The doublecortin (DCX) gene encodes DCX, a microtubule-associated protein that plays a crucial role in brain development. DCX variants can disrupt microtubule binding and stabilization, interfere with intracellular transport, and affect post-translational modifications. A correlation exists between variant types and clinical severity. Animal models and induced pluripotent stem cell (iPSC) models simulating DCX deficiency revealed the dynamic progression of the disease, which has provided a powerful tool for investigating disease mechanisms and screening therapeutic agents. Currently there is no cure for DCX variants, with treatment primarily relying on anti-epileptic drugs and symptom management. Basic research is now offering new avenues for future therapeutic approaches. This article has summarized the potential pathogenic mechanisms and therapeutic strategies for the DCX variants, with an aim to provide insights for clinical treatment.
Humans ; Doublecortin Protein ; Doublecortin Domain Proteins ; Animals ; Neuropeptides/metabolism* ; Microtubule-Associated Proteins/metabolism* ; Mutation

OBJECTIVE: To explore the changes of CKLF-like MARVEL transmembrane domain-containing 6 (CMTM6) and programmed death-ligand 1 (PD-L1) expression in gastric mucosal epithelial cells after Helicobacter pylori infection and the regulation of CMTM6 on PD-L1, and to analyze the mRNA expression differences before and after CMTM6 gene knock-out in helicobacter pylori infected gastric epithelial cells by microarray analysis. METHODS: The standard Helicobacter pylori strain ATCC 26695 was co-cultured with human gastric epithelial cell GES-1 for 6, 24 and 48 hours, and the mRNA and protein levels of CMTM6 and PD-L1 were detected by real-time quantitative PCR and Western blot. Using CRISPR/Cas9 to construct CMTM6 gene knockout plasmid and knockout CMTM6 gene of GES-1 cells. Helicobacter pylori was co-cultured with CMTM6 gene knockout and wild type GES-1 cells for 48 hours to detect PD-L1 transcription and protein level changes, and CMTM6 gene knockout GES-1 cells were treated with the proteasome inhibitor MG-132 to detect the changes in PD-L1 protein levels. Agilent Human ceRNA Microarray 2019 was used to detect the differentially expressed genes in CMTM6 gene knockout and wild-type GES-1 cells co-cultured with Hp for 48 hours, and the signal pathway of differentially expressed genes enrichment was analyzed by Kyoto Encyclopedia of Genes and Genomes (KEGG) database. RESULTS: The mRNA and protein levels of CMTM6 and PD-L1 in GES-1 cells were significantly up-regulated after Helicobacter pylori infection, and CMTM6 mRNA was most significantly up-regulated 48 hours after infection. After CMTM6 gene knockout, the CD274 gene transcription level of Helicobacter pylori infected GES-1 cells did not change significantly, but PD-L1 protein level was significantly down-regulated, and the PD-L1 level increased after the application of proteasome inhibitor MG-132. After CMTM6 gene knockout, 67 genes had more than two times of differential expression. The transcription levels of TMEM68, FERMT3, GPR142, ATP6V1FNB, NOV, UBE2S and other genes were significantly down-regulated. The transcription levels of PCDHGA6, CAMKMT, PDIA2, NTRK3, SPOCK1 and other genes were significantly up-regulated. After CMTM6 gene knockout, ubiquitin-conjugating enzyme E2S (UBE2S) gene expression was significantly down-regulated, which might affect protein ubiquitination degradation. After CMTM6 gene knockout, adrenoceptor alpha 1B (ADRA1B), cholinergic receptor muscarinic 1 (M1), CHRM1, platelet activating factor receptor (PTAFR) gene expression was significantly up-regulated. CONCLUSION Helicobacter pylori infection up-regulates the expression level of CMTM6 in gastric mucosa cells, and CMTM6 can stabilize PD-L1 and maintain the protein level of PD-L1. CMTM6 gene knockout may affect biological behaviors such as protein ubiquitination and cell surface receptor expression.
