New labdane diterpenoids from Alpinia galanga: A new type of GLP-1 secretagogues targeting the PKA-CREB and PI3K-Akt signaling axes.

Glucagon-like peptide-1 (GLP-1) secretagogues are fascinating pharmacotherapies to overcome the defects of GLP-1 analogs and dipeptidyl peptidase-4 (DPP-4) inhibitors in treating diabetes and obesity. To discover new GLP-1 secretagogues from natural sources, alpigalangols A-Q (1-17), 17 new labdane diterpenoids including four unusual nor-labdane and N-containing ones, were isolated from the fruits of Alpinia galanga. Most of the isolates showed GLP-1 promotive effects in NCl-H716 cells, of which compounds 3, 4, 12, and 14-17 were revealed with high promoting rates of 246.0%-413.8% at 50 µM. A mechanistic study manifested that the most effective compound 12 upregulated the mRNA expression of Gcg and Pcsk1, and the protein phosphorylation of PKA, CREB, and GSK3ß, but was inactive on GPBAR and GPR119 receptors. Network pharmacology analysis indicated that the PI3K-Akt pathway was involved in the GLP-1 stimulation of 12, which was highly associated with AKT1, CASP3, PPARG, and ICAM1 proteins. This study suggests that A. galanga is rich in diverse labdane diterpenoids with GLP-1 promoting effects, representing a new type of antidiabetic candidates from natural sources.

Whole fresh fruit intake and risk of incident diabetes in different glycemic stages: a nationwide prospective cohort investigation.

PURPOSE: Fruit intake is beneficial to several chronic diseases, but controversial in diabetes. We aimed to investigate prospectively the associations of whole fresh fruit intake with risk of incident type 2 diabetes (T2D) in subjects with different glucose regulation capacities. METHODS: The present study included 79,922 non-diabetic participants aged ≥ 40 years from an ongoing nationwide prospective cohort in China. Baseline fruit intake information was collected by a validated food frequency questionnaire. Plasma HbA1c, fasting and 2 h post-loading glucose levels were measured at both baseline and follow-up examinations. Cox proportional hazards models were used to calculate hazard ratio (HR) and 95% confidence intervals (CI) for incident diabetes among participants with normal glucose tolerance (NGT) and prediabetes, after adjusted for multiple confounders. Restricted cubic spline analysis was applied for dose-response relation. RESULTS: During a median 3.8-year follow-up, 5886 (7.36%) participants developed diabetes. Overall, we identified a linear and dose-dependent inverse association between dietary whole fresh fruit intake and risk of incident T2D. Each 100 g/d higher fruit intake was associated with 2.8% lower risk of diabetes (HR 0.972, 95%CI [0.949-0.996], P = 0.0217), majorly benefiting NGT subjects with 15.2% lower risk (HR 0.848, 95%CI [0.766-0.940], P = 0.0017), while not significant in prediabetes (HR 0.981, 95%CI 0.957-4.005, P = 0.1268). Similarly, the inverse association was present in normoglycemia individuals with a 48.6% lower risk of diabetes when consuming fruits > 7 times/week comparing to those < 1 time/week (HR 0.514, 95% CI [0.368-0.948]), but not in prediabetes (HR 0.883, 95% CI [0.762-1.023]). CONCLUSION: These findings suggest that higher frequency and amount of fresh fruit intake may protect against incident T2D, especially in NGT, but not in prediabetes, highlighting the dietary recommendation of higher fresh fruit consumption to prevent T2D in normoglycemia population.

New diarylheptanoid dimers as GLP-1 secretagogues and multiple-enzyme inhibitors from Alpinia katsumadai.

