OBJECTIVE: To study the in vitro and in vivo antitumor effects of the polysaccharide of Alocasia cucullata (PAC) and the underlying mechanism. METHODS: B16F10 and 4T1 cells were cultured with PAC of 40 µg/mL, and PAC was withdrawn after 40 days of administration. The cell viability was detected by cell counting kit-8. The expression of Bcl-2 and Caspase-3 proteins were detected by Western blot and the expressions of ERK1/2 mRNA were detected by quantitative real-time polymerase chain reaction (qRT-PCR). A mouse melanoma model was established to study the effect of PAC during long-time administration. Mice were divided into 3 treatment groups: control group treated with saline water, positive control group (LNT group) treated with lentinan at 100 mg/(kg·d), and PAC group treated with PAC at 120 mg/(kg·d). The pathological changes of tumor tissues were observed by hematoxylin-eosin staining. The apoptosis of tumor tissues was detected by TUNEL staining. Bcl-2 and Caspase-3 protein expressions were detected by immunohistochemistry, and the expressions of ERK1/2, JNK1 and p38 mRNA were detected by qRT-PCR. RESULTS: In vitro, no strong inhibitory effects of PAC were found in various tumor cells after 48 or 72 h of administration. Interestingly however, after 40 days of cultivation under PAC, an inhibitory effect on B16F10 cells was found. Correspondingly, the long-time administration of PAC led to downregulation of Bcl-2 protein (P<0.05), up-regulation of Caspase-3 protein (P<0.05) and ERK1 mRNA (P<0.05) in B16F10 cells. The above results were verified by in vivo experiments. In addition, viability of B16F10 cells under long-time administration culture in vitro decreased after drug withdrawal, and similar results were also observed in 4T1 cells. CONCLUSIONS Long-time administration of PAC can significantly inhibit viability and promote apoptosis of tumor cells, and had obvious antitumor effect in tumor-bearing mice.
Mice ; Animals ; Alocasia/metabolism* ; MAP Kinase Signaling System ; Caspase 3/metabolism* ; Apoptosis ; RNA, Messenger/metabolism*

Objective To investigate the effects of rhynchophylline on methamphetamine-dependent SH-SY5Y cells model and microRNA-375-3p(miR-375-3p)/embryonic lethal abnormal vision drosophila-like 4(Elavl4)expression.Methods A methamphetamine-dependent cell model by maximum safe dose induction was established.The cells were divided into normal group(complete culture medium),control group(complete culture medium+400 μmol·L-1 rhynchophylline incubated for 48 h),model group(complete culture medium+100 μmnol·L-1 methamphetamine incubated for 48 h)and experimental group(complete culture medium+400 μmol·L-1 rhynchophylline incubated for 15 min,then 100 μmol·L-1 methamphetamine incubated for 48 h).The cyclic adenosine monophosphate(cAMP)and 5-hydroxytryptamine(5-HT)expression were detected by enzyme-linked immunosorbent assay(ELISA),miR-375-3p expression was detected by quantitative real time polymerase chain reaction(qRT-PCR),and Elavl4 expression was detected by Western blot.Results The cAMP expression levels of normal group,control group,model group and experimental group were(6.33±0.93),(6.57±1.12),(10.89±1.03)and(7.81±1.32)pmol·mg-1;5-HT were(682.46±17.32),(690.31±15.09),(510.11±27.67)and(649.99±21.42)pg·mL-1;miR-375-3p expression were 1.00±0.13,1.13±0.24,3.48±0.18 and 1.58±0.19;Elavl4 expression were 1.00±0.05,0.89±0.10,0.50±0.09 and 0.90±0.11,respectively.The differences between above indicators in model group and normal group were statistically significant(P＜0.01,P＜0.05);the differences between above indicators in experimental group and model group were statistically significant(P＜0.01,P＜0.05).Conclusion This study preliminarily established a methamphetamine-dependent cell model,and also found that rhynchophylline may regulate miR-375-3p/Elavl4 expression to antagonize methamphetamine addiction.

