Effects of Traditional Herbal Formulae on Human CYP450 Isozymes

Objective:To assess the effects of traditional herbal formulae Sijunzi Decoction(四君子汤,Sagunja-tang,SJZD),Siwu Decoction(四物汤,Samul-tang,SWD),Bawu Decoction(八物汤,Palmul-tang,BWD)and Shiquan Dabu Decoction(十全大补汤,Sipjeondaebo-tang,SDD) on the activities of human cytochrome P450(CYP450),a drug-metabolizing enzyme.Methods:Herbal formula water extracts were filtered and lyophilized after the powder extracts were dissolved in distilled water.The activities of major human CYP450isozymes(CYP3A4,CYP2C19,CYP2D6 and CYP2E1) were measured using in vitro fluorescence-based enzyme assays.The inhibitory effects of the herbal formulas on the activities of CYP450 were characterized as half maximal inhibition concentration(IC_(50)) values.Results:All the tested herbal formulae inhibited CYP2C19activity(IC_(50):SJZD,83.28 μg/mL;SWD,235.54 μg/mL;BWD,166.82 μg/mL;SDD,178.19 μg/mL);SJZD(lC_(50) = 196.46 μ g/mL),SWD(IC_(50) = 333.42 μ g/mL) and SDD(IC_(50) = 163.42 μg/mL) inhibited CYP2E1-mediated metabolism;whereas BWD exhibited comparatively weak inhibition of CYP2E1(IC_(50) = 501.78 μg/mL).None of the four herbal formulas significantly affected CYP3A4 or CYP2D6.Conclusions:These results suggest that SJZD,SWD,BWD and SDD could potentially inhibit the metabolism of co-administered synthetic drugs whose primary route of elimination is via CYP2C19.In addition,clinically relevant pharmacokinetic interactions could occur when SJZD,SWD or SDD is co-administered with drugs metabolized by CYP2E1.Our findings provide information for the safety and effective clinical use of these four classic herbal formulas.     

Astragaloside Ⅳ Protects Against Aβ1-42-induced Oxidative Stress,Neuroinflammation and Cognitive Impairment in Rats

Objective To investigate the neuroprotective action of astragaloside Ⅳ (AS-Ⅳ) on spatial learning and memory impairment induced by amyloid-beta 1-42 (Aβ1-42) in rats and elucidate its underlying molecular mechanisms. Methods Adult-male Sprague-Dawley rats (230-250 g) were divided into six groups randomly: control, Aβ1-42, AS-Ⅳ, Aβ1-42 plus 5 mg/kg·d AS-Ⅳ, Aβ1-42 plus 25 mg/kg·d AS-Ⅳ, and Aβ1-42 plus 50 mg/kg·d AS-Ⅳgroups. Aβ1-42 were delivered by intracerebroventricular injection under the guidance of a brain stereotaxic apparatus. The Morris water maze test (hidden platform test, probe trials, visible platform test) was performed one week after Aβ1-42 injection to obtain the ability of rat spatial learning and memory. AS-Ⅳ (5, 25 and 50 mg/kg·d) was administrated intraperitoneally once per day from the 8th day after Aβ1-42 injection for 5 consecutive days. Average escape latencies, distances for searching for the platform under water and the percentage of total time elapsed and distance swam in the right quadrant after removing platform were determined by behavior software system. The vision and swim speeds of rats were also determined to exclude the effect of these factors on the parameters of learning and memory. After behavioral tests, the rats were sacrificed immediately by decapitation. Hippocampus were collected. The enzyme activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-px) and catalase (CAT) in the hippocampus obtained from different-treated rat brain were measured by following the manufacturer's instructions. The levels of interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) in tissue lysates were assayed with ELISA. Results The water maze test results indicated that chronic treatments with AS-Ⅳ effectively protected the rats from Aβ1-42-induced spatial learning and memory impairment. Furthermore, the activities of SOD, GSH-px and CAT decreased by Aβ1-42 were also restored by AS-Ⅳ treatment in the hippocampus of rats. In addition, AS-Ⅳsignificantly decreased the levels of IL-1β and TNF-α in the hippocampus of Aβ1-42-induced amnesia's rats. Conclusion Our findings suggest that AS-Ⅳ might be a useful chemical in improving the spatial memory and relieving the oxidative stress and neuroinflammation in Alzheimer patients.     