Ten new diarylheptanoid dimers, katsumadainols C1 - C10 (1-10), were isolated from the seeds of Alpinia katsumada and elucidated by extensive spectroscopic methods, ECD calculations, and single-crystal X-ray diffraction. Their antidiabetic effects were evaluated by the stimulation of GLP-1 secretion in STC-1 cells and inhibition against four diabetes-related enzymes, GPa, α-glucosidase, PTP1B, and DPP4. Compounds 1-5 and 7-10 significantly stimulated GLP-1 secretion by 267.5-433.1% (25.0 µM) and 117.8-348.2% (12.5 µM). Compounds 1-4 exhibited significant inhibition on GPa with IC50 values of 18.0-31.3 µM; compounds 1-5 showed obvious inhibition on α-glucosidase with IC50 values of 6.9-18.2 µM; compounds 1-5 and 10 possessed PTP1B inhibitory activity with IC50 values ranging from 35.5 to 80.1 µM. This investigation first disclosed compounds 1-4 as intriguing GLP-1 secretagogues and GPa, α-glucosidase, and PTP1B inhibitors, which provided valuable clues for searching multiple-target antidiabetic candidates from Zingiberaceae plants.

[Terpenoids from fruits of Amomum villosum and their hypoglycemic activity].

Eight terpenoids were isolated from the fruits of Amomum villosum by silica gel, Sephadex LH-20, Rp-C_(18), MCI GEL CHP20 P column chromatography, preparative TLC, and HPLC. Their structures were identified by HR-ESI-MS, ~1H and ~(13)C-NMR, IR, UV, [α]_D, and ECD spectroscopic data as kravanhin A 3-O-ß-D-glucopyranoside(1), kravanhin B(2), 6-eudesmene-1ß,4ß-diol(3), oplodiol(4), vicodiol(5),(1R,2S,4R,7S)-vicodiol 9-O-ß-D-glucopyranoside(6),(1R,2S,4S,5R)-angelicoidenol 2-O-ß-D-glucopyranoside(7), and(1S,2S,4R,6S)-bornane-2,6-diol 2-O-ß-D-glucopyranoside(8). Compound 1 was a new compound, and compounds 2-5 were isolated from A. villosum for the first time. Their hypoglycemic activity was tested based on STC-1 cell model and two enzymatic models(GPa and PTP1 B). The results showed that compounds 1, 7, and 8 could stimulate GLP-1 with the secretion rates of 692.8%, 398.6%, and 483.3% at 25.0 µmol·L~(-1), and compound 6 showed inhibitory activity against GPa with an IC_(50) value of 78.6 µmol·L~(-1).

Dithiolation of [70]Fullerene with Aliphatic Primary Thiols in the Presence of n-Butylamine via Aerobic Oxidation Reaction.

Aerobic oxidation reaction of [70]fullerene with aliphatic primary thiols in the presence of n-butylamine was studied. The reaction afforded both the fullerene dithiol (2) and dithiol epoxide (3) compounds, which were characterized by 1H and 13C NMR, HRMS and UV-vis spectroscopies. In situ vis-NIR spectral measurement showed absorption bands due to anionic fullerene species, indicating that the reaction was likely initiated with the addition of thiolate anions to C70. Computational calculations were performed to rationalize the structure of the dithiol epoxide compounds. The electrochemical property of 2,5-( n-C5H11S)2C70 (2a) was compared with two analogue compounds of 2,5-Bn2C70 and 2-MeO-5-BnC70 that had identical addition pattern and number of addends. The result showed that the dithiol compound was more electron deficient than the other two derivatives.

Copper-Catalyzed Aerobic Oxidative Reaction of C60 with Aliphatic Primary Amines and CS2.

A novel type of fullerene derivatives, [60]fullerothiazolidinethiones (2), were obtained from the copper-catalyzed aerobic oxidative reaction of C60 with aliphatic primary amines and CS2 in 4:1 v/v DMF and o-DCB. The obtained products were fully characterized with the X-ray single-crystal diffraction and spectroscopic methods. Control experiment with maleic anhydride, an analogue to C60, also afforded thiazolidinethione product, but via a mechanism different from that of C60 judging from the structure difference between the two types of thiazolidinethione compounds, demonstrating the unique reactivity of C60.

