Influence of ganglioside combined with methylprednisolone sodium succinate on efficacy and neurological function in patients with acute myelitis.

BACKGROUND: Acute myelitis (AM) can lead to sudden sensory, motor and autonomic nervous dysfunction, which negatively affects their daily activities and quality of life, so it is necessary to explore optimization from a therapeutic perspective to curb the progression of the disease. AIM: To investigate the effect of ganglioside (GM) combined with methylprednisolone sodium succinate (MPSS) on the curative effect and neurological function of patients with AM. METHODS: First, we selected 108 AM patients visited between September 2019 and September 2022 and grouped them based on treatment modality, with 52 patients receiving gamma globulin (GG) + MPSS and 56 patients receiving GM + MPSS, assigned to the control group (Con) and observation group (Obs), respectively. The therapeutic effect, neurological function (sensory and motor function scores), adverse events (AEs), recovery (time to sphincter function recovery, time to limb muscle strength recovery above grade 2, and time to ambulation), inflammatory factors (IFs) [interleukin (IL)-6, C-reactive protein (CRP), and tumor necrosis factor (TNF)-α] and other data of the two groups were collected for evaluation and comparison. RESULTS: The Obs had: (1) A significantly higher response rate of treatment than the Con; (2) Higher scores of sensory and motor functions after treatment that were higher than the baseline (before treatment) and higher than the Con levels; (3) Lower incidence rates of skin rash, gastrointestinal discomfort, dyslipidemia, osteoporosis and other AEs; (4) Faster posttreatment recovery of sphincter function, limb muscle strength and ambulation; and (5) Markedly lower posttreatment IL-6, CRP and TNF-α levels than the baseline and the Con levels. CONCLUSION: From the above, it can be seen that GM + MPSS is highly effective in treating AM, with a favorable safety profile comparable to that of GG + MPSS. It can significantly improve patients' neurological function, speed up their recovery and inhibit serum IFs.

Retraction: Activation of endoplasmic reticulum stress promotes autophagy and apoptosis and reverses chemoresistance of human small cell lung cancer cells by inhibiting the PI3K/AKT/mTOR signaling pathway.

[This retracts the article DOI: 10.18632/oncotarget.12718.].

Activation of endoplasmic reticulum stress promotes autophagy and apoptosis and reverses chemoresistance of human small cell lung cancer cells by inhibiting the PI3K/AKT/mTOR signaling pathway.

OBJECTIVE: This study aims to investigate the effects of endoplasmic reticulum stress (ERS) on autophagy, apoptosis and chemoresistance of human small cell lung cancer (SCLC) cells via the PI3K/AKT/mTOR signaling pathway. RESULTS: The expressions of ERS-related proteins (PEAK, eIF2α and CHOP) up-regulated, autophagy-related proteins (LC3, LC3-II and Beclin1) and apoptosis-related proteins (Bax and procaspase-3) down-regulated in NCI-H446 and H69 cells after tunicamycin treatment for 24 h. Compared with the blank group, the tunicamycin, BEZ235 and tunicamycin + BEZ235 groups exhibited decreased expressions of p-PI3K, p-AKT and p-mTOR, and increased expressions of autophagy-related proteins (LC3, LC3-II and Beclin1) and apoptosis proteins (Bax and procaspase-3), and the most obvious changes were observed in the tunicamycin + BEZ235 group. MATERIALS AND METHODS: CCK-8 assay was applied to select the best cell line from five SCLC cell lines (NCI-H446, H69, H526, H146 and H209). Finally, NCI-H446 and H69 cells were selected for further experiments. NCI-H446/CDDP and H69/CDDP were selected and divided into the blank group, tunicamycin (an ESR inducer) group, BEZ235 (inhibitors of PI3K/AKT/mTOR pathway) group and tunicamycin + BEZ235 group. Cell apoptosis was detected by flow cytometry. Autophagy was observed by fluorescence microscopy and flow cytometry. Western blotting was used to detect the expressions of ERS-related proteins, autophagy-related proteins, apoptosis-related proteins and PI3K/AKT/mTOR pathway-related proteins. CONCLUSIONS: Our findings provide evidence that the activation of ERS could promote autophagy and apoptosis and reverse chemoresistance of human SCLC cells by inhibiting the PI3K/AKT/mTOR pathway.

Differentially expressed microRNAs and affected signaling pathways in placentae of transgenic cloned cattle.