Aim To investigate the mechanism of Banxia Baizhu Tianma Decoction(BBTD)in interfer-ing methamphetamine(MA)addiction using network pharmacology.Methods The mechanism of BBTD intervention in MA addiction was analyzed using net-work pharmacology,and MA-dependent SH-SY5Y cell model was further constructed to observe the effects of BBTD on cell model and PI3K-Akt pathway.Results A total of 88 active ingredients and 583 potential tar-gets of BBTD were screened.KEGG analysis showed that BBTD might intervene in MA addiction through PI3K-Akt,cAMP and other pathways.The molecular docking results showed that key active ingredients ex-hibited strong binding ability with core targets of PI3K-Akt pathway.In vitro experiments showed that MA-de-pendent model cells had shorter synapses,tended to be elliptical in morphology,had blurred cell boundaries,showed typical cell damage morphology,and had high intracellular expression of cAMP(P＜0.01)and low expression of 5-HT(P＜0.05).BBTD intervention could counteract the above morphology,cAMP,and 5-HT changes,suggesting that it had therapeutic effects on MA-dependent model cells.Western blot showed that MA modeling elevated the p-PI3K/PI3K(P＜0.05)and p-Akt/Akt(P＜0.01);BBTD inter-vention decreased their relative expression.Conclu-sions Gastrodin and other active ingredients in BBTD have therapeutic effects on MA addiction,and the mechanism may be related to regulation of PI3K-Akt pathway relevant targets.

Aim To study the effect of three kinds of active ingredients of traditional Chinese medicine (sinomenine, rhynchophylline and isorhynchophylline) on dre-miR-723-5p expression in morphine-induced zebrafish brain. Methods Morphine was injected intraperitoneally to zebrafish, conditional position preference (CPP) was trained and then the behavioral of animals were observed; the miRNA expression profiles of morphine-additive zebrafish were determined by small RNA sequencing; qRT-PCR was used to verify the expression of dre-miR-723-5p, three target gene databases (miRanda, miRDB, andRNAhybrid) were used to predict the target genes of dre-miR-723-5p; Kobas 3.0 was used to perform Gene Ontology (GO) and KEGG pathway analysis of these target genes. Results Morphine-induced CPP model was established successfully. Compared with control group, the resident time and movement map in drug-pair box of zebrafish in model group significantly increased. After drug administration, the resident time and movement map in drug-pair box of zebrafish decreased. The verification results of qRT-PCR were consistent with the results of small RNA sequencing. Ninety-nine putative target genes of dremiR-723-5p that were common to all three target gene databases, which were mainly enriched in biological process, cell composition and molecular function, involved in the positive regulation of MAPK signaling pathway, lysosome, cytokine-cytokine receptor interaction, and apoptosis. Conclusion Morphine can increase the expression of dre-miR-723-5p in the zebrafish brain, which can be reversed by sinomenine, isorhynchophylline, and rhynchophylline treatment, and dre-miR-723-5p may participate in the mechanism underlying morphine-induced damage of brain.

ObjectiveThere is still controversy about which internal fixation method should be used in oblique lateral interbody fusion (OLIF). This paper aims to compare the biomechanical stability of OLIF with different internal fixation methods.MethodsA 31-year-old healthy male volunteer was selected to have a 64-slice spiral CT scan of his lumbar spine. Mimics 19.0, Geomagic Studio 2013, SolidWorks 2017 and other software were used to build a three-dimensional model of L3-L5, and OLIF surgery was simulated to build OLIF finite element models with five different fixation methods: pedicle screw (PS), lateral single rod screw (LSRS), lateral double rod screw (LDRS), lateral single rod screw+ipsilateral translaminar facet screw (LSRS+ITLFS), lateral single rod screw+contralateral translaminar facet screw (LSRS+CTLFS). After validating the validity of the model, the motion modes of spinal flexion, extension, lateral bending and rotation were simulated, and the fixed segment activity and stress distribution characteristics of each model were compared.ResultsIn terms of fixed segment activity, PS had the best fixation effect, and its range of motion (ROM) was the smallest in all 6 modes. The ROM of the vertebral body was maximized when the LSRS was fixed in all directions. LSRS+ITLFS, LSRS+CTLFS and PS had the similar ROM. In terms of maximum stress of cage, PS had the minimum one except in the left bending. LSRS+ITLFS had little stress in all directions except in flexion; LSRS+CTLFS had little stress in all directions except in extension. In terms of the maximum stress in internal fixation, PS had the least one in all directions; LSRS+CTLFS followed, and the maximum stress appeared in extension and right bending (123.05MPA and 91.74MPA, respectively).ConclusionIn OLIF surgery, PS has the best biomechanical effect. LSRS+CTLFS has the similar effect and its clinical operation is simple with relatively small surgical injury, thus providing a reference for clinical choice.