Reductive Activation of C70 Equatorial Carbons and Structurally Characterized C70 δ-Adduct with Closed [5,6]-Ring Fusion.

The C70 δ-adducts with closed [5,6]-ring fusion are an important type of compound in classifying bond delocalization in the equatorial belt of C70. However, the formation of such compounds is severely restricted due to the low reactivity of the carbon atoms in the flat equatorial region. Such a restriction is lifted when reduced anionic C70 species are used, where the inert equatorial carbon atoms are activated.

Necrolytic migratory erythema as the first manifestation of pancreatic neuroendocrine tumor.

Necrolytic migratory erythma (NME) is an obligatory paraneoplastic syndrome. Here we describe a woman admitted to the dermatology ward with NME which was later found to be associated with glucagonoma, a slow-growing, rare pancreatic neuroendocrine tumor. Even more rarely, the tumor was located in the pancreas head, while most of such lesions are located in the distal pancreas. The diagnosis of this rare tumor requires an elevated serum glucagon level and imaging confirming a pancreatic tumor. After surgical removal of the tumor, the patient's cutaneous and systemic features resolved. It is therefore imperative that clinicians recognize NME early in order to make an accurate diagnosis and to provide treatment for this rare tumor.

BACE1 inhibitors from the fruits of Alpinia oxyphylla have efficacy to treat T2DM-related cognitive disorder.

The fruits of Alpinia oxyphylla (Alpiniae Oxyphyllae Fructus, AOF) are one of the "Four Famous South Medicines" in China. In this study, beta-site amyloid protein precursor cleaving enzyme 1 (BACE1) was applied to explore the active components in AOF responsible for type 2 diabetes mellitus (T2DM)-related cognitive disorder. As a result, 24 compounds including three unreported ones (1, 3, 4) were isolated from AOF. Compound 1 is an unusual carbon­carbon linked diarylheptanoid dimer, and compound 4 is the first case of 3,4-seco-eudesmane sesquiterpenoid with a 5/6-bicyclic skeleton. Four diarylheptanoids (3, 5-7), one flavonoid (9) and two sesquiterpenoids (14 and 20) showed BACE1 inhibitory activity, of which the most active 6 was revealed to be a non-competitive and anti-competitive mixed inhibitor. Docking simulation suggested that OH-4' of 6 played important roles in maintaining activity by forming hydrogen bonds with Ser36 and Ile126 residues. Compounds 3, 5, 9 and 20 displayed neuroprotective effects against amyloid ß (Aß)-induced damage in BV2 cells. Mechanism study revealed that compounds 5 and 20 downregulated the expression of BACE1 and upregulated the expression of Lamp2 to exert effects. Thus, the characteristic diarylheptanoids and sesquiterpenoids in AOF had the efficacy to alleviate T2DM-related cognitive disorder by inhibiting BACE1 activity and reversing Aß-induced neuronal damage.

Sesquiterpenoids from Alpinia oxyphylla with GLP-1 Stimulative Effects through Ca2+/CaMKII and PKA Pathways and Multiple-Enzyme Inhibition.

Six new sesquiterpenoids (1-6), a pair of enantiomers (7a and 7b), and six known ones (8-13) were isolated from the fruits of Alpinia oxyphylla. The structures of the new compounds were elucidated by extensive spectroscopic data and ECD calculations. The stereochemistry of 7a and 7b was reported for the first time. All compounds showed significant GLP-1 stimulation in NCI-H716 cells with promoting ratios ranging from 90.4 to 668.9% at 50 µM. Mechanism study indicated that compound 6 stimulated GLP-1 secretion mainly by regulating the transcription and the shearing process of proglucagon, while compound 13 exerted its effects through up-regulating prkaca levels. Interestingly, the GLP-1 stimulative effects of 6 and 13 were both closely related with Ca2+/CaMKII and PKA pathways but irrelevant to TGR5 and GPR119 receptors. Moreover, most compounds exhibited inhibitory activity against α-glucosidase and PTP1B at concentrations of 100 and 200 µM, while showing no activity against GPa. Compounds 3, 9, 11, and 13 could suppress α-glucosidase with IC50 values of 190.0, 204.0, 181.8, and 159.6 µM, equivalent to acarbose (IC50 = 212.0 µM). This study manifests that A. oxyphylla contains diverse sesquiterpenoids with multiple activities.