Placental deficiencies are related to the developmental abnormalities of transgenic cattle produced by somatic cell nuclear transfer, but the concrete molecular mechanism is not very clear. Studies have shown that placental development can be regulated by microRNAs (miRNAs) in normal pregnancy. Thus, this study screened differentially expressed miRNAs by the next-generation sequencing technology to reveal the relationship between miRNAs expression and aberrant development of placentae produced by the transgenic-clone technology. Expressions of miRNAs and mRNAs in different placentae were compared, the placentae derived from one natural pregnancy counterpart (PNC), one natural pregnancy of a cloned offspring as a mother (PCM), and two transgenic (human beta-defensin-3) cloned pregnancy: one offspring was alive after birth (POL) and the other offspring was dead in 2 days after birth (POD). Further, signaling pathway analysis was conducted. The results indicated that 694 miRNAs were differentially expressed in four placental samples, such as miR-210, miR-155, miR-21, miR-128, miR-183, and miR-145. Signaling pathway analysis revealed that compared with PNC, significantly upregulated pathways in POL, POD, and PCM mainly included focal adhesion, extracellular matrix-receptor interaction, pathways in cancer, regulation of actin cytoskeleton, endosytosis, and adherens junction, and significantly downregulated pathways mainly included malaria, nucleotide binding oligomerization domain-like receptor signaling, cytokine-cytokine receptor interaction, Jak-STAT signaling pathway. In conclusion, this study confirmed alterations of the expression profile of miRNAs and signaling pathways in placentae from transgenic (hBD-3) cloned cattle (PTCC), which could lead to the morphologic and histologic deficiencies of PTCC. This information would be useful for the relative research in future.

Inhibition of hepatitis B Virus replication by hepatocyte nuclear factor 4-alpha specific short hairpin RNA.

BACKGROUND: Previous studies showed that hepatocyte nuclear factor 4α (HNF4α) may play a critical role in hepatitis B virus (HBV) replication. AIMS: This study aimed to investigate the effect of knocking down of HNF4α with RNA interference technique on HBV replication in a HBV replication mouse model. METHODS: Four HNF4α, specific short hairpin RNA (shRNA)-producing plasmids were constructed. HBV mRNA and DNA replication intermediates were analysed using Northern and Southern blot respectively. The expression of HNF4α and HBV core antigen (HBcAg) was detected using immunohistochemistry technique. RESULTS: One of the HNF4α shRNAs, HNF4α shRNA1, efficiently inhibited the expression of HNF4α in HepG2 cells and mice liver. HBV RNA transcripts and DNA replication intermediates in HNF4α shRNA1 group were decreased 67.3 and 76%, respectively, in HepG2 cells, and 68.1 and 70.6% in mice liver respectively. The expression level of HBcAg in the liver was also decreased with the inhibition of HNF4α expression. CONCLUSIONS: These results suggested that decreasing of HNF4α expression was associated with the reduced level of HBV replication in HepG2 cells and mice liver. These data indicated that HNF4α played a critical role in HBV replication in vivo, and HNF4α shRNA could inhibit HBV replication in vivo.

Establishment of cell lines using a doxycycline-inducible gene expression system to regulate expression of hepatitis B virus X protein.

The hepatitis B virus (HBV) X gene plays an important role in HBV-associated pathogenesis, especially hepatocarcinogenesis. Establishment of a stable and regulable HBx expression system will allow study of the function of this gene. Here, we describe the development of a doxycycline-inducible recombinant plasmid (pBPSTR3-FlagX) with the full-length HBV X gene and all components of the tetracycline-on ("Tet-on") gene expression system. This vector exhibited dose-dependent doxycycline-dependent induction of the Flag-HBx protein in HepG2 and Hep3B cells. We also observed dose-dependent doxycycline transactivation of HBx in HepG2 cells. After transfecting HepG2 cells with the pBPSTR3-FlagX plasmid, we isolated five puromycin-resistant cell clones with stable HBx expression, two of which exhibited stable and tight control of HBx expression by doxycycline. This new system has great potential for functional studies of the HBV X gene.

[Gene transfection of endostatin synergizes with doxorubicin to suppress human hepatocellular carcinomas: experiment with mice].

OBJECTIVE: To investigate whether endostatin, a potent antiangiogenic agent, synergizes with doxorubicin to suppress human hepatocellular carcinoma (HCC). METHODS: An endostatin expression plasmid, Endo-cDNA3.1, was constructed and transfected into COS-1 cells. Human HCC cells of the line HepG2 and human umbilical vein endothelial cells of the line HUVEC were cultured and stimulated by the supernatant of the CoS-1 cells transfected with Endo-pcDNA3.1 and doxorubicin of different concentrations. MTT method was used to detect the proliferation of the cells. (How many) BALB/c mice were inoculated with HepG2 cells to establish HCC models, and then divided into 4 groups to undergo intratumoral injection of pcDNA3.1, End-pcDNA3.1, doxorubicin, or doxorubicin + Endo-pcDNA3.1. Other mice were used as untreated control group. Two weeks later 5 mice from each group were killed with the tumors taken out. Immunostaining was used to calculate the microvessel density and Western blotting was sued to detect the expression of endostatin, hypoxia-inducible factor (HIF) and vascular endothelial growth factor (VEGF). RESULTS: The proliferation of the HUVEC cells, but not that of the HepG2 cells, transfected with Endo-pcDNA3.1 + doxorubicin was inhibited. Doxorubicin dose-dependently inhibited the proliferation of both HUVEC and HepG2 cells. Endostatin was strongly expressed in the cells treated with Endo-pcDNA3.1 the tumor size of the Endo-pcDNA3.1 and doxorubicin groups were (1545 +/- 180) mm(3) and (953 +/- 250) mm(3) respectively, both significantly lower than that of the untreated and pcDNA3.1 groups [(2360 +/- 330) mm(3) and (2235 +/- 268) mm(3), respectively, all P < 0.01], and the tumor size of the Endo-pcDNA3.1 + doxorubicin group was (426 +/- 87) mm(3), significantly lower than any other groups (all P < 0.01). The number of microvessels of the Endo-pcDNA, doxorubicin, and doxorubicin + Endo-pcDNA3.1 groups were all significantly less than those of the pcDNA3.1 and untreated groups (P < 0.05 or P < 0.01). The expression of HIF-1alphawas downregulated in the Endo-pcDNA3.1 and Endo-pcDNA3.1 + doxorubicin groups, but not in the doxorubicin group. The VEGF expression was down-regulated in the Endo-pcDNA3.1, doxorubicin, and Endo-pcDNA3.1 + doxorubicin groups, especially in the latter. CONCLUSION: Endostatin gene therapy synergizes with doxorubicin to suppress HCC.