Reduction in bile acid pool causes delayed liver regeneration accompanied by down-regulated expression of FXR and c-Jun mRNA in rats

The present study attempted to examine the effects of bile acid pool size on liver regeneration after hepatectomy.The rats were fed on 0.2% cholic acid(CA)or 2% cholestyramine for 7 days to induce a change in the bile acid size,and then a partial hepatectomy(PH)was performed.Rats fed on the normal diet served as the controls.Measurements were made on the rate of liver regeneration,the labeling indices of PCNA,the plasma total bile acids(TBA),and the mRNA expression of cholesterol 7alpha-hydroxylase(CYP7A1),...     

Effect of Yuxingeng Fluid (愈心梗液) on Myocardial Energy Metabolism in Wistar Rats with Acute Myocardial Infarction

Objective: To examine the effect of Yuxingeng fluid (愈心梗液, YXGF) on myocardial energy metabolism in Wistar rats with acute myocardial infarction (AMI) by observing the ultrastructure of mitochondria and the enzyme activities of rat myocardial adenosine triphosphate (ATP), succinate dehydrogenase (SDH), acid phosphatase (ACP), alkaline phosphatase (ALP) and the content of glycogen. Methods; AMI models were established by ligature of left anterior descending coronary artery and then the rats with AMI were randomly divided into 7 groups: namely, blank group, model group, sham-operated group, captopil group, high-dose YXGF group, middle-dose YXGF group and low-dose YXGF group. From the next day after modeling, the rats were given YXGF through gastrogavage which lasted for 4 weeks. And then, the ultra-structure of mitochondria was observed by electronic microscope and the enzyme activities of ATP, SDH, ACP, ALP and the content of glycogen were determined. Results: Compared with model group, the other thr     

The signal transduction pathway in the proliferation of airway smooth muscle cells induced by urotensin Ⅱ

Background Human urotensin Ⅱ (UⅡ) is the most potent mammalian vasoconstrictor identified so far. Our previous study showed that UⅡ is a potent mitogen of airway smooth muscle cells (ASMC) inducing ASMC proliferation in a dose-dependent manner. The signal transduction pathway of UⅡ mitogenic effect remains to be clarified. This study was conducted to investigate the signal transduction pathway in the proliferation of ASMC induced by UⅡ. Methods In primary cultures of rat ASMCs, activities of protein kinase C (PKC), mitogen-activated protein kinase (MAPK) and calcineurin (CaN) induced by UⅡ were measured. The effect of CaN on PKC and MAPK was studied by adding cyclosporin A (CsA), a specific inhibitor of CaN. Using H7 and PD98059, inhibitors of PKC and MAPK, respectively, to study the effect of PKC and MAPK on CaN. The cytosolic free calcium concentration induced by UⅡ was measured using Fura-2/AM. Results UⅡ 10-7 mol/L stimulated ASMC PKC and MAPK activities by 44% and 24% (P<0.01), respectively, after incubating for 20 minutes. It increased CaN activity in a time-dependent manner, being 1.68 times as that of control for 24 hours (P<0.01). It promoted the cytosolic free calcium concentration increase of 18% (P<0.01). CsA 10-6 mol/L and H7 50 μmol/L inhibited UⅡ-stimulated CaN activity by 45% (P<0.01) and 21% (P<0.05), respectively, while PD98059 50 μmol/L had no effect on CaN activity (P>0.05). CsA 10-6 mol/L inhibited UⅡ-stimulated PKC activity by 14% (P<0.05), while having no effect on MAPK activity (P>0.05). Conclusions UⅡ increases cytosolic free calcium concentration and activates PKC, MAPK and CaN. The signal transduction pathway between PKC and CaN has cross-talk.     