Diarylheptanoids with hypoglycemic potency from the rhizomes of Kaempferia galanga.

Five new diarylheptanoids, kaemgalangins A-E (1-5), and seven known ones were isolated from the rhizomes of Kaempferia galanga. The structures of new compounds were identified by spectroscopic analyses involving 1D and 2D NMR, HRESIMS, IR, UV, [α]D, ECD calculations, and chemical methods. All compounds were tested for their hypoglycemic effects against α-glucosidase, Gpa and PTP1B enzymes, and stimulative effects on GLP-1 secretion. Kaemgalangins A (1) and E (5) showed significant inhibition on α-glucosidase with IC50 values of 45.3 and 116.0 µM; renealtin B (8) showed inhibition on GPa with an IC50 value of 68.1 µM; whereas all compounds were inactive to PTP1B. Docking study manifested that 1 well located in the catalytic pocket of α-glucosidase and OH-4″ played important roles in maintaining activity. Moreover, all compounds showed obviously stimulative effects on GLP-1 with promoting rates of 826.9%-1738.3% in NCI-H716 cells. This study suggests that the diarylheptanoids in K. galanga have antidiabetic potency by inhibiting α-glucosidase and Gpa enzymes, and promoting GLP-1 secretion.

Oligostilbenes from the seeds of Paeonia lactiflora as potent GLP-1 secretagogues targeting TGR5 receptor.

One unusual stilbene trimer-flavonoid hybrid, paeonilactiflobenoid (1), together with six known stilbenes (2-7) were isolated from the seeds of Paeonia lactiflora. The structure of 1 was elucidated with the aid of HRESIMS, 1D and 2D NMR, [α]D spectroscopic data and ECD calculation. Compounds 2-7 showed stimulative effects on GLP-1 secretion with promoting rates of 79.8%-880.4% (25 µM) and 217.6%-1089.4% (50 µM), more potent than the positive control, oleoylethanolamide (250.2% at 50 µM). Moreover, compounds 4 and 6 exhibited agonistic activity on the G protein-coupled receptor (GPCR) TGR5 with stimulative ratios of 40.2% and 40.5% at 50 µM, and 54.2% and 49.1% at 100 µM, respectively. Docking study manifested that 6 well located in the catalytic pocket of TGR5 by hydrogen-bond and hydrophobic interactions. The GLP-1 promotion of 6 could be attenuated by IP3, Ca2+/CaMKII and MEK/ERK pathway inhibitors, suggesting that these pathways played important roles in GLP-1 secretion. Thus, stilbenes in peony seeds maybe regarded as potential GLP-1 secretagogues through TGR5-IP3-Ca2+/CaMKII-MEK/ERK pathways.

Norlignans as potent GLP-1 secretagogues from the fruits of Amomum villosum.

The dried fruit of Amomum villosum (Amomi Fructus) is an important spices and traditional Chinese medicine. In this study, the EtOH extract of Amomi Fructus was revealed with hypoglycemic effects on db/db mice by increasing plasma insulin levels. After extracted with EtOAc, the EtOAc fraction showed increased activity in stimulating glucagon-like peptide-1 (GLP-1) secretion compared with the EtOH extract. In order to clarify the antidiabetic constituents, four undescribed norlignans, amovillosumins A‒D, were isolated from the EtOAc fraction, and the subsequent chiral resolution yielded three pairs of enantiomers. Their structures were determined by extensive spectroscopic data (1D and 2D NMR, HRESIMS, IR, UV and [α]D) and ECD calculations. Amovillosumins A and B significantly stimulated GLP-1 secretion by 375.1% and 222.7% at 25.0 µM, and 166.9% and 62.7% at 12.5 µM, representing a new type of GLP-1 secretagogues.