Objective To study the mechanism of change of the electrical conductivity （EC） of rat skeletal muscle impregnating solution that occurs with the change of postmortem interval （PMI）. Methods Healthy Sprague-Dawley rats were killed and kept at about 25 ℃. Skeletal muscles were extracted at different PMI--immediate （0 d）, 1 d, 2 d, 3 d, 4 d, 5 d, 6 d, and 7 d, then mixed with deionized water to make impregnating solution with a mass concentration of 0.1 g/mL. The solution's EC and nine common chemicals in it, such as potassium ion, calcium ion, and chloride ion, were determined. Results EC increased gradually with the extending of PMI （P=0.024） during the 7 days after the rats' death. The content of uric acid （P=0.032）, urea nitrogen （P=0.013） and phosphorus （P=0.022） also increased during the extension. However, the content of magnesium ions decreased with extending of PMI （P=0.047）. The correlation between potassium ion, sodium ion, chlorine ion, calcium ion, creatinine and PMI were weak （P>0.05）. Conclusion The molecular basis of skeletal muscle EC change in rats after their death is the changes of uric acid, urea nitrogen, inorganic phosphorus and other chemical components. Furthermore, combine use of various indicators can improve the accuracy of the EC method to infer PMI.
Animals ; Electric Conductivity ; Forensic Pathology ; Muscle, Skeletal ; Postmortem Changes ; Rats ; Rats, Sprague-Dawley ; Time Factors

OBJECTIVETo establish osteoblast-osteoclast cell co-culture system in a Transwell chamber, and detect cell viability of osteoblasts and osteoclasts in system.

METHODSOsteoblast MC3T3-E1 and mouse monocytes RAW264.7 were cultivated in vitro. RANKL-induced mouse RAW264.7 monocytes differentiated into mature osteoclasts, osteoblast-osteoclast cell co-culture system was established in Transwell chamber. Cell activity of osteoblasts and osteoclasts were detected by CCK-8 experimenting, Alizarin Red staining, TRAP staining. The expression of OPG, ALP, RANKL, TGF-b1 gene and RANKL protein in osteoblast MC3T3-E1 were detected by PCR, Western-Blot methods. Also, the expression of RANK, NF-κB in gene and protein level in osteoclast were measured through the same method respectively.

RESULTSThe co-culture system of Mouse MC3T3-E1 cells and RAW264.7 cell were established in Transwell chamber. Co-culture system affected cell division activities of osteoblasts and osteoclasts. Differentiation of osteoblasts were increased, while differentiation of osteoclast division were slight decreased under microscope observation. OPG (0.65±0.08) and ALP (0.16±0.01) gene expression of co-culture system were less than single culture OPG(1.00±0.08) and ALP (1.01±0.16); TGF-b1(4.42±0.21) and RANKL(4.12±1.04) of osteoblasts in co-culture system were higher than TGF-b1(1.00±0.10) and RANKL(1.00±0.09) under single culture. However, gene expression of RANK(0.63±0.06) and NF-κB(0.64±0.08) in co-culture system were decreased than RANK(1.00±0.08) and NF-κB(1.00±0.09), in single culture, and had significant differences. Similarly, protein expression of OPG(0.43±0.05) and NF-κB(0.59±0.05) of co-culture system were less than OPG(0.84±0.06) and NF-κB(1.13±0.03) of single culture. While RANKL protein expression (0.54±0.03)of co-culture system was more than single culture RANKL(0.31±0.03), and had statistically differences, which was in agreement of the trend of gene expression change.

