OBJECTIVE:To investigate the effect of aqueous extraction of Lonicerae Flos (SYHW) on anti-influenza A virus H1N1 (H1N1 virus) in vitro. METHODS:Using Madin-Darby canine kid ney (MDCK) cells cultured in vitro by H1N1 virus, half of the tissue culture infection dose(TCID50)was calculated. Culturing MDCK cells for 24 h with different mass concentrations of SYHW,the maximum non-toxic concentration was investigated. And then test was divided into normal cell group,virus control group,SYHW preventive administration group,therapeutic administration group and direct killing group (given SYHW of maxi-mum non-toxic concentration,infecting cells by 100 TCID50 H1N1 virus),and antiviral effective rate (ER) of SYHW was deter-mined. Test was divided into normal cell group,virus control group,SYHW therapeutic group and direct killing group (the same administration and infection as above),changes of cell proliferation index (PI) and cell apoptosis rate were respectively deter-mined. RESULTS:100 TCID50 of H1N1 virus was 1.26×10-7,and the maximum non-toxic concentration of SYHW on MDCK cells was 50 μg/mL(cell survival rate was 91.3%). ERs of preventive administration group,therapeutic administration group and direct killing group were 0,80.3% and 52.7%,respectively. Compared with normal cell group,PI value in virus control group was sig-nificantly reduced (P<0.05),early and late apoptotic rates were significantly increased (P<0.05). Compared with virus control group,PI value in directly killing group was significantly increased(P<0.05),and early apoptotic rate was significantly reduced (P<0.05);early apoptotic rates in therapeutic administration group were significantly reduced (P<0.05). CONCLUSIONS:SY-HW shows anti-H1N1 virus effect in vitro,therapeutic administration and directly killing are preferred in antiviral effect.

OBJECTIVETo observe the prevention and therapeutic effects of tanshitone (TAN) on retinoic acid induced osteoporosis in mice.

METHODThe mice osteoporosis was induced by given retinoic acid intragastrically for two weeks. The histomorphological features of bone were observed and biochemical indexes in serum (Ca, P, ALP, TRAP, E2, BGP) were determined after the mice were given TAN at the dose of 40, 80, 160 mg x kg(-1) respectly.

RESULTTanshinone can induce high conversion of osteoporosis. The levels of P, ALP, TRAP and BGP in the TAN groups were lower than the model group, while the E2 level was higher than the model group.

CONCLUSIONTanshitone can prevent the loss bone in the experimental mice. The mechanism may be that it improves the level of estrogenic hormone and inhibits the high bone turnover.

Alkaline Phosphatase ; blood ; Animals ; Bone Density ; drug effects ; Disease Models, Animal ; Diterpenes, Abietane ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Humans ; Male ; Mice ; Osteoporosis ; blood ; chemically induced ; drug therapy ; prevention & control ; Phenanthrenes ; administration & dosage ; Tretinoin ; adverse effects

Objective To investigate the mechanism of Roscovitine, an inhibitor of cyclin-dependent kinase (CDK), in inhibiting HBV replication.Methods Recombiant expression plasmids of SAMHD1 (sterile alpha motif and histidine/aspartic acid domain-containing protein 1) mutants that were defective in dNTPase (deoxynucleoside triphosphate triphosphohydrolase) activity and phosphorylation at the threonine (T) 592 residue were constructed.Huh7.0 cells were respectively co-transfected with different SAMHD1 mutants in combiantion with HBV replication plasmid to analyze whether the retroviral restriction ability of SAMHD1 was regulated by phosphorylation.The cytotoxicity of Roscovitine to Huh7 cells was evaluated by MTT assay.HBV core-associated DNA levels and phosphorylation of SAMHD1 in transfected Huh7.0 cells which were treated with different concentrations of Roscovitine were measured by Southern blot and Western blot assays.Results The SAMHD1 mutant that was defective in the dNTPase active site of D207N lost its ability to restrict HBV replication.Dephosphorylation of SAMHD1 at T592 enhanced its restriction on HBV.The median toxic concentration (TC50) of Roscovitine was 11.20 μmol/L.Both the HBV core-associated DNA levels and the phosphorylation of SAMHD1 were down-regulated by Roscovitine.Conclusion The anti-HBV function of SAMHD1 in dividing cells is regulated by phosphorylation.Roscovitine can inhibit the replication of HBV through reducing the phosphorylation of SAMHD1.

