Upregulation of microRNA-320 decreases the risk of developing steroid-induced avascular necrosis of femoral head by inhibiting CYP1A2 both in vivo and in vitro.

Steroid-induced avascular necrosis of femoral head (SANFH) occurs frequently in patients receiving high-dose steroid treatment for these underlying diseases. The target of this study is to investigate the effect of microRNA-320 (miR-320) on SANFH by targeting CYP1A2. CYP1A2 expression was detected using immunohistochemistry. Specimens were collected from patients with SANFH and femoral neck fracture. Seventy rats were assigned into seven groups. The targeting relationship between miR-320 and CYP1A2 was verified by bioinformatics website and dual luciferase reporter gene assay. RT-qPCR and Western blot analysis were used to detect miR-320 and CYP1A2 expressions. The enzymatic activity of CYP1A2 was detected by fluorescence spectrophotometry. Hemorheology and microcirculation were measured in rats. MiR-320 expression decreased and CYP1A2 expression and enzymatic activity increased in SANFH patients compared to those with femoral neck fracture. CYP1A2 was the target gene of miR-320. Hemorheology and microcirculation results showed that up-regulated expression of CYP1A2 promoted the development of SANFH while increased expression of miR-320 inhibited the development of SANFH. Compared with the SANFH group, the SANFH + miR-320 mimic group showed increased miRNA-320 expression, and decreased CYP1A2 expression and enzymatic activity. Opposite results were found in the SANFH + miR-320 inhibitor group. The SANFH + miR-320 inhibitor + pCR-CYP1A2_KO group showed decreased miRNA-320 expression and the SANFH + pCR-CYP1A2_KO group showed decreased CYP1A2 expression and enzymatic activity. Our findings provide evidences that miR-320 might inhibit the development of SANFH by targeting CYP1A2.

[Small interfering RNA-mediated α-enolase knockdown suppresses glycolysis and proliferation of human glioma U251 cells in vitro].

OBJECTIVE: To investigate the role of α-enolase (ENO1) in regulating glucose metabolism and cell growth in human glioma cells. METHODS: Glucose uptake and lactate generation were assessed to evaluate the changes in glucose metabolism in human glioma U251 cells with small interfering RNA (siRNA)-mediated ENO1 knockdown. MTT assay and 5-ethynyl-2'-deoxyuridine (EdU) staining were used to examine the cell growth and cell cycle changes following siRNA transfection of the cells. RESULTS: Transfection of U251 cells with siRNA-ENO1 markedly reduced glucose uptake (P=0.023) and lactate generation (P=0.007) in the cells and resulted in significant suppression of cell proliferation (*P<0.05) since the second day following the transfection. Transfection with siRNA-ENO1 also obviously suppressed cell cycle G1/S transition in the cells (P=0.0425). The expressions of HK2 and LDHA, the marker genes for glucose metabolism, were significantly down-regulated in the cells with siRNA-mediated ENO1 knockdown. CONCLUSION: ENO1 as a potential oncogene promotes glioma cell growth by positively modulating glucose metabolism.

[Antioxidating and energy metabolism improving effects of Qiangjing Decoction on oligospermia and asthenospermia: An experimental study].