Inverted U-Shaped Associations between Glycemic Indices and Serum Uric Acid Levels in the General Chinese Population: Findings from the China Cardiometabolic Disease and Cancer Cohort (4C) Study.

OBJECTIVE: The relationship between serum uric acid (SUA) levels and glycemic indices, including plasma glucose (FPG), 2-hour postload glucose (2h-PG), and glycated hemoglobin (HbA1c), remains inconclusive. We aimed to explore the associations between glycemic indices and SUA levels in the general Chinese population. METHODS: The current study was a cross-sectional analysis using the first follow-up survey data from The China Cardiometabolic Disease and Cancer Cohort Study. A total of 105,922 community-dwelling adults aged ≥ 40 years underwent the oral glucose tolerance test and uric acid assessment. The nonlinear relationships between glycemic indices and SUA levels were explored using generalized additive models. RESULTS: A total of 30,941 men and 62,361 women were eligible for the current analysis. Generalized additive models verified the inverted U-shaped association between glycemic indices and SUA levels, but with different inflection points in men and women. The thresholds for FPG, 2h-PG, and HbA1c for men and women were 6.5/8.0 mmol/L, 11.0/14.0 mmol/L, and 6.1/6.5, respectively (SUA levels increased with increasing glycemic indices before the inflection points and then eventually decreased with further increases in the glycemic indices). CONCLUSION: An inverted U-shaped association was observed between major glycemic indices and uric acid levels in both sexes, while the inflection points were reached earlier in men than in women.

[Study of the microwave emissivity characteristics over different land cover types].

The microwave emissivity over land is very important for describing the characteristics of the lands, and it is also a key factor for retrieving the parameters of land and atmosphere. Different land covers have their emission behavior as a function of structure, water content, and surface roughness. In the present study the global land surface emissivities were calculated using six month (June, 2003-August, 2003, Dec, 2003-Feb, 2004) AMSR-E L2A brightness temperature, MODIS land surface temperature and the layered atmosphere temperature, and humidity and pressure profiles data retrieved from MODIS/Aqua under clear sky conditions. With the information of IGBP land cover types, "pure" pixels were used, which are defined when the fraction cover of each land type is larger than 85%. Then, the emissivity of sixteen land covers at different frequencies, polarization and their seasonal variation were analyzed respectively. The results show that the emissivity of vegetation including forests, grasslands and croplands is higher than that over bare soil, and the polarization difference of vegetation is smaller than that of bare soil. In summer, the emissivity of vegetation is relatively stable because it is in bloom, therefore the authors can use it as its emissivity in our microwave emissivity database over different land cover types. Furthermore, snow cover can heavily impact the change in land cover emissivity, especially in winter.

Serum from radiofrequency-injured livers induces differentiation of bone marrow stem cells into hepatocyte-like cells.