Humans ; MARVEL Domain-Containing Proteins/metabolism* ; Helicobacter pylori/physiology* ; B7-H1 Antigen/genetics* ; Helicobacter Infections/metabolism* ; Epithelial Cells/metabolism* ; Gastric Mucosa/metabolism* ; Chemokines/metabolism* ; Cell Line ; Gene Knockout Techniques ; Myelin Proteins

OBJECTIVE: To investigate the biological activity and antitumor effect of pegylated recombinant human interleukin 2 (PEG-rhIL-2) obtained by site-specific conjugation of polyethylene glycol (PEG) with non-natural amino acids, and to explore its antitumor mechanism. METHODS: The binding activities of PEG-rhIL-2 at three different sites (T41, Y45, and V91) to human interleukin 2 receptors α (IL-2R_α) and β (IL-2R_β) and were detected by surface plasmon resonance (SPR) technology. Western blot was used to detect the levels of the Janus kinase-signal transducer and activator of transcription 5 (JAK-STAT5) signaling pathway activated by different doses of rhIL-2 and PEG-rhIL-2 in CTTL-2 and YT cells. Blood was collected after a single administration in mice to detect the drug concentration at different time points and evaluate the pharmacokinetic parameters of Y45-PEG-rhIL-2. Mouse hepatoma cell line Hepa1-6, pancreatic cancer cell line Pan-02, and colon cancer cell line MC-38 were selected. Tumor models were constructed in C57BL/6 mice. Different doses of Y45-PEG-rhIL-2 and excipient control were administrated respectively to evaluate the tumor suppression effect of the drug. In the MC-38 colon cancer model, the tumor suppression effect of Y45-PEG-rhIL-2 combined with anti-programmed death-1 (PD-1) monoclonal antibody was evaluated. Hepa1-6 mouse tumor models were constructed and rhIL-2, Y45-rhIL-2 and Y45-PEG-rhIL-2 were administrated respectively. The proportion of tumor-infiltrating lymphocytes was analyzed by flow cytometry. RESULTS: The SPR detection results showed that the binding activities of PEG-rhIL-2 to IL-2R_α/IL-2R_β were both reduced. The affinity of Y45-PEG-rhIL-2 to IL-2R_α was reduced to approximately 1/250, and its affinity to IL-2R_β was reduced to 1/3. Western blot results showed that the activity of Y45-PEG-rhIL-2 in stimulating JAK-STAT5 signaling in CTLL-2 cells expressing heterotrimeric IL-2 receptor complex IL-2R_αβγwas reduced to approximately 1/300, while its activity in YT cells expressing heterodimeric IL-2 receptor complex IL-2R_βγwas reduced to approximately 1/3. The pharmacokinetic evaluation after a single dose in the mice showed that the elimination half-life of Y45-PEG-rhIL-2 was 17.7 h. Y45-PEG-rhIL-2 has pharmacokinetic characteristics superior to those of rhIL-2. Y45-PEG-rhIL-2 showed dose-dependent tumor suppression activity, and the combination of Y45-PEG-rhIL-2 and anti-PD-1 antibody had a better tumor-inhibiting effect than the single use of Y45-PEG-rhIL-2 or anti-PD-1 antibody. Flow cytometry analysis demonstrated that 72 h after the administration of Y45-PEG-rhIL-2, the proportion of tumor-infiltrating cytotoxic T lymphocytes (CD8⁺T cells) increased by 86.84%. At 120 h after administration, the ratio of CD8⁺T cells to regulatory T cells (Treg) increased by 75.10%. CONCLUSION Y45-PEG-rhIL-2 obtained by site-specific conjugation via non-natural amino acids changed its receptor binding activity and inhibited tumor growth in dose-dependent manner in multiple tumor models by regulating CD8⁺T cells.