[The application of co-culture system on the in vitro development of bovine somatic nuclear transferred embryos].

To establish a co-culture system of nuclear transferred embryos in bovine, effects of co-culture cell types, passages and cryopreservation as well as addition of BFF or FBS were investigated. The results showed that embryos co-cultured with oviductal epithelial cell and granulosa cell achieved significantly higher blastocyst rate compared with the control group (P < 0.05) and co-cultured with oviductal epithelial cell had more embryo cell number than those with granulosa cell. Passages of co-culture cells significantly affected the blastocyst rate and embryo cell number (P < 0.05), and cryopreservation decreased the blastocyst rate and embryo cell number remarkably. Supplemention of BFF increased blastocyste rate significantly (P < 0.05). In conclusion, co-cultured with fresh primary oviductal epithelial cell along with addition of 10% BFF in SOFaa could improve development of nuclear transferred bovine embryo in vitro.

[Gene transfer of von Hippel-Lindau inhibits the growth of transplanted solid tumors].

OBJECTIVE: To explore the effect of von Hippel-Lindau(VHL) gene on growth of EL-4 solid tumors in vivo. METHODS: C57BL/6 mice model of solid tumors was established by subcutaneous injection of EL-4 lymphoma cells. Mice were randomly divided into two groups as treatment group (n=6) and control group (n=6) when tumor diameter increased to 0.1 cm and 0.4 cm respectively. Plasmid pcDNA3-VHL was injected into solid tumor in treatment group, empty pcDNA3 vector in control group. The growth of tumor was observed. Immunohistochemistry and Western blot analysis were used to examine the transgenic expression of VHL, hypoxia inducible factor-1alpha (HIF)-1alpha, bcl-2 and VEGF. Microvessel density (MVD) and apoptosis index (AI) of tumors were also detected. RESULTS: VHL gene transfer eradicated tumors with small size (0.1 cm diameter), but it only retarded the growth of large tumors (0.4 cm diameter). VHL was overexpressed, the expression levels of VEGF, HIF-1alpha and bcl-2 were reduced in treatment group compared with those in the control group. The level of MVD was significantly lower in treatment group (P< 0.05), but AI was higher in treatment group compared with those in the control group (P< 0.01). CONCLUSION: VHL gene therapy can inhibit the growth of EL-4 solid tumor in vivo.

[Study on anti-HBV effects of genetically engineered replication-defective hepatitis B virus expressing dominant negative mutants of core protein].

BACKGROUND: To explore the possibility of using HBV as a gene delivery vector, and to test the anti-HBV effects by intracellular expression of dominant negative mutants of core protein. METHODS: Two kinds of full length mutant HBV genome, which express Core-partial P and Core-S fusion protein, were transfected into HepG 2.2.15 cell lines. Positive clones were selected and mixed in respective groups with hygromycin in the culture medium. HBsAg and HBeAg, which exist in the culture medium, were tested by ELISA and intracellular HBc related HBV DNA was examined by dot blot hybridization. The existence of recombinant HBV virion in the culture medium was examined by PCR. RESULTS: The mean inhibitory rates of HBsAg were 2.74+/-3.83%, 40.08+/-2.05% (P less than 0.01) and 52.94+/-1.93% (P less than 0.01) in group 2.2.15-pMEP4, 2.2.15-CP and 2.2.15-CS, respectively. The mean inhibitory rates of HBeAg were 4.46+/-4.25%, 52.86+/-1.32% (P less than 0.01) and 41.60+/-1.65% (P less than 0.01), respectively. The inhibitory rates of HBc related HBV DNA were 15.3%, 82.0% and 67.2%, respectively. Recombinant HBV virion was detectable in the culture medium of only group 2.2.15-CP. CONCLUSION: Dominant negative mutants of core protein can efficiently suppress wt-HBV replication and the expressions of HBV antigens. With the help of wild-type HBV, the recombinant HBV genome can form and secret HBV like particles, which provides evidence that the antiviral gene will be hepatotropic expression and the antiviral effects will be amplified.