Aim To investigate the effects of astragalo-side IV on apoptosis of PC12 cells inducedby hypoxia/hypoglycemia and reoxygenation. Metheds PC12 cells were randomly divided into 4 groups: normal control group,hypoxia/hypoglycemia and reoxygenation group, astragaloside Ⅳ group and vehicle group. Hypoxia/hy-poglycemia and reoxygenation group, astragaloside Ⅳgroup and vehiclegroup were exposed to reoxygenation (12 h) after 3 h of oxygen and glucose deprivation, and astragaloside Ⅳ was added into cells at the same time. Inverted microscope was used to observe the morphological changes ofPC12 cells and MTT method to detect the activities of PC12 cells, and Annexin V-FITC/PI assay and TUNEL staining were used to meas-ure the apoptosis of PC12 cells. Results Compared with normal control group, cells became round or swol-len and its cellula processes were retracted or disap-peared in hypoxia/hypoglycemia and reoxygenation group;a large number of apoptotic cells could also be observed,whose nucleus were shrinkaged, fragmented or deep-stained. The activities of hypoxia/hypoglycemia and reoxygenation group were decreased markedly than those in normalcontrol group(P<0. 05),while the ap-optotic rates of hypoxia/hypoglycemia and reoxygen-ation group were increased obviously than those in nor-malcontrol group( P<0. 05 ) . Compared with hypoxia/hypoglycemia and reoxygenation group, a good cell growth state could be observed and cellula processes could also be observed significantly in astragaloside Ⅳgroup. The activities of astragaloside Ⅳ group were in-creased than those in hypoxia/hypoglycemia and reoxy-genation group(P<0. 05),while the apoptotic rates of astragalosideⅣgroup were decreased than those in hy-poxia/hypoglycemia and reoxygenation group ( P <0. 05 ) . There was no obvious difference between vehi-clegroup and hypoxia/hypoglycemia and reoxygenation group( P >0. 05 ) . Conclusion Astragaloside IV can reduce the damage of PC12 cells induced by hypoxia/hypoglycemia and reoxygenation, increase cell activity and inhibit cell apoptosis.

[ ABSTRACT] AIM:To investigate the relationship between morphological changes of autophagy and apoptosis in the PC12 cells induced by oxygen-glucose deprivation and reoxygenation .METHODS:The PC12 cells were randomly di-vided into normal control group , oxygen-glucose deprivation and reoxygenation group , autophagy inhibitor group and auto-phagy activator group .The cells in oxygen-glucose deprivation and reoxygenation group , autophagy inhibitor group and au-tophagy activator group were exposed to reoxygenation (12 h) after 3 h of oxygen-glucose deprivation, and autophagy inhib-itor 3-methyladenine and autophagy activator rapamycin were added into the cells at the same time .Using transmission elec-tron microscope and monodansylcadaverine fluorescence staining , the morphological changes of autophagosome were ob-served.The apoptosis of the PC12 cells were analyzed by flow cytometry with Annexin V-FITC/PI staining and TUNEL method.RESULTS: Compared with normal control group , the numbers of autophagosomes and the apoptotic rates in-creased in oxygen-glucose deprivation and reoxygenation group (P<0.05).Compared with oxygen-glucose deprivation and reoxygenation group , the numbers of autophagosomes decreased obviously ( P<0.05 ) and the apoptotic rates increased markedly in autophagy inhibitor group (P<0.05).The numbers of autophagosomes increased obviously (P<0.05), the apoptotic rates decreased markedly ( P<0.05 ) , the autophagosomes became bigger in size , and autolysosomes was also found in autophagy activator group .CONCLUSION: Oxygen-glucose deprivation and reoxygenation induce autophagy in PC12 cells, and autophagy inhibits cell apoptosis to play a protective role .