Inhibitive effect of cremophor RH40 or tween 80-based self-microemulsiflying drug delivery system on cytochrome P450 3A enzymes in murine hepatocytes

This study examined the effect of self-microemulsiflying drug delivery system (SMEDDS) containing Cremophor RH40 or Tween 80 at various dilutions on cytochrome P450 3A (CYP3A) enzymes in rat hepatocytes, with midazolam serving as a CYP3A substrate.The particle size and zeta potential of microemulsions were evaluated upon dilution with aqueous medium.In vitro release was detected by a dialysis method in reverse.The effects of SMEDDS at different dilutions and surfactants at different concentrations on the metabolism of MDZ were investigated in murine hepatocytes.The cytotoxicity of SMEDDS at different dilutions was measured by LDH release and MTT technique.The effects of SMEDDS on the CYP3A enzymes activity were determined by Western blotting.Our results showed that dilution had less effect on the particle size and zeta potential in the range from 1:25 to 1:500.The MDZ was completely released in 10 h.A significant decrease in the formation of 1’-OH-MDZ in rat hepatocytes was observed after treatment with both SMEDDS at dilutions ranging from 1:50 to 1:250 and Cremophor RH 40 or Tween 80 at concentrations ranging from 0.1% to 1% (w/v), with no cytotoxicity observed.A significant decrease in CYP3A protein expression was observed in cells by Western blotting in the presence of either Cremophor RH40 or Tween 80-based SMEDDS at the dilutions ranging from 1:50 to 1:250.This study suggested that the excipient inhibitor-based formulation is a potential protective platform for decreasing metabolism of sensitive drugs that are CYP3A substrates.     

Inhibitory Effect of TanshinoneⅡA on TGF-β1-induced Cardiac Fibrosis

This study examined the effect of tanshinoneⅡA (TSNⅡA) on the cardiac fibrosis induced by transforming growth factor β1 (TGF-β1) and the possible mechanisms. Cardiac fibroblasts were isolated from cardiac tissues of neonatal Sprague-Dawley (SD) rats by the trypsin digestion and differential adhesion method. The cells were treated with 5 ng/mL TGF-β1 alone or pretreated with TSNⅡA at different concentrations (10–5 mol/L, 10–4 mol/L). Immunocytochemistry was used for cell identification, RT-PCR for detection of the mRNA expression of connective tissue growth factor (CTGF) and collagen type Ⅰ (COLⅠ), Western blotting for detection of the protein expression of Smad7 and Smad3, and immunohistochemistry and immunofluorescence staining for detection of the protein expression of phosphorylated Smad3 (p-Smad3), CTGF and COLⅠ. The results showed that TGF-β1 induced the expression of CTGF, COLⅠ, p-Smad3 and Smad7 in a time-dependent manner. The mRNA expression of CTGF and COLⅠ was significantly increased 24 h after TGF-β1 stimulation (P<0.01 for all). The protein expression of p-Smad3 and Smad7 reached a peak 1 h after TGF-β1 stimulation, much higher than the baseline level (P<0.01 for all). Pretreatment with high concentration of TSNⅡA resulted in a decrease in the expression of p-Smad3, CTGF and COLⅠ (P<0.01). The protein expression of Smad7 was substantially upregulated after pretreatment with two concentrations of TSNⅡA as compared with that at 2h post TGF-β1 stimulation (P<0.05 for low concentration of TSNⅡA; P<0.01 for high concentration of TSNⅡA). It was concluded that TSNⅡA may exert an inhibitory effect on cardiac fibrosis by upregulating the expression of Smad7, suppressing the TGF-β1-induced phosphorylation of Smad3 and partially blocking the TGF-β1-Smads signaling pathway.     

Linkage of the cholesterol 7α-hydroxylase gene and low-density lipoprotein cholesterol conditional on apolipoprotein E association: the National Heart, Lung, and Blood Institute Family Heart Study

Background Genetic factors account for approximately 50% of the individual variation in plasma low-density lipoprotein cholesterol (LDL-C) concentrations in the general population. Several candidate genes have been proposed but their relative contributions to the variance in LDL-C are not known, except for apolipoprotein E (apoE). We report here an investigation of the relationship between LDL-C and cholesterol 7α-hydroxylase (CYP7), as well as apoE and low-density lipoprotein receptor (LDLR), three pixotal genes in LDL metabolism.Methods Our study population included more than 200 nuclear families with increased coronary heart disease (CHD) risk from the National Heart, Lung, and Blood Institute (NHLBI) Family Heart Study. Variance-component linkage methods, a measured genotype approach, and a variance-component linkage analysis conditional on a measured genotype association were used.Results The results showed significant linkage between a genetic determinant of plasma LDL-C concentrations and a polymorphism near CYP7 with its allelic variation accounting for 27% of the total LDL-C variation. There is significant association between plasma LDL-C concentrations and apoE genotypes. Conditional on the apoE association, the total LDL-C variation accounted by allelic variation of a polymorphism near CYP7 was increased significantly.Conclusion Our results suggest the apoE and CYP7 may be two important genes accounting for the genetic variation of plasma LDL-C concentrations in a population with cardiovascular diseases.     

