gel and incubated respectively in DMEM/F12 alone (control group), DMEM/F12 plus epidermal growth factor (EGF), DMEM/F12 plus EGF and cholera toxin (CT). Transdifferentiation was proved by microscopy, RT-PCR, immunohistochemistry and RIA. RESULTS: Islets embedded in collagen gel plus EGF and CT were cystically transformed and could express new gene cytokeratin 19 while still maintaining the expression of insulin and Pdx-1 genes. Immunohistochemistry demonstrated that the protein of cytokeratin 19 was only expressed in the third group. The insulin content secreted by islets in the third group decreased significantly during the transdifferentiation. CONCLUSION: CT is a crucial factor for the islet-ductal cell transdifferentiation.

[Effects of long-term estrogen replacement treatment on the blood pressure and expression of IR and IRS-1 in myocardium].

OBJECTIVE: To determine the effects of long-term estrogen replacement treatment on blood pressure and expressions of insulin receptor (IR) and insulin receptor substrate-1 ( IRS-1) in myocardium. METHODS: Fifty female SD rats were randomly divided into 3 groups. And then sham ( n = 16), ovariectomy (OVX, n = 17), and estrogen replacement treatment group (OVX + E2, n = 17) were established. Systolic blood pressure of tail artery was determined by tail-cuff technique before the operation and on week 12 after the operation. The expressions of IR and IRS-1 were measured by RT-PCR in myocardium of SD rats. RESULTS: Blood pressure [ (118.75+/-2.77) mmHg] in OVX was significantly higher than that in the sham [ ( 103.86+/-1.84) mmHg, P < 0.05 ] and OVX + E2 [( 107.83+/-3.24) mmHg, P < 0.05 ] rats. Expression of IRS-1 in OVX group was significantly lower ( 1.2588+/-0.1045)than that in the sham(2.2089+/-0.0988, P <0.05) and OVX + E2 groups ( 1.9100+/-0.1230, P <0.05 ). However, there was no difference on blood pressure and expression of IRS-1 between sham and OVX + E2 groups (P > 0.05 ). The difference of IR expression has no statistical significance among the 3 groups. CONCLUSION: Long-term estrogen replacement treatment might protect cardiovascular system through decreasing the blood pressure and inducing the expression of IRS-1 in myocardium. However, plasma estrogen level doesn't significantly influence the IR expression.

[Effects of long-term estrogen replacement treatment on the expression of bcl-2 and H-ras in rat endometrium].

OBJECTIVE: To investigate the effects of long-term estrogen replacement treatment (ERT) on the expression of bcl-2 and H-ras in rat endometrium. METHODS: Thirty 5-month-old SD female rats were randomly divided into 3 groups: Control group ( sham operated and vehicle injected, n 10) , OVX group (OVX operated and vehicle injected, n = 10) , and ERT group (OVX operated and 17 beta-estradiol injected, n = 10). The rats were killed in the 13th week and the uteri were isolated and weighed, pathologically analyzed, and we measured the thickness of the endometrium. Immunochemistry and in situ hybridization analysis were used to examine the changes of bcl-2 and H-ras mRNA and Bcl and H-ras protein expression in the endometrium of the rats. RESULTS: Uterine weight and endometrial thickness of OVX decreased much more than those of the control (P <0.01 ) and ERT rats (P < 0.01). One simple hyperplasia and one squamous metaplasia of endometrium were found in ERT rats. Quantitatively, bcl-2 and H-ras mRNA and Bcl and H-ras protein level of ERT were higher than those of OVX rats (P < 0.01 ), and there were no statistical significances between the ERT group and the control rats. CONCLUSION: Long-term estrogen replacement can keep the endometrium from atrophy, and lead to the genesis of simple hyperplasia and squamous metaplasia of the endometriun, which can increase the risk of endometrial carcinomas. Estrogen may inhibit apoptosis and promote the proliferation of endometrial cells through increasing the expression of bcl-2 and H-ras mRNA and Bcl-H-ras proteins.

Influence of ovariectomy and 17beta-estradiol treatment on insulin sensitivity, lipid metabolism and post-ischemic cardiac function.