Interleukin-2/pharmacokinetics* ; Animals ; Mice ; Humans ; Recombinant Proteins/pharmacology* ; Polyethylene Glycols/chemistry* ; Cell Line, Tumor ; Antineoplastic Agents/pharmacokinetics* ; Signal Transduction/drug effects* ; STAT5 Transcription Factor/metabolism* ; Interleukin-2 Receptor alpha Subunit/metabolism* ; Interleukin-2 Receptor beta Subunit/metabolism*

OBJECTIVE: To investigate the hepatotoxicity of alkaloids from Euodiae Fructus combined with berberine (BBR) in Zuojin Pill, and to preliminarily explore the possible detoxification mechanism of the combination components. METHODS: The combination ratio of components was determined by the maximum concentration (Cmax) of the chemical components in Zuojin Pill. HepG2 cell model was used to investigate the combined toxicity of the hepatotoxic components from Euodiae Fructus, such as evodiamine (EVO) or dehydroevodiamine (DHED), with BBR for 48 h. The experimental groups were set as follows: the vehicle control group, the EVO group, the DHED group, the BBR group, and the combination group of EVO or DHED with BBR. The cell counting kit-8 (CCK-8) method was used to determine the cell viability, and the combination index (CI) was used to determine the combined toxicity of the components. The alanine transaminase (ALT), aspartate aminotransferase (AST), lactate dehydroge-nase (LDH), and alkaline phosphatase (ALP) activities as well as total bilirubin (TBIL) content in the cell culture supernatant were detected. The protein expression levels of bile acid transporters, such as bile salt export pump (BSEP) and multidrug resistance-associated protein 2 (MRP2), were detected by Western blot. The intracellular malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in HepG2 cells were detected. RESULTS: Compared with EVO or DHED group, the combination of EVO 1 μmol/L with BBR 10 μmol/L or DHED 50 μmol/L with BBR 35 μmol/L significantly increased cell viability of HepG2 cells (P < 0.01), with CI values of 77.89 or 4.49, respectively, much greater than 1. Significant decreases in the activities of ALT, AST, LDH, ALP, and TBIL content in the cell culture supernatant were found in both combination groups (P < 0.05, P < 0.01). Compared with the EVO group, the combination of EVO with BBR upregulated the protein expression levels of BSEP and MRP2. Compared with the DHED group, the combination of DHED with BBR significantly downregulated the protein expression levels of BSEP and MRP2 (P < 0.01). Compared with EVO or DHED group, the combination of EVO or DHED with BBR significantly reduced the MDA content in HepG2 cells (P < 0.05, P < 0.01). CONCLUSION A certain ratio of BBR combined with EVO or DHED had an antagonistic effect on HepG2 cytotoxicity, which might be related to regulating the expression of bile acid transpor-ters, and reducing lipid peroxidation damage.
Humans ; Hep G2 Cells ; Berberine/pharmacology* ; Drugs, Chinese Herbal/toxicity* ; Evodia/chemistry* ; Alkaloids/pharmacology* ; Cell Survival/drug effects* ; Multidrug Resistance-Associated Proteins/metabolism* ; Multidrug Resistance-Associated Protein 2 ; Quinazolines

OBJECTIVE: To investigate the biological effect of medical recombinant human-derived collagen functional dressings in wound healing. METHODS: MTT assay and RTCA assay were used to detect cell toxicity and proliferation. Scratch assay and Transwell cell migration assay were used to detect cell motility and migration ability. Enzyme-linked immunosorbent assay was used to detect the contents of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and platelet-endothelial cell adhesion molecule (CD31) in the supernatant of four types of cells. After animal surgery, the surgical wound was taken at 1 week, 4 weeks and 13 weeks, respectively, for hematoxylin eosin (HE) staining and immunohistochemistry to observe the inflammatory response and CD31 expression of the wound. RESULTS: Medical recombinant human-derived collagen functional dressing promotes cell proliferation and migration, enhances wound angiogenesis by upregulating the expression of VEGF, FGF, and CD31 in human dermal vascular endothelial cells (HDVEC) and human vascular endothelial cells (HVEC), thereby improving local blood supply to the wound, regulating the inflammatory response of the wound, and accelerating wound healing. CONCLUSION Recombinant type Ⅲ humanized collagen plays an important role in wound healing.