Shenling Baizhu Powder (参苓白术散) Ameliorates Pi (Spleen)-Deficiency-Induced Functional Diarrhea in Rats

Objective To explore the mechanism of Pi(Spleen)-deficiency-induced functional diarrhea(FD)model rats treated by Shenling Baizhu Powder(参苓白术散,SBP).Methods Thirty male Sprague-Dawley rats were randomly divided into 5 groups including control,model,low-,medium-,and high-dose SBP groups(SBPLDG,SBPMDG,SBPHDG),6 rats in each group,respectively.Pi-deficiency-induced FD rats model was developed through Radix et Rhizoma Rhei gavage for 7 days.After modeling,the rats were treated with 3 doses of SBP[0.93,1.86,and 3.72 g/(kg·d)],and the rats in the control and model groups were given pure water for 7 days.The diarrhea index was calculated.On the 7th and 14th days,the traveled distance of rat was measured by the open field test.Serum D-xylose content was determined by the phloroglucinol method and interleukin(IL)-10 and IL-17 levels were measured using an enzyme-linked immunosorbent assay kit.The content of Treg cells was determined by flow cytometry.Results Compared with the control group,the diarrhea index and IL-17 level in the model group were significantly higher and the total exercise distance and D-xylose content significantly decreased(P>0.05).The expression of IL-10 in the SBPHDG group was significantly up-regulated,and serum D-xylose level and Treg cells increased significantly compared with the model group(P>0.05).Conclusion High-dose SBP exhibited ameliorating effects against Pi-deficiency induced FD,which might be attributed to its modulations on intestinal absorption function as well as adaptive immunity in mesenteric lymph nodes of rat.     

雷公藤甲素对大鼠体内环磷酰胺药代动力学的影响：对细胞色素P3A4可能的抑制作用

Objective:To evaluate the effect of a 10-day course of triptolide(TP) on rat cytochrome(CY)P3A4 activity,and on the pharmacokinetics of cyclophosphamide(CPA).Methods:In the pharmacokinetics experiment,rats were orally given 0.9%NaCI solution(n=5) and TP[1.2(mg/kg·d)]for 10 days and a single dose of CPA was administered intravenously(100 mg/kg) to rats on day 11.Blood samples were collected up to 4 h at predetermined time intervals,the plasma concentration of CPA was determined by high performance liquid chromatography(HPLC) and pharmacokinetic parameters were determined.In the in vitro CYP3A4 activity inhibition research,rat blank liver microsomes were divided into 3 groups:a control group,a TS(5 μ L,200 μmol/L) with TP(5 μL,12.5 μmol/L) group,a TS with ketoconazole(5 μL,1 μmol/L) group.Concentration of 6 β-hydroxylated testosterone(6 β-OHT) in liver microsomes was measured by HPLC and the activity of CYP 3A4 was calculated through the following formula:E_(inhibitor)/E_(control)×100%=C_(inhibitor)/C_(control)× 100%.Results:Compared with the control group,the area under the plasma concentration-time curve(AUC_(0-∞)) of CPA was significantly increased by 229.05%pretreated with TP(P0.01).Peak plasma concentrations(C_(max))of CPA was significantly increased and plasma half-life was correspondingly extended.The CYP3A4 activity was significantly inhibited by ketoconazole 93.5%±0.2%and TP 84.6%±0.3%compared with the control group(P0.01 and P0.05,respectively).Conclusion:Our results strongly suggested that long-term oral intake of TP can distinctly inhibit the CYP3A4 activity and this inhibition evidently decrease the formation of toxic metabolites of CPA.     