Objective To investigate the regulatory role of host restriction factor Moloney leukemia virus 10 (MOV10) protein in HBV replication. Methods Firstly, a HBV replication-expression plasmid was transfected into Huh7 cells to investigate the effect of HBV replication on MOV10 expression. Secondly, HBV DNA was extracted and measured by quantitative PCR ( qPCR) after knocking down the expression of endogenous MOV10 or enhancing the expression of exogenous MOV10. Furthermore, MOV10 conserved do-mainⅡenzyme active mutants (D645A, E646Q and G648A) were constructed and analyzed regarding their antiviral activities. The HBV replication plasmid and MOV10 expression plasmid were co-transfected into hu-man renal epithelial cells (HEK293) to investigate whether MOV10 could bind to HBV mRNA using RNA binding protein immunoprecipitation ( RIP) . Results The expression of MOV10 was increased after trans-fection of HBV replication plasmid into Huh7 cells. After knocking down the expression of endogenous MOV10 by siRNA in Huh7 cells, HBV replication was increased about 1. 5 times compared with control group, while the viral DNA level was significantly decreased in Huh7 cells that overexpressed MOV10. MOV10 domain Ⅱ mutants also significantly inhibited HBV replication. MOV10 could bind to 3. 5 kb HBV RNA. Conclusion In liver cancer cells, the expression of the host restriction factor MOV10 was associated with HBV replication. Its inhibitory effect against HBV replication was independent of its helicase activity, but might be associated with its binding activity with 3. 5kb HBV RNA.

Objective To investigate the potential of polylactic acid glycolic acid copolymer (PLGA) composite ordered multi tunnel collagen scaffold in fabricating a biomimetic artificial nerve graft to repair the sciatic nerve defects in rats.Methods The ordered multi tunnel collagen scaffold was prepared by vacuum freeze-drying and directional drawing method to simulate the epineurium;the outer conduit was prepared by PLGA to simulate the epineurium;and then,the ordered multi tunnel collagen scaffolds were loaded in the PLGA conduit (5∶1) under a stereomicroscope to develop a novel biomimetic artificial nerve.Sixty-four rats were randomly divided into four groups:artificial nerve group,PLGA group,peripheral nerve group,and non-graft control group (n=16);rats in the artificial nerve group,PLGA group,and peripheral nerve group were repaired with artificial nerve graft,hollow PLGA conduit and allogeneic sciatic nerve to bridge the sciatic nerve defect,while the sciatic nerve with the gap in rats of the control group was without any grafting.After 11 weeks of operation,the hind limbs of rats in each group were detected by behavioral test,electrophysiological examination and Fluoro-Gold retrograde tracing method.The changes of muscle tissues (gastrocnemius) were observed by hematoxylin staining and TMR-α-BTX staining,and the regenerated axons were observed by immunohistochemical staining with NF200 and the regenerative spinal anterior horn motor neurons were observed by Nissl fluorescence staining 12 weeks after operation.Results After 11 weeks of operation,the recoveries of the motor functions (the distance between the injured hindlimb and forelimb,the rotation angle of the injured foot) in the peripheral nerve group,artificial nerve group,PLGA group and control group were significantly deteriorated in turn,and the differences were statistically significant (P＜0.05).Electrophysiological examination showed that the recovery effect of peripheral nerve group was the best in both latency and amplitude of the compound muscle action potential,followed by artificial nerve group.The latency of PLGA group was the longest and the amplitude of compound action potential was the smallest;significant differences were noted between each two groups (P2＜0.05).At 12 weeks after operation,the wet weight ratio of muscle fibers,area of muscle fibers and neuromuscular junction area were significantly different between each two groups (P＜0.05);the degree of gastrocnemius atrophy in the artificial nerve group was significantly improved than that in the PLGA group,but not yet reached the level of peripheral nerve group.NF200 immunohistochemical staining showed that a large number of NF200-positive axons were seen in the grafts of the artificial nerve group,but the number was slightly smaller than that of the peripheral nerve group;the number of regenerated axons in the PLGA group was the smaller and mainly distributed near the proximal side.In the PLGA group,only (19.33 ±6.73)％ regenerated spinal anterior horn motor neurons were labeled with Fluoro-Gold,while the positive rates of Fluoro-Gold in the artificial nerve group and peripheral nerve group were (42.67±7.45)％ and (50.13±4.33)％;the differences between each two groups were statistically significant (P＜0.05).Conclusion The biomimetic artificial nerve made of PLGA conduit and ordered multi tunnel collagen scaffold can efficiently reconstruct the defected peripheral nerve with guiding axonal regeneration and promoting functional restoration in rats;however,its effect is poor than peripheral nerve grafting.

