R-CHOP and consolidation radiotherapy for limited-stage and low-IPI high-grade b-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements: A single-center case series and review of literature

@#High-Grade B-Cell Lymphoma (HGBCL) with gene rearrangements in MYC and BCL2 and/or BCL6 is an aggressive malignancy usually presenting in advanced stages. Current recommendations suggest the use of regimens more intensive than R-CHOP (rituximab, cyclophosphamide, vincristine, doxorubicin, prednisone), which are based on retrospective studies and single-arm prospective trials that included patients who are mostly in the advanced stage, and did not receive consolidation radiotherapy. The optimal approach and treatment of HGBCL, whether limited-stage (LS) or advanced-stage, remains to be determined. Here we describe the promising outcomes of three patients with LS and low IPI HGBCL with the use of R-CHOP as induction chemotherapy regimen, which was followed by consolidation radiotherapy. Three women, 54-, 60-, and 64-years of age diagnosed to have HGBCL with MYC, and BCL2 and/or BCL6 rearrangements, with Ann Arbor stages I-IIE were included in this case series. All three patients had complete metabolic response to 6 cycles of R-CHOP and was subsequently treated with consolidation involved site radiotherapy (ISRT; total dose 30-36 Gy). Chemotherapy and radiotherapy were tolerated very well. All patients remain to be in remission, with the longest being at 23 months. Outcomes of patients with HGBCL generally remain to be poor, but this may not be the case for patients with limited-stage disease and favorable clinicopathologic risk profile. Nevertheless, the treatment of HGBCL is currently evolving and more studies are needed to determine the ideal approach and preferred chemotherapy regimen. Also, more studies are needed to elucidate the potential role of consolidation radiotherapy in patients with limited-stage HGBCL to improve survival outcomes. Findings of this case series suggest that patients with LS HGBCL may still derive benefit from R-CHOP followed by consolidation ISRT, but prospective trials are needed to confirm this.

Polysaccharide of Alocasia cucullata Exerts Antitumor Effect by Regulating Bcl-2, Caspase-3 and ERK1/2 Expressions during Long-Time Administration / 中国结合医学杂志

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.

Mechanism of Fufang Congrong Yizhi Capsules in treatment of mild cognitive impairment based on network pharmacology / 中国药理学通报

Aim To predict the mechanism of Fufang Congrong Yizhi Capsules (FCYC) in the treatment of mild cognitive impairment (MCI) by network pharmacology method, and further validate it in combination with cellular experiments. Methods TCMSP, Gene-Cards, OMIM and TTD databases, Chinese Pharmacopoeia and related literature were used to screen the active ingredients of FCYC and the targets of MCI treatment. The TCM-compound-target-disease network and PPI of intersection targets were constructed, and the GO and KEGG analysis were performed by the Ehamb bioinformation platform. GO and KEGG analysis were performed through Yihanbo biological information platform. Cell model of MCI was established by PC-12 injury induced by Aβ

Salvianolic Acid F Regulates Bax/Caspase-3/GSDME Signaling Pathway to Inhibit Pyroptosis of HK-2 Cells / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of salvianolic acid F （Sal F） in repairing the high glucose-induced injury in human kidney-2 （HK-2） cells via the B-cell lymphoma-2 （Bcl-2）-associated X protein （Bax）/cysteinyl aspartate-specific proteinase 3 （Caspase-3）/gasdermin-E （GSDME） pathway. MethodThe cell counting kit-8 （CCK-8） was used to measure the relative viability of HK-2 cells exposed to high glucose and different concentrations （2.5， 5， 10， 20 μmol·L-1） of Sal F and the relative viability of HK-2 cells treated with Sal F for different time periods. The levels of lactate dehydrogenase （LDH） and interleukin-1β （IL-1β） in the supernatant of the cell culture were measured by the LDH assay kit and enzyme-linked immunosorbent assay （ELISA） kit， respectively. Flow cytometry combined with Annexin V-FITC/propidium iodide （PI） and Hoechst 33342/PI staining was employed to reveal the proportion of PI-positive HK-2 cells exposed to high glucose. Western blotting was employed to determine the protein levels of Bax， Bcl-2， cytochrome C， cysteinyl aspartate-specific proteinase （Caspase）-9， Caspase-3， and GSDME in the HK-2 cells exposed to high glucose and treated with Sal F. The 2，7-dichlorodihydrofluorescein diacetate fluorescence probe （DCFH-DA） and mitochondrial membrane potential assay kit （JC-1） were used to determine the production of reactive oxygen species （ROS） and the mitochondrial membrane potential in the HK-2 cells exposed to high glucose and treated with Sal F. ResultCompared with the blank group， the model group showed decreased cell viability （P<0.01）， elevated levels LDH and IL-1β， increased proportion of PI-positive cells （P<0.01）， up-regulated protein levels of Bax， cytochrome C， Caspase-9， Caspase-3， and GSDME （P<0.01）， down-regulated protein level of Bcl-2 （P<0.01）， decreased mitochondrial membrane potential