The role of vasoactive substances in hyperhemodynamics after orthotopic liver transplantation in cirrhotic rats

Objective To evaluate the role of endogenous vasoactive substances in hyperdynamic circulation after orthotopic liver transplantation (OLT) in cirrhotic rats. Methods Male SD rats were randomly divided into 4 groups: normal controls (NL, n=10), rats with intrahepatic portal hypertension (IHPH, n=10), normal rats with OLT (NL-OLT, n=9), and IHPH rats with OLT (IHPH-OLT, n=16). IHPH-OLT rats were divided into 2 subgroups: Group A (3 days after OLT, n=9) and Group B (7 days after OLT, n=7). IHPH was induced by injection of CCI(4) and OLT was performed using cuffs for the anastomosis of suprahepatic inferior vena cava, infrahepatic vena cava and portal vein. Radioactive microsphere method was used for hemodynamic study. The concentrations of plasma glucagon (Glu), nitric oxide (NO), prostaglandin (PGI(2)), thromboxaneA(2) (TXA(2)) and endothelin (ET) were measured by radioimmunoassay. Results No significant difference in hemodynamic changes was observed between NL-OLT and NL rats, except for mean arterial blood pressure. No significant changes in NO and PGI(2) were seen between NL-OLT and NL rats. Glu, ET and TXA(2) were significantly elevated in NL-OLT rats compared with NL rats (P&lt;0.05). Characteristics of systemic and splanchnic hyperdynamic circulatory states were observed in IHPH, IHPH-OLT A, IHPH-OLT B rats. Both the magnitude of hyperhemodynamics and increasing concentrations of Glu and NO occurred in the order of IHPH&gt;IHPH-OLT A&gt;IHPH-OLT B rats. The level of plasma PGI(2) in IHPH rats was significantly elevated compared with NL rats, while PGI(2) in IHPH-OLT A and B rats was found to be lower than in IHPH rats (P&lt;0.05). There was no obvious difference in PGI(2) between IHPH-OLT B and NL rats. Vasoconstrictors including ET and TXA(2) were found elevated in IHPH-OLT rats. Conclusions OLT does not induce postoperative hyperhemodynamics per se. Vasodilators including NO and Glu, especially NO, play an important role in the hyperhemodynamics of IHPH and IHPH-OLT rats. The results of the present study demonstrate that the persistence of systemic and splanchnic hyperkinetic circulation in the early stages after OLT may result from those non-eliminated factors that caused hyperhemodynamics in liver cirrhosis patients with portal hypertension before OLT.     

Ping-tang Recipe (平糖方) improves insulin resistance and attenuates hepatic steatosis in high-fat diet-induced obese rats

Objective:To investigate the therapeutic effects of Ping-tang Recipe(平糖方,PTR) on highfat diet(HFD)-induced insulin resistance and non-alcoholic fatty liver disease(NAFLD),and to elucidate the underlying mechanisms.Methods:Forty male SD rats were included in the study.Ten rats were fed on normal diet as normal control,and thirty rats were fed on HFD for 8 weeks to induce obesity,followed with low dose (0.42 g/kg) or high dose(0.84 g/kg) of PTR or vehicle for 8 weeks with 10 animals for each group.Glucose metabolism and insulin sensitivity were evaluated by oral glucose tolerance test and insulin tolerance test. Hepatic steatosis was measured by immunohistochemistry.Liver lipid metabolic genes were analyzed by quantitative real-time polymerase chain reaction,while AMP-activated protein kinase(AMPK) expression was examined by Western blot.Results:Rats fed on HFD developed abdominal obesity,insulin resistance and NAFLD.PTR treatment reduced visceral fat(peri-epididymal and peri-renal) accumulation,improved glucose metabolism,and attenuated hepatic steatosis.The expressions of the key lipolytic regulating genes,including peroxisome proliferators-activated receptorγco-activator 1α(PGC-1α),peroxisome proliferator-activated receptorγ(PRAR-γ) andα(PRAR-α),were up-regulated(P<0.05 or P<0.01),while the expressions of lipogenic genes such as sterol regulatory element-binding protein 1c(SREBP-1c),fatty acid synthase (FAS) and liver fatty acid-binding protein(L-FABP) were down-regulated(P<0.05 or P<0.01).In addition, PTR activated AMPK and promoted acetyl-CoA carboxylase phosphorylation in the liver.Conclusions:PTR improves insulin resistance and reverse hepatic steatosis in the rat model of HFD-induced obesity through promotion of lipolysis and reduction of lipogenesis,which involves the AMPK signaling pathway,thus representing a new therapeutic intervention for obesity related insulin resistance and NAFLD.     