The purpose of this study was to test whether the insulin sensitivity, lipid metabolism and the susceptibility of the heart to ischemia/reperfusion injury are modulated by the chronic estrogen status. Rats were ovariectomized (OVX), not ovariectomized (sham) or ovariectomized and treated with subcutaneous 17 -estradiol (30 mug/kg/day, OVX+E2) (n=14-17 per group). Within 3 months after operation, body weight, the serum levels of estrogen, glucose, insulin, total cholesterol (T-chol), HDL-chol, LDL-cholesterol (LDL-chol), triglycerides (TG) and lipoprotein a (Lp(a)) were monitored. Three months after operation, hearts of partial rats (n=6-8 per group) were isolated and allowed an initial 20-min stabilization period, and then cardiac function was recorded and creatine kinase (CK) release in the coronary effluent was measured after 4 h of hypothermic ischemia in isolated rat hearts. The experimental results showed that from 2 weeks after ovariectomy to the end of the study, body weights of OVX were significantly higher compared with the other two groups (p<0.05). On weeks 5 and 9, insulin level of OVX was significantly higher than that of the other two groups (p<0.05), whereas it was not different among the three groups on weeks 12 and 13 (p>0.05). Blood glucose on week 13 was significantly higher in OVX (p<0.05). Consequently, Insulin Sensitivity Index (ISI) of OVX was lower than that of the other two groups on weeks 5 and 9 (p<0.05), but not on weeks 12 and 13. Serum values for T-chol, HDL-chol and LDL-chol were not significantly different among the three groups within the observing period. On week 13, TG level in ovariectomized group was significantly lower than in the sham- and E2-treated groups (p<0.05). Compared with sham, Lp(a) level was slight increased in OVX rats (p<0.05), while it was further increased in E2-treated rats (p<0.05). Cardiac function (left ventricular pressure (LVP) and +/-dp/dtmax) of hearts removed from OVX rats was depressed, and CK release was markedly increased (p<0.05). However, treatment with E2 significantly improved cardiac function, as shown by increasing left ventricular pressure,+dp/dtmax and -dp/dtmax, and decreased CK release. In conclusion, chronic E2 treatment has some beneficial effects on cardiovascular disease (CVD), which come from the results of improvement of insulin sensitivity and post-ischemia cardiac function. However, the mechanism did not include changes in lipids and lipoproteins. The change in Lp(a) level shows that estrogen does not confer cardiovascular protection and may increase the risk of stroke.

Oestrogen improves glucose metabolism and insulin signal transduction in HepG2 cells.

1. Many clinical studies have suggested a relationship between oestrogen and insulin sensitivity. In the present study, HepG2 cells were divided into four groups: (i) control, incubated with 1 nmol/L insulin; (ii) the HI group, which was incubated with 100 nmol/L insulin to induce insulin resistance; (iii) the E2 group, in which control cells were incubated with 1 nmol/L insulin plus 1 nmol/L oestradiol; and (iv) the HI + E2 group, in which insulin-resistant cells were incubated with 100 nmol/L insulin + 1 nmol/L oestradiol. 2. A high concentration of insulin decreased the activity of phosphofructo-1-kinase (PFK), pyruvate dehydrogenase (PDH) and glycogen synthase (GS), as well as decreasing the expression of insulin receptor (IR) and insulin receptor substrate-2 (IRS-2). High insulin had no effect on glucose transport or the expression of insulin receptor-1 (IRS-1). 3. The addition of oestradiol to control cells increased glucose transport, the activity of PFK, PDH and GS and the expression of IRS-1 and IRS-2, but had no effect on the expression of IR. 4. Treatment of insulin-resistant HepG2 cells with oestradiol attenuated HI-induced decreases, except for IR, and the expression of IRS-1 was significantly higher than control, attaining levels seen in group 3. The expression of IRS-2 was significant higher than in insulin-resistant cells, but did not reach control levels. Changes in the activity of PFK, PDH and GS were the same as the changes seen in the expression of IRS-2. 5. These results suggest that high concentrations of insulin induce insulin resistance in HepG2 cells, whereas oestradiol improves glucose metabolism and insulin signal transduction of cells by enhancing the activity of key enzymes involved in glucose metabolism and the expression of IRS-1 and IRS-2.