Humans ; Wound Healing/drug effects* ; Recombinant Proteins/pharmacology* ; Animals ; Cell Proliferation ; Cell Movement ; Collagen/pharmacology* ; Vascular Endothelial Growth Factor A/metabolism* ; Bandages ; Platelet Endothelial Cell Adhesion Molecule-1/metabolism* ; Endothelial Cells ; Fibroblast Growth Factors/metabolism*

OBJECTIVE: To investigate the anti-tumor effects of cinobufacini (CINO) on hepatocellular carcinoma (HCC) induced by des-gamma-carboxy-prothrombin (DCP) and to uncover the underlying mechanisms. METHODS: The inhibitory effect of CINO on HCC cell proliferation was evaluated using the cell counting kit-8 method, and the apoptosis rate was quantified using flow cytometry. Immunofluorescence and Western blot analyses were used to investigate the differential expression of proteins associated with cell growth, apoptosis, migration, and invasion pathways after CINO treatment. The therapeutic potential of CINO for HCC was confirmed, and the possibility of combining cinobufacini with c-Met inhibitor for the treatment of primary HCC was further validated by in vivo experiments. RESULTS: Under the induction of DCP, CINO inhibited the activity of HCC cells, induced apoptosis, and inhibited migration and invasion. Upon the induction of DCP, CINO regulated c-Met activation and the activation of the phosphatidylinositol-3 kinase/protein kinase B (PI3K/AKT) and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) pathways. In a mouse model of HCC, CINO exhibited significant antitumor effects by inhibiting the phosphorylation of c-Met and the downstream PI3K/AKT and MEK/ERK pathways in tumor tissues. CONCLUSIONS CINO inhibited HCC cell growth, promoted apoptosis, and suppressed HCC cell invasion and migration by targeting c-Met and PI3K/AKT and MEK/ERK signaling pathways under DCP induction.
Carcinoma, Hepatocellular/drug therapy* ; Proto-Oncogene Proteins c-met/metabolism* ; Liver Neoplasms/drug therapy* ; Signal Transduction/drug effects* ; Animals ; Humans ; Cell Movement/drug effects* ; Apoptosis/drug effects* ; Cell Proliferation/drug effects* ; Amphibian Venoms/therapeutic use* ; Cell Line, Tumor ; Neoplasm Metastasis ; Cell Survival/drug effects* ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Neoplasm Invasiveness ; Mice, Inbred BALB C ; Mice, Nude ; Mice ; Male ; Bufanolides/therapeutic use* ; Protein Precursors ; Prothrombin ; Biomarkers

OBJECTIVE: To investigate the mechanism of electroacupuncture (EA) in treating diabetic gastroparesis (DGP) by inhibiting the activation of Nod-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome and pyroptosis mediated via NLRP3/cysteinyl aspartate specific proteinase-1 (caspase-1)/gasdermin D (GSDMD) signaling pathway. METHODS: Forty Sprague-Dawley rats were randomly divided into 4 groups including the control, DGP model, EA, and MCC950 groups. The DGP model was established by a one-time high-dose intraperitoneal injection of 2% streptozotocin and a high-glucose and high-fat diet for 8 weeks. EA intervention was conducted at Zusanli (ST 36), Liangmen (ST 21) and Sanyinjiao (SP 6) with sparse-dense wave for 15 min, and was administered for 3 courses of 5 days. After intervention, the blood glucose, urine glucose, gastric emptying, and intestinal propulsive rate were observed. Besides, HE staining was used to observe histopathological changes in gastric antrum tissues, and TUNEL staining was utilized to detect DNA damage. Protein expression levels of NLRP3, apoptosis-associated speck-like protein containing CARD (ASC), pro-caspase-1, caspase-1 and GSDMD were measured by Western blot. Immunofluorescence staining was employed to assess the activity of GSDMD-N. Lactate dehydrogenase (LDH) levels were detected by using a biochemical kit. RESULTS: DGP rats showed persistent hyperglycemia and a significant decrease in gastrointestinal motility (P<0.05 or P<0.01), accompanied by pathological damage in their gastric antrum tissues. Cellular DNA was obviously damaged, and the expressions of NLRP3, ASC, pro-caspase-1, caspase-1 and GSDMD proteins were significantly elevated, along with enhanced fluorescence signals of GSDMD-N and increased LDH release (P<0.01). EA mitigated hyperglycemia, improved gastrointestinal motility in DGP rats and alleviated their pathological injury (P<0.05). Furthermore, EA reduced cellular DNA damage, lowered the protein levels of NLRP3, ASC, pro-caspase-1, caspase-1 and GSDMD, suppressed GSDMD-N activity, and decreased LDH release (P<0.05 or P<0.01), demonstrating effects comparable to MCC950. CONCLUSION EA promotes gastrointestinal motility and repairs the pathological damage in DGP rats, and its mechanism may be related to the inhibition of NLRP3 inflammasome and pyroptosis mediated by NLRP3/caspase-1/GSDMD pathway.