引经药独活根合二妙方可促进骨关节炎软骨损伤大鼠骨髓造血干细胞归巢

Objective:To investigate the effect of Ermiao Recipe(二妙方,EMR)with medicinal guide Angelicae Pubescentis Radix(APR)on the homing of bone marrow stem cells(BMSCs)to focal zone in osteoarthritis(OA)rats.Methods:Forty-eight Sprague-Dawley rats were randomly assigned to the sham-operated,model,EMR,and EMR plus APR groups(12 rats in each group).The OA rat model was induced by anterior cruciate ligament transection and medial meniscus resection.All rats were injected with recombinant human granulocyte colonystimulating factor[rhG-CSF,30μg/(kg-d)for continuous 7 days],and rats in the EMR and EMR plus APR groups were treated with EMR or EMR plus APR at 1.6 or 1.9 g/(kg·d)for 3 or 6 weeks,respectively.Cartilage histopathologic changes were observed by hematoxylin and eosin staining.Chondrocytes apoptosis and cartilage matrix components were tested by transferase-mediated deoxyuridine triphosphate-biotin nick end labeling assay and special staining,lnterleukin-1 B(IL-1β),tumor necrosis factorα(TNF-α),bone morphogenetic protein 2(BMP-2),and transforming growth factor beta-1(TGF-β_1)in serum were detected by enzyme-linked immunosorbent assay or radioimmunoassay assay.Matrix metalloproteinase(MMP)-13,tissue inhibitors of metalloproteinase(TIMP)-1,bromodeoxyuridine(BrdU),cluster of differentiation 34(CD34),and stromal cell derived factor 1(SDF-1)were measured by immunohistochemistry assay.Results:EMR and EMR plus APR significantly inhibited articular cartilage damage and synovium inflammation in OA rats at 3 or 6 weeks of treatment,the most obvious changes in these parameters were found in the EMR plus APR group.At 6 weeks,compared with EMR treatment,EMR plus APR remarkably inhibited chondrocytes apoptosis and the release of IL-1βand TNF-α,obviously decreased MMP-13 expression,and significantly increased expressions of proteoglycan,collagen,typeⅡcollagen and TIMP-1,serum levels of BMP-2 and TGF-β_1 as well as expressions of BrdU,CD34 and SDF-1 in cartilage articular(P0.01 or P0.05).Conclusion:The medicinal guide APR improved the therapeutic effects of EMR on OA rats by promoting directional homing of BMSCs to focal zone.     

生脉注射液对接受阿霉素治疗大鼠膈肌收缩力的作用

Objective:To explore the diaphragmatic toxicity in doxorubicin(DOX)-treated rats and the related mechanisms,as well as the effects of Shengmai Injection(SMI,生脉注射液)on the diaphragmatic dysfunction.Methods:Thirty Sprague-Dawley male rats were randomly divided into three groups:control,DOX-treated and DOX+SMI treated groups.DOX was given to rats in DOX and DOX+SMI groups in 6 equal doses[2.5 mg/kg,intraperitoneal injection(i.p.)],on alternate days,over a period of 2 weeks for a cumulative dose of 15 mg/kg.SMI was given to DOX+SMI rats in 12 doses(3 mL/kg,i.p.)for a period of 2 weeks before the administration of DOX and 2 weeks during the administration of DOX.The rats in the control group received equal volume of normal saline.Subsequently,the twitch and tetanic characteristics and force-frequency relationships,and the malondialdehyde(MDA)levels and the superoxide dismutase(SOD)activities,as well as the mRNA content and proteins of inducible nitric oxide synthase(iNOS)were determined.Results:The DOX-treated rats had decreased the peak twitch tension(Pt),maximal tetanic tension(P_0)and force-frequency relationship as compared with the control rats(P0.01),while the diaphragm contractility in rats treated with SMI were significantly higher than that in DOX-treated rats(P0.01).The DOX-treated rats had increased MAD levels and decreased SOD activities(P0.05),and SMI decreased the MDA levels and increased the SOD activities in DOX-treated rats(P0.05).Ultrastructure of diaphragm in the DOX-treated rats revealed typical alterations including fracture of diaphragm fibers,and edema and degeneration of mitochondria;these changes were relieved by SMI treatment.The mRNA content and protein of iNOS in DOX-treated rats were remarkably higher than those in control rats(P0.01),while SMI decreased the mRNA expression level of iNOS in DOX-treated rats(P0.05).Conclusions:Lipid peroxidation is responsible for DOX-induced diaphragm toxicity.SMI protects diaphragm muscles and their function from DOX impairment,and these beneficial effects may be somehow correlated with the decrease in expression of iNOS and lipid peroxidation.     