Objective To study the inhibitory effect of astragaloside IV (AST) on the apoptosis of PC12 cells induced by oxygen-glucose deprivation/oxygen-glucose rehabilitation (OGD/OGR) through activating autophagy.Methods PC12 cells in logarithmic phase were collected and randomly divided into normal group, OGD/OGR group (model group), AST solvent control group (DMSO group), AST group, AST+autophagy inhibitor group (AST+3-MA group), 3-MA group and autophagy activator group (RAPA group).Except of normal group, all other groups were given treating of OGD for 3 h and then OGR for 12 h and simultaneous medication treating.The morphological changes of autophagosomes were observed by using transmission electron microscope (TEM), expressions of LC3II, Beclin1 and Caspase-3 were detected by using Western blotting, and apoptosis of PC12 cells were detected by using flow cytometry after TUNEL staining.Results Compared with normal group, the typical autophagosomes were observed, expressions of LC3II, Beclin1 and Caspase3 increased, and apoptosis rate and apoptosis increased (P<0.05).Compared with model group, the quantity of autophagosomes and expressions of LC3II and Beclin1 increased (P<0.05), and expression of Caspase3 and apoptosis rate and apoptosis decreased in AST group and RAPA group (P<0.05).Compared with model group, the quantity of autophagosomes and expressions of LC3II and Beclin1 decreased (P<0.05), and expression of Caspase3 and apoptosis rate and apoptosis increased in 3-MA group (P<0.05), while DMSO group and model group had no significant difference (P>0.05).Compared with AST group, the quantity of autophagosomes and expressions of LC3II and Beclin1 decreased (P<0.05), and expression of Caspase3 and apoptosis rate and apoptosis increased in AST+3-MA group and 3-MA group (P<0.05).Conclusion AST play a neuroprotective role through activating autophagy and inhibiting apoptosis of PC12 cells induced by OGD/OGR.

Objective To develop a multi-modal deep learning method for automatic classification of immune-mediated glomerular diseases based on images of optical microscopy(OM),immunofluorescence microscopy(IM),and transmission electron microscopy(TEM).Methods We retrospectively collected the pathological images from 273 patients and constructed a multi-modal multi-instance model for classification of 3 immune-mediated glomerular diseases,namely immunoglobulin A nephropathy(IgAN),membranous nephropathy(MN),and lupus nephritis(LN).This model adopts an instance-level multi-instance learning(I-MIL)method to select the TEM images for multi-modal feature fusion with the OM images and IM images of the same patient.By comparing this model with unimodal and bimodal models,we explored different combinations of the 3 modalities and the optimal methods for modal feature fusion.Results The multi-modal multi-instance model combining OM,IM,and TEM images had a disease classification accuracy of(88.34±2.12)%,superior to that of the optimal unimodal model[(87.08±4.25)%]and that of the optimal bimodal model[(87.92±3.06)%].Conclusion This multi-modal multi-instance model based on OM,IM,and TEM images can achieve automatic classification of immune-mediated glomerular diseases with a good classification accuracy.

Objective To develop a multi-modal deep learning method for automatic classification of immune-mediated glomerular diseases based on images of optical microscopy(OM),immunofluorescence microscopy(IM),and transmission electron microscopy(TEM).Methods We retrospectively collected the pathological images from 273 patients and constructed a multi-modal multi-instance model for classification of 3 immune-mediated glomerular diseases,namely immunoglobulin A nephropathy(IgAN),membranous nephropathy(MN),and lupus nephritis(LN).This model adopts an instance-level multi-instance learning(I-MIL)method to select the TEM images for multi-modal feature fusion with the OM images and IM images of the same patient.By comparing this model with unimodal and bimodal models,we explored different combinations of the 3 modalities and the optimal methods for modal feature fusion.Results The multi-modal multi-instance model combining OM,IM,and TEM images had a disease classification accuracy of(88.34±2.12)%,superior to that of the optimal unimodal model[(87.08±4.25)%]and that of the optimal bimodal model[(87.92±3.06)%].Conclusion This multi-modal multi-instance model based on OM,IM,and TEM images can achieve automatic classification of immune-mediated glomerular diseases with a good classification accuracy.