CONCLUSIONSCo-culture system of mouse MC3T3-E1 cells and RAW264.7 cell was viable in Transwell chamber, and the activity of osteoblasts is higher than osteoclasts in co-culture system.

3T3 Cells ; Animals ; Cell Differentiation ; Coculture Techniques ; Mice ; NF-kappa B ; metabolism ; Osteoblasts ; cytology ; Osteoclasts ; cytology ; Osteoprotegerin ; metabolism ; RANK Ligand ; metabolism ; RAW 264.7 Cells ; Receptor Activator of Nuclear Factor-kappa B ; metabolism ; Transforming Growth Factor beta1 ; metabolism

Objective To detect 715 bp sequence of 28S rRNA in sarcosaphagous flies, and to identify their common species for solving the problem of morphological identification, as well as providing technical support for postmortem interval (PMI) estimation. Methods Twenty-nine common sarcosaphagous flies were collected in Luoyang and classified by morphological characteristics. The DNA was extracted from the fly's legs by Chelex-100 method and then the fragments of 28S rRNA were amplified and sequenced. The results were compared with twenty-eight corresponding fly species of GenBank and EMBL databases. All the sequences were analyzed by MEGA7.0 software, and sequence alignment was performed by the searching in BLAST. The nucleotide composition was analysed, and the intraspecific and interspecific ge-netic distance and phylogenetic tree were established. Results Twenty-nine sarcosaphagous flies were classified into 6 species of 5 genera, 3 families by morphological characteristics. In the obtained 715 bp sequence of 28S rRNA , the comparison result of online BLAST showed that the similarity was 100％. Five species were well clustered by a phylogenetic tree. Between different groups, the interspecific and intraspecific differences ranged from 0.007 to 0.045 and 0 to 0.001, respectively. Conclusion The 28S rRNA target gene sequences shows a good identification capability, which can be a new genetic marker for the identification of sarcosaphagous flies.

Exosomes are nanoscale vesicles produced and secre-ted into extracellular fluid by all cells. They mediate cell com-munication through carrying and transferring informational car-goes ( proteins, nucleic acids, lipids and so on ) to recipient cells. In central nervous system, exosomes can be released from all cell types including neurons, neural stem cells and neuroglia cells. These exosomes shuttle nucleic acids ( miRNAs, mRNAs and so on) and play an important role in nervous system devel-opment and function as well as diseases including Alzheimer's disease and drug addiction. Furthermore, the functional effects and targeting characteristics of exosomes-shuttle-RNAs suggest that exosomes-shuttle-RNAs can be diagnostic and therapeutic targets. In this review, we elaborate the effects, functions and mechanisms of exosomes-shuttle-RNAs in order to gain a new recognition of CNS development and diseases.

Aim To explore the effect of rhynchophyl-line on the expression of tyrosine hydroxylase ( TH) in hippocampus of methamphetamine-induced condition place preference ( CPP) mice. Methods Metham- phetamine was injected intraperitoneally to mice, and the expression of TH was observed by immunohisto-chemistry and Western blot. Results The CPP mouse model was established successfully by methamphet-amine ( 4 mg·kg-1) . Ketamine ( 15 mg·kg-1) , rhynchophylline low dosage group (40 mg·kg-1) and rhynchophylline high dosage group ( 80 mg·kg-1) could remove the effect of methamphetamine on CPP mice. The result of immunohistochemistry showed that methamphetamine ( 4 mg·kg-1) could increase the number of TH positive cells in hippocampus while ket-amine (4 mg·kg-1), rhynchophylline (40, 80 mg· kg-1) group could attenuate the change. Western blot-ting indicated the expression of TH of model group in-creased significantly, whereas ketamine ( 15 mg· kg-1) , rhynchophylline ( 40, 80 mg·kg-1) group presented less expression. Conclusions The CPP in-duced by methamphetamine in mice may be inhibited to some extent by rhynchophylline, and its mechanism may be associated with the expression of TH.