Animals ; Electroacupuncture ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Pyroptosis ; Rats, Sprague-Dawley ; Caspase 1/metabolism* ; Gastroparesis/physiopathology* ; Signal Transduction ; Male ; Diabetes Mellitus, Experimental/physiopathology* ; Phosphate-Binding Proteins/metabolism* ; Gastrointestinal Motility ; Rats ; Intracellular Signaling Peptides and Proteins/metabolism* ; Diabetes Complications/physiopathology* ; Gasdermins

OBJECTIVE: To explore the mechanism of action of asperosaponin VI (AVI) in the treatment of rheumatoid arthritis (RA) and validate it in ex vivo experiments using network pharmacology and molecular docking methods. METHODS: The predicted targets of AVI were obtained from PharmMaper, UniProt and SwissTarget Prediction platforms, the disease targets were collected from Online Mendelian Inheritance in Man, Therapeutic Target Database and GeneCards databases, the intersection targets of AVI and RA were obtained from Venny 2.1.0, and the protein-protein interaction (PPI) network was obtained from STRING database, which was analyzed by Cytoscape software and screened to obtain the core targets. Cytoscape software was used to analyze PPI network and screen the core targets. Based on the Database for Annotation, Visualization and Integrated Discovery database, Gene Ontology functional and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were performed, and Cytoscape software was used to construct the "Disease-Pathway-Target-Drug" network, which was finally verified by molecular docking and animal experiments. RESULTS: Network pharmacological studies showed that AVI was able to modulate 289 targets, with 102 targets for the potential treatment of RA, with the core pathway being the AKT/PI3K signaling pathway, and the core targets being the epidermal growth factor receptor (EGFR) and matrix metalloproteinase 9 (MMP9). Molecular docking results showed that AVI could produce strong binding with both of the 2 core targets. In vitro cellular experiments showed that AVI reduced nitric oxide, prostaglandin E₂, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and IL-1 β levels (P<0.05) and inhibited cyclooxygenase-2, nitric oxide synthase, EGFR, MMP9, phosphorylated phosphoinositide 3-kinase (p-PI3K), and phosphorylated serine-threonine kinase (p-AKT) proteins (P<0.05). The results of in vivo studies showed that AVI improved RA score and foot swelling thickness and decreased TNF-α, IL-6, p-PI3K and p-AKT levels in RA rats (P<0.05). CONCLUSION AVI exerts anti-inflammatory and anti-RA effects which might be related to the EGFR/MMP9/AKT/PI3K pathway.
Saponins/chemistry* ; ErbB Receptors/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Signal Transduction/drug effects* ; Molecular Docking Simulation ; Animals ; Arthritis, Rheumatoid/drug therapy* ; Proto-Oncogene Proteins c-akt/metabolism* ; Matrix Metalloproteinase 9/metabolism* ; Protein Interaction Maps/drug effects* ; Humans ; Network Pharmacology ; Male ; Rats

OBJECTIVE: To elucidate the mechanism of Banxia Houpo Decoction (BHD) in treating gastroesophageal reflux disease (GERD) by integrating and utilizing the compound analysis, network pharmacology, and empirical verification. METHODS: Ultra-high performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) was utilized to identify the primary compounds in BHD. Network pharmacology was employed to retrieve target genes. A GERD rat model was developed and 32 SD rats were randomly divided into model, BHD-L (3 g/kg), BHD-H (6 g/kg), and mosapride (0.75 mg/kg) groups using a random number table, 8 rats in each group. Eight rats without the construction of a GERD model were selected as the blank group. Esophageal damage was evaluated through visualization and histopathology evaluation. 5-hydroxytryptamine (5-HT) levels in serum and lower esophageal sphincter (LES) were determined by ELISA. LES contractility was measured with a force transducer, and serotonin transporter (SERT) and 5-HT4R expressions in LES were assessed by RT-PCR, Western blot, and immunofluorescence staining, respectively. RESULTS: UPLC-HRMS analysis identified 37 absorption peaks and 157 compounds in BHD. Functional enrichment identified SERT as a significant target for LES contractility. Histopathological findings indicated less severe esophageal mucosal damage in the BHD-H group compared with the model group. Although serum 5-HT levels showed no significant difference, 5-HT concentration in LES tissue was notably higher in the BHD-H group (P<0.05). Within the range from 10^-10 to 10^-7 mmol/L, LES contractility in the BHD-H and mosapride groups was significantly increased (P<0.05). Within the range from 3 × 10^-7 to 3 × 10^-6 mmol/L 5-HT, LES contractility in the BHD-H group was increased (P<0.05). No significant difference was detected within the range from 10^-5 to 10^-4 mmol/L 5-HT. Notably, SERT expression in the BHD-H group assessed by RT-PCR, Western blot, and immunofluorescence staining were significantly lower than that in the model group (all P<0.01); while 5-HT4R expression remained unchanged. CONCLUSION BHD may increase LES contractility by inhibiting SERT expression in LES tissue.