Possible Role of Mast Cells and Neuropeptides in the Recovery Process of Dextran Sulfate Sodium-induced Colitis in Rats

Objective To clarify the role of mast cells and neuropeptides substance P(SP),somatostatin(SS),and vasoactive intestinal peptide(VIP) in dextran sulfate sodium(DSS)-induced colitis in rats.Methods Experimental colitis was induced in Sprague-Dawley rats(180-200 g,n=20) by oral ingestion of 4%(w/v) DSS in drinking water for 7 days.Control rats(n=5) drank water and were sacrificed on day 0.Mast cell number,histamine levels in whole blood and tissue,tissue levels of SP,SS and,VIP in the distal colon of the rats were measured on day 8,day 13,and day 18 of experimentation.Results Oral administration of 4% DSS solution for 7 days resulted in surface epithelial loss and crypt loss in the distal colon.Mast cell count increased on day 8(1.75±1.09/mm vs.0.38±0.24/mm,P<0.05) and day 13(1.55±1.01/mm vs.0.38±0.24/mm,P<0.05) after DSS treatment.Whole blood histamine levels were increased on day 8(266.93±35.62 ng/mL vs.76.87±32.28 ng/mL,P<0.01) and gradually decreased by day 13 and day 18 after DSS treatment.Histamine levels in the distal colon were decreased on day 8(1.77±0.65 ng/mg vs.3.06±0.87 ng/mg,P<0.05) and recovered to control levels by day 13 after DSS treatment.SP level in the distal colon gradually increased and were raised significantly by day 13(8777.14±3056.14 pg/mL vs.4739.66±3299.81 pg/mL,P<0.05) after DSS treatment.SS and VIP levels in the distal colon were not changed.Conclusions Mast cell degranulation followed by histamine release may play an important role in the pathogenesis of colitis induced by DSS.SP may be a significant substance in the progression of inflammation and the recovery process of DSS-induced colitis.     

Short-andLong-Term Effects of Xuezhikang(血脂康),An Extract of Cholestin,on Serum Proprotein Convertase Subtilisin/Kexin Type 9 Levels

Objective:To investigate the short- and long-term effects of Xuezhikang(血脂康,XZK),an extract of Cholestin,on proprotein convertase subtilisin/kexin type 9(PCSK9) level.Methods:Thirty rats were randomly divided into three groups and were given saline,XZK 1,200 mg/kg or lovastatin 10 mg/kg respectively by daily gavage for 3 days(n=10 for each).Sixteen patients without previous iipid-lowering drug treatment for dyslipidemia received XZK 1,200 mg daily for 8 weeks.Fasting blood samples and liver tissue were collected at day 3 for rats,while the blood samples were obtained at baseline and week 8 from patients.The serum PCSK9 and lipid profile were measured.The expression of hepatic low density lipoprotein(LDL) receptor and sterol regulatory element binding protein 2(SREBP-2) were measured by real time-PCR.Results:PCSK9 levels in rats were significantly increased in the XZK and lovastatin groups(P=0.002,P=0.003 vs.control) at day 3,while no significant differences were found in the levels of lipid parameters.PCSK9 levels in patients increased by34%(P=0.006 vs.baseline) accompanied by total cholesterol and LDL-cholesterol decreased by 22%and 28%(P=0.001,P=0.002 vs.baseline).The hepatic mRNA levels of LDL-receptor and SREBP-2 were significantly increased in the XZK and lovastatin groups.Conclusion:XZK has significant impact on PCSK9 in a short- and long-term manner in both rats and humans.Moreover,the data indicated that as lovastatin,XZK increased PCSK9 levels through SREBP-2 pathway.     