BACKGROUND: Roles that bone marrow stem cells (BMSCs) play in liver repair after liver injury and the cell therapy for liver diseases are widely accepted. However, the availability of hepatocyte-like cells from BMSCs and possible animal diseases association with culturing in fetal calf serum (FCS) are the major limitations of clinical therapy. Therefore, this study was designed to search for a new cell source for the treatment of liver injuries through investigating whether serum from radiofrequency ablation-injured rabbit livers can induce the differentiation of BMSCs into hepatocyte-like cells. METHODS: Serum was collected from rabbits 36 h after radiofrequency ablation (RFA) treatment of the liver. BMSCs were isolated from rabbit bone marrow and were cultured in the collected serum. Cellular morphology and cell cycle were observed. Hepatocyte markers of the differentiated cells were detected by immunohistochemistry. RESULTS: After induction for 7 d, spindle-shaped BMSCs turned into round cells that resembled the morphology of hepatocyte-like cells. Flow cytometry showed that the percentage of cells in the S/G2/M phase was higher in the RFA group than that in the FCS group and HGF groups. This result suggests that BMSC can transform into mature cells from stem cell phase. Albumin and CK18 were considered as typical marker of hepatocytes. Following induction for 14 d, the differentiated cells expressed immunofluorescence of FITC-labeled albumin and TRITC-labeled CK18. CONCLUSION: BMSCs treated with serum collected from radiofrequency ablation-injured livers can differentiate into hepatocyte-like cells providing a cell source to cell therapy.

Effect of resveratrol on NF-kappaB activity in rat peritoneal macrophages.

This study was to investigate the inhibitive effect of resveratrol (RESV) on nuclear factor kappa B (NF-kappaB) expression and activity induced by lipopolysaccharide (LPS) in rat peritoneal macrophages (PMA). Male Sprague-Dawley (SD) rats were randomly divided into 7 groups, including control group, LPS group and RESV I-V group. In the LPS group, PMA were incubated in DMEM containing LPS (10 microg/ml), whereas in control group, PMA were incubated in DMEM only. In the RESV I-V groups, PMA were incubated in DMEM containing LPS (10 microg/ml) and different concentrations of RESV. After 24 hours of incubation, NF-kappaB activity in PMA, and the levels of tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1) and nitric oxide (NO) in the culture medium were measured. In the concentrations of 1.25-5 microg/ml, RESV had a dose- dependent inhibitive effect on NF-kappaB activity in PMA as well as the expressions of TNF-alpha, IL-1 and NO in the culture medium contrasted with the LPS group. There was no significant difference in the levels of these pro-inflammatory factors between the groups of 5 microg/ml and 10 microg/ml RESV. In conclusion, RESV has the potential for the future application of preventing inflammatory diseases involving PMA.

Effects of emodin and double blood supplies on liver regeneration of reduced size graft liver in rat model.

AIM: To study the influences of emodin and reconstruction of double blood supplies on liver regeneration of reduced size graft liver in rat model. METHODS: A total of 45 SD-SD rat reduced size liver transplantation models were randomly divided into three groups (A-C). The conventional reduced size liver transplantation was performed on rats in group A, while the hepatic artery blood supply was restored in groups B and C. The emodin (1.5 mg/kg/d) was given by intraperitoneal route in group C only. The recipients were killed on the seventh day after the operation. The proliferative cell nuclear antigen (PCNA), TBil and ALT of serum were detected, and the pathological changes of liver cell were observed. RESULTS: The numbers of the rats that survived in A, B, and C group on the seventh day after operation were 14, 13, 13, respectively. The levels of TBil (31.5+/-5.2 micromol/L, 23.2+/-3.1 micromol/L vs 38.6+/-6.8 micromol/L), and ALT (5 351+/-1 050 nKat, 1300+/-900 nKat vs 5779+/-1202 nKat) in serum in groups B and C were lower than those in group A (P<0.05), while the expression of PCNA in groups B or C was higher than that in group A (22.0+/-3.5%, 28.2+/-4.2% vs 18.6+/-3.2%, P<0.05). The deeper staining nuclei, double nuclei, multi-nuclei and much glycogen were observed in liver cells of groups B and C, especially in group C, while fewer were found in liver cells of group A. CONCLUSION: The reconstruction of arterial blood supply is very important for rat liver regeneration after reduced size liver transplantation. Emodin has the effect of promoting liver regeneration and improving liver function in rats after reduced size transplantation. The possible mechanism is improving proliferation of liver cell and protecting liver cells from injury.

Resveratrol prolongs allograft survival after liver transplantation in rats.