Animals ; Gastroesophageal Reflux/physiopathology* ; Drugs, Chinese Herbal/chemistry* ; Rats, Sprague-Dawley ; Network Pharmacology ; Male ; Serotonin/metabolism* ; Rats ; Disease Models, Animal ; Serotonin Plasma Membrane Transport Proteins/metabolism* ; Esophagus/drug effects*

OBJECTIVE: To explore the molecular mechanism of Shenmai Injection (SMI) against doxorubicin (DOX) induced cardiomyocyte apoptosis. METHODS: A total of 40 specific pathogen-free (SPF) male Sprague Dawley (SD) male rats were divided into 5 groups based on the random number table, including the control group, the model group, miR-30a agomir group, SMI low-dose (SMI-L) group, and SMI high-dose (SMI-H) group, with 8 rats in each group. Except for the control group, the rats were injected weekly with DOX (2 mg/kg) in the tail vein for 4 weeks to induce myocardial injury, and were given different regimens of continuous intervention for 2 weeks. Cardiac function was detected by echocardiography and myocardial pathological changes were observed by Van Gieson (VG) staining. Myocardial injury serum markers, including creatine kinase (CK), lactate dehydrogenase (LDH), troponin T (cTnT), N-terminal pro-brain natriuretic peptide (NT-proBNP), soluble ST2 (sST2), and growth differentiation factor-15 (GDF-15) were detected by enzyme linked immunosorbent assay (ELISA). Cardiomyocyte apoptosis was observed by terminal deoxynucleotidyl transferase-mediated biotinylated dUTP triphosphate nick end labeling (TUNEL) and transmission electron microscopy, and the expressions of target proteins and mRNA were detected by Western blot and quantitative real time polymerase chain reaction (qRT-RCR), respectively. RESULTS: The treatment with different doses of SMI reduced rat heart mass index and left ventricular mass index (P<0.05), significantly improved the left ventricular ejection fraction (P<0.05), decreased the levels of serum CK, LDH, cTnT, and NT-proBNP (P<0.05 or P<0.01), reduced the levels of serum sST2 and GDF-15 (P<0.05 or P<0.01), decreased the collagen volume fraction, reduced the expressions of rat myocardial type I and type III collagen (P<0.05 or P<0.01), and effectively alleviated myocardial fibrosis. And the study found that SMI promoted the expression levels of miR-30a and Bcl-2 in myocardium, and down-regulated the expression of Bax, which inhibited the activation of Caspase-3 and Caspase-9 (P<0.05 or P<0.01), and improved myocardial cell apoptosis. CONCLUSIONS SMI can alleviate myocardial injury and apoptosis caused by DOX, and its mechanism possibly by promoting the targeted expression of myocardial Bcl-2 protein through miR-30a.
Animals ; Myocytes, Cardiac/metabolism* ; Apoptosis/drug effects* ; MicroRNAs/genetics* ; Rats, Sprague-Dawley ; Male ; Drugs, Chinese Herbal/administration & dosage* ; Doxorubicin/pharmacology* ; Proto-Oncogene Proteins c-bcl-2/genetics* ; Drug Combinations ; Injections ; Rats