OBJECTIVE: To explore the mechanisms of Qianjing Decoction in the treatment of oligoasthenospermia (OAS). METHODS: We randomly divided 100 SPF male rats into five groups of equal number: normal, model, Huangjingzanyu, levocarnitine, and Qiangjing. OAS models were established in the animals followed by intragastrical administration of normal saline, ornidazole, Huangjingzanyu Capsules (200 mg per kg body weight per day), levocarnitine (100 mg per kg body weight per day), and Qianjing Decoction (10 g per kg body weight per day), respectively, qd, for 4 successive weeks. Then, we detected the concentration and motility of the epididymal sperm, obtained the contents of superoxide dismutase (SOD), malonaldehyde (MDA), glutathione peroxidase (GSH-Px), lactate dehydrogenase (LDH), α-glucosidase, and fructose in the epididymis, and determined the mRNA expressions of nuclear factor erythroid 2-related factor 2 (Nrf2) and succinate dehydrogenase (SDH) in the epididymal tissue of the rats by real-time PCR. RESULTS: The concentration and motility of the epididymal sperm in the model, Huangjingzanyu, levocarnitine, and Qianging groups were (35.34 ± 4.22) x 10(6)/ml and (40.04 ± 7.05)%, (48.12 ± 5.56) x 10(6)/ml and (62.46 ± 7.12)%, (47.14 ± 4.87) x 10(6)/ml and (63.23 ± 6.34)%, and (50.25 ± 5.08) x 10(6)/ml and (66.34 ± 7.58)%, respectively, all significantly lower than in the normal group ([53.05 ± 4.55] x 10(6)/ml and [70.20 ± 8.54]%) (P < 0.05), but remarkably higher in the Huangjingzanyu, levocarnitine, and Qiangjing groups than in the model rats (P < 0.05). Compared with the thinned epididymal lumen walls, decreased sperm count, and disorderly and loose arrangement of the lumens in the OAS models, the rats in the Huangjingzanyu, levocarnitine, and Qiangjing groups showed evidently thicker epididymal lumen walls, with the lumens full of sperm cells and arranged regularly and compactly, similar to those of the normal rats. The levels of SOD and GSH-Px were significantly lower but that of MDA markedly higher in the model rats ([84.12 ± 23.25], [10.56 ± 3.02], and [14.04 ± 2.06] nmol/mg) than in the normal group ([110.04 ± 19.56], [17.25 ± 3.56], and [8.87 ± 1.35] nmol/mg) (P < 0.05), while the former two indexes remarkably higher and the latter one significantly lower in the animals treated with Qiangjing Decoction ([120.56 ± 23.68], [16.34 ± 3.12], and [8.45 ± 1.56] nmol/mg), Huangjingzanyu Capsules ([115.34 ± 21.35], [15.23 ± 3.67], and [8.33 ± 1.54] nmol/mg), and levocarnitine ([116.67 ± 22.67], [15.35 ± 3.45], and [8.05 ± 1.78] nmol/mg) than in the models (P < 0.05). The levels of fructose, LDH and α-glucosidase were decreased markedly in the OAS models ([100.22 ± 12.12] mg/[ ml x g], [322 ± 46.13] U/[ ml x g], and [10.48 ± 2.33] U/[ml x g]) as compared with the normal rats ([128.12 ± 13.45] mg/[ml x g], [428 ± 35.12] U/[ml x g], and [15.34 ± 3.12] U/[ ml x g]) (P < 0.05), remarkably higher in the rats treated with Qiangjing ([130.23 ± 13.67] mg/[ml x g] [455 ± 51.50] U/[ml x g], and [18.56 ± 4.67] U/[ml x g]), Huangjingzanyu ([124.16 ± 14.02] mg/[ml x g], [ 419 ± 43.14] U/[ml x g], and [17.64 ± 4.08] U/[ml x g]), and levocarnitine ([123.34 ± 14.02] mg/[ml x g], [430 ± 31.80] U/ [ml x g], and [16.85 ± 5.55] U/[ml x g]) than in the models (P < 0.05). The Nrf2 mRNA expression was significantly reduced in the models as compared with the normal rats (P < 0.05) but remarkably increased in the Huangingzanyu, Qiangjing and levocarnitine groups as compared with the model and normal animals (P < 0.05). The SDH mRNA expression was significantly lower in the model than in the normal rats (P < 0.05) but markedly elevated in the Huangjingzanyu, Qiangjing and levocarnitine groups as compared with the model and normal animals (P < 0.05), remarkably higher in the Qiangjing than in the Huangjingzanyu group (P < 0.05). CONCLUSION: Ornidazole induces OAS in rats, which is closely associated with excessive oxidation and energy metabolism dysfunction. Qiangjing Decoction can improve and even reverse ornidazole-induced OAS in rats as well as improve the ultrastructure of their testicular and epididymal tissues. Antioxidation and improvement of energy metabolism are probably the action mechanisms of Qiangjing Decoction in the treatment of OAS.