AIM: To study the immuno-modulatory effect of resveratrol (RES) on allograft rejection after liver transplantation in rats. METHODS: Male Sprague-Dawley (SD) rats were selected as donors and male Wistar rats as recipients for a rejection model. The recipients were divided into four groups after orthotopic liver transplantation (OLTx). In the RES A, B, and C groups, RES was given intra-peritoneally once a day (25, 50, and 100 mg/kg, respectively) after OLTx, whereas in the control group, vehicle buffer was given intra-peritoneally once a day. The survival time, serum chemistry, production of cytokines, activation of transcription factor NF-kappaB, and histopathologic findings were then compared among these groups. RESULTS: The mean survival time after OLTx in the RES C group was significantly longer than that in the control group (16.7+/-1.2 d vs 9.3+/-0.6 d, P<0.01). On the 7th post-transplant day the serum albumin level significantly improved in the RES C group, the serum total bile acid and alanine aminotransferase (ALT) levels were significantly lower in the RES C group, the serum IL-2 and INF-gamma levels were significantly lower in the RES C group, and the activation of transcription factor NF-kappaB in peripheral blood T lymphocytes was significantly suppressed in the RES A, B, and C groups in comparison to those in the control group. On the 7th post-transplant day, a histological examination revealed apparent difference in the severity of rejection between the RES C group and control group. CONCLUSION: RES has an immuno-suppressive property as well as protective effect on hepatocytes under allograft rejection. It might serve as a novel agent for reducing the severity of hepatic allograft rejection in rats.

Rat bone marrow mesenchymal stem cells differentiate into hepatocytes in vitro.

AIM: To investigate the mechanism and regulation of differentiation from bone marrow mesenchymal stem cells (MSCs) into hepatocytes and to find a new source of cell types for therapies of hepatic diseases. METHODS: MSCs were isolated by combining gradient density centrifugation with plastic adherence. The cells were cultured in osteogenic or adipogenic differentiation medium and determined by histochemical staining. MSCs were plated in plastic culture flasks that were not coated with components of extracellular matrix (ECM). When MSCs reached 70% confluence, they were cultured in low glucose Dulbecco's modified Eagle's medium supplemented with 10 mL/L fetal bovine serum, 20 ng/mL hepatocyte growth factor (HGF) and 10 ng/mL fibroblast growth factor-4 (FGF-4). The medium was changed every 3 d and stored for albumin, alpha-fetoprotein (AFP) and urea assay. Glycogen store of hepatocytes was determined by periodic acid-Schiff staining. RESULTS: By combining gradient density centrifugation with plastic adherence, we isolated a homogeneous population of cells from rat bone marrow and differentiated them into osteocytes and adipocytes. When MSCs were cultured with FGF-4 and HGF, approximately 56.6% of cells became small round and epithelioid on d 24 by morphology. Compared with the control, levels of AFP increased significantly from d 12 to 15.5+/-1.4 microg/L (t = 2.31, P<0.05) in MSCs cultured with FGF-4 and HGF, and were higher (46.2+/-1.5 microg/L) on d 21 (t = 41.926, P<0.01), then decreased to 24.8+/-2.2 microg/L on d 24 (t = 10.345, P<0.01). Albumin increased significantly on d 21 (t = 3.325, P<0.01) to 1.4+/-0.2 microg/mL, and to 2.1+/-0.7 microg/mL on d 24 (t = 3.646, P<0.01). Urea (2.3+/-0.4 mmol/L) was first detected on d 21 (t = 6.739, P<0.01), and continued to increase to 2.6+/-0.9 mmol/L on d 24 (t = 4.753, P<0.01). Glycogen storage was first seen on d 21. CONCLUSION: The method combining gradient density centrifugation with plastic adherence can isolate MSCs. Rat MSCs may be differentiated into hepatocytes by FGF-4 and HGF. Cytokines may play a more important role in differentiation from rat MSCs into hepatocytes.