The bcl-2 mRNA Expression in GCDC-induced Obstructive Jaundice in Rats and Its Implication in Hepatocellular Apoptosi

The modulatory role of bcl-2 gene in hepatocellular apoptosis of rats with glycochenodeoxycholate (GCDC)-induced obstructive jaundice was investigated. The hepatocytes in normal rats and those with bile duct-ligation for 7 days, 14 days and 21 days were isolated and obtained by in situ collagenase perfusion and primary culture. The expression of bcl-2 mRNA in the hepatocytes was detected by RT-PCR. Primary culture was performed on the hepatocytes from normal rats and those with bile duct-ligation for 14 days. 100 μmol/L GCDC was added to the hepatocytes for incubation for 24 h. The hepatocellular apoptotic ratio was measured by using FCM and hepatocellular apoptosis detected in situ by using TUNEL technique. Results showed that the expression of bcl-2 mRNA was not detectable in the hepatocytes of normal rats by RT-PCR technique, while detectable in the hepatocytes of those with bile duct ligation (BDL) for 7, 14 and 21 days. Hepatocellular apoptosis in the BDL group was obviously decreased as compared with normal control group after addition of 100μmol/L GCDC to the cells for 24 h. It was concluded that the hepatocytes in the BDL rats expressed bcl-2. During obstructive jaundice, expression of bcl-2 from the hepatocytes can inhibit the bile saltinduced hepatocellular apoptosis.     

Effects of heme precursors on CYP1A2 and POR expression in the baculovirus/Spodoptera frugiperda system

Objective: CYP1A2 and NADPH-CYP450 oxidoreductase (POR) were expressed in the baculovirus/Spodoptera frugiperda (sf9) system. The aim of this study was to investigate the effects of heme precursors on the expression of CYP1A2 and POR. Methods: The heme precursors [δ-Aminolaevulinic Acid (5-ALA), Fe3+ and hemin] were introduced into the system to evaluate their effects on the expression of CYP1A2, POR and their co-expression. All the proteins were identified using immunoblotting, CO-difference spectroscopy, or cytochrome c assay. Results: In the present study, functional CYP1A2 and POR were successfully expressed in the baculovirus/sf9 system, and both of them showed high activities. Co-addition of 5-ALA and Fe3+ significantly improved expression of CYP1A2 by about 50% compared with the addition of 5-ALA, Fe3+ or hemin alone. Either co-addition of 5-ALA and Fe3+ or addition of 5-ALA or Fe3+ alone improved the POR expression level 2 fold and its activity 7-10 fold compared with control (no addition). However, unlike CYP1A2, there was no difference between the co-addition and addition of these heme precursors alone. Different ratios of BvCYP1A2 to BvPOR also affected the co-expression of CYP1A2 and POR, with a 3:1 ratio of BvCYP1A2 / BvPOR significantly increasing their co-expression. Surprisingly, the addition of 0.1 mM 5-ALA or Fe3+ alone, but not their co-addition, could significantly improve the CYP1A2 and POR co-expression (P < 0.05). Conclusion: 5-ALA and Fe3+ increased the expression of CYP1A2 and POR in a baculovirus/sf9 system, but the pattern of their expression was different between their expression alone and co-expression.     

Study on the Analgesic Effect and Mechanism of Zhitong Capsule (止痛胶囊)in Adjuvant Arthritis Rats

Objective:To observe the analgesic effect of Zhitong Capsule (止痛胶囊, ZTC) and study its mechanism in adjuvant arthritis (AA) rats. Methods: Forty-eight male Sprague-Dawley rats were randomly divided into six groups with 8 rats in each group. On the first day, except to those in the normal group that were treated with normal saline, the same amount of Freund's complete adjuvant (FCA) was given through intradermal injection into the right hind paw to all the rats in the other groups. From the 17th day of the modeling on, the rats in groups of ZTC were administered daily through gastrogavage with a dose of 1000, 500,250 mg/kg respectively, while equal volume of normal saline was given to those in the normal group and model group, and an equal volume of aspirin (ASA) solution was given to rats in the ASA group through gastrogavage for 10 days, once per day, and on the 27th day, the analgesic effect of ZTC was measured with heat withdraw method. The activities and contents of superoxide dismutase (SOD) and lipid peroxides (LPO) in serum were observed by spectrophotometry, and the level of beta-endorphin (β-EP) in hypothalamus were determined by the assay of immunohistochemistry. Results: ZTC showed significant effects on enhancing the pain threshold and at the same time it increased the activities of SOD and reduced the contents of LPO in serum. ZTC could also increase the level of β-EP in hypothalamus. Conclusion: ZTC has analgesic effect and its mechanism is probably related with its effect in inhibiting the level of oxygen free radicals in serum and increasing the level of β-EP of hypothalamus in rats.