Electroacupuncture relieves labour pain and influences the spinal dynorphin/κ-opioid receptor system in rats.

BACKGROUND: The dynorphin (DYN)/κ-opioid receptor (KOR) system plays a key role in the control of labour pain. Our previous clinical study reported that electroacupuncture (EA) provided intrapartum analgesia, but the underlying mechanisms of action have not been fully elucidated. AIMS: To observe the effect of EA on labour pain and to explore the underlying mechanisms of action in a rat model. METHODS: Copulation-confirmed pregnant rats (n=120) were given castor oil to induce labour. Rats remained untreated (control group, n=20) or received either meperidine (an opioid that is commonly used to treat labour pain, n=20) or EA at SP6, LI4, SP6+LI4 or SP10 (four groups, n=20 each). Labour pain was evaluated by the warm water tail-flick test. Serum DYN values were measured by ELISA. Protein and mRNA expression of prodynorphin (PDYN, the precursor protein of DYN) and KOR were analysed by Western blotting and real-time PCR, respectively. RESULTS: EA treatment at all acupuncture point combinations studied significantly relieved labour pain and increased serum DYN concentrations, to a degree similar to that achieved with meperidine. EA notably enhanced protein expression of KOR and PDYN and mRNA expression in the lumbar spinal cord but not in the cerebral cortex. The size of effect varied by EA group in the order: SP6>LI4>SP6+LI4>SP10 for all parameters measured, indicating differential effects relating to acupuncture point selection/combination. CONCLUSIONS: The present study indicates that EA relieves labour pain, at least in part, by regulation of the spinal DYN/KOR system in a rat model.

[Mechanism of Dahuang Lingxian Capsule for Regulating and Controlling Expression of Hepatocyte Transporters and Bile Metabolism Spectrum in Gallstone Mice].

Objective To observe the effects of Dahuang Lingxian Capsule (DLC) for regulating and controlling expression of hepatocyte transporters and bile metabolism spectrum in gallstone mice u- sing Western blot and gas chromatography-mass spectrometer ( GC-MS) , and to explore its possible mechanism. Methods Fifty male C57BL/6 mice were randomly divided into five groups, i.e., the normal control group (N), the model group (M) , the ursodeoxycholic acid (UDCA) control group (U) , the drug control group (Y) ,the DLC treatment group (D) , 10 in each group. Mice in group N and group Y were fed with common forage. Forage with high fat, high calorie, high cholesterol was fed to mice in group M, U, and D to induce cholecystolithiasis. Meanwhile, UDCA solution (130 mg kg⁻¹ - d⁻¹) was administered to mice in group U by gastrogavage. DLC decoction (13 g - kg⁻¹ - d1) was administered to mice in group Y and D by gastrogavage. Equal volume of normal saline was administered to mice in group N and M by gastrogavage. After 8-week intervention, gallstone formation rate was observed. Changes of ATP binding cassette subfamily B member 11 (Abcb1l ) and ATP binding cassette subfamily C member 2 (Abcc2) were observed using Western blot. Metabonomic features were detected by GC-MS. Results Improper operation occurred during modeling. One mouse died in group U since its esophagus was pricked by lavage needle. The gallstone formation rate was 100. 00% , higher than that in group N and Y (0.00%, 0.00%; x² =20. 00,P <0. 01). Compared with group M, the gallstone formation rate was reduced in group U and D (44. 44%, x² =7. 54,P = 0. 011 ; 30. 00%, X² = 10. 77, P 0. 003). Cholesterol characteristic absorption peak could be seen at 2 939, 1 446, 1 382, and 1 056 cm - after infrared spec- trum analysis. There was statistical difference in Abcbl1 and Abcc2 among the 5 groups (F =41. 89, P < 0. 01; F=90. 01 , P <0. 01). Compared with group N, expressions of Abcb1 1 and Abcc2 transporters sig- nificantly decreased in group M (P <0. 01). Compared with group M, expressions of Abcb11 and Abcc2 transporters significantly increased in group U, Y, D (P <0. 01). Compared with other groups, concentrations of endogenous metabolites such as alanine, citric acid, lysine, methionine, phenylalanine, tyrosine, cholesterol, low-density lipoprotein (LDL), glycerine, malic acid, acetone increased; concentrations of lactic acid, glutamine, amine glycine, choline, and taurine decreased (P <0. 05, P <0. 01). Con- clusion DLC could stabilize expression of Abcb1 1 and Abcc2, change or improve pathological secretion of bile and its metabolites, thereby achieving the effect of gallstone prevention and treatment.

Moist exposed burn ointment promotes cutaneous excisional wound healing in rats involving VEGF and bFGF.

Cutaneous delayed wounds are a challenging clinical problem, and vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) exhibit key roles in wound healing. Moist exposed burn ointment (MEBO), a Chinese burn ointment with a USA patented formulation, has been reported to promote chronic ischemic and neurogenic ulcer healing in patients; however, the underlying mechanisms remain unclear. In the present study, MEBO significantly promoted the formation of granulation tissue in cutaneous excisional wounds, shortened the time of wound healing, and increased neovascularization and the number of fibroblasts. Furthermore, as well as enhancing the protein expression, MEBO application also increased the gene expression of VEGF and bFGF. The results indicate that MEBO promotes cutaneous excisional wound healing by at least partially enhancing VEGF and bFGF production, implicating the potential uses of MEBO for delayed cutaneous wound healing.

GPR40: a therapeutic target for mediating insulin secretion (review).

G-protein-coupled receptor 40 (GPR40), known as free fatty acid receptor 1, is mainly expressed in pancreatic ß-cells and activated by medium- and long-chain fatty acids. Increasing evidence indicates that the activation of GPR40 in cells causes insulin secretion, and GPR40 has become an attractive therapeutic target for type 2 diabetes. Recently, certain novel GPR40 agonists have been identified that regulate glucose-stimulated insulin secretion, leading to the development of new drugs for the treatment of type 2 diabetes. In this review, we focus on progress in the physiological role of GPR40 and potential drugs targeting GPR40 over the past decade.

Response surface modeling and optimization of accelerated solvent extraction of four lignans from fructus schisandrae.

A new method based on accelerated solvent extraction (ASE) combined with response surface methodology (RSM) modeling and optimization has been developed for the extraction of four lignans in Fructus Schisandrae (the fruits of Schisandra chinensis Baill). The RSM method, based on a three level and three variable Box-Behnken design (BBD), was employed to obtain the optimal combination of extraction condition. In brief, the lignans schizandrin, schisandrol B, deoxyschizandrin and schisandrin B were optimally extracted with 87% ethanol as extraction solvent, extraction temperature of 160 ° C, static extraction time of 10 min, extraction pressure of 1,500 psi, flush volume of 60% and one extraction cycle. The 3D response surface plot and the contour plot derived from the mathematical models were applied to determine the optimal conditions. Under the above conditions, the experimental value of four lignans was 14.72 mg/g, which is in close agreement with the value predicted by the model.

Yidiyin, a Chinese herbal decoction, improves erectile dysfunction in diabetic patients and rats through the NO-cGMP pathway.

The nitric-oxide (NO)-cyclic-guanosine-monophosphate (cGMP) pathway plays a key role in penile erection. Erectile dysfunction (ED) is a complication in male diabetic patients that impacts their quality of 1ife. Recently, Yidiyin, a Chinese herbal decoction, is used to treat diabetic ED, but convincing evidence is lacking, and the potential mechanisms remain uncertain. In the study, diabetic ED patients had low scores on international index of erectile function-5 (IIEF-5), and administration of Yidiyin and hypoglycemic drugs for 16 weeks ameliorated patients' scores on IIEF-5 more than the hypoglycemic drug alone. Moreover, streptozotocin-induced diabetes severely impaired rats' erectile function and the activity of the NO-cGMP pathway in the corpora cavernosum, and treatment with Yidiyin for 4 weeks obviously increased the rats' erectile function, remarkably enhanced the activity of nitric oxide synthase (NOS), and elevated the contents of NO and cGMP. Our findings indicate that Yidiyin improves diabetic ED probably by enhancing the NO-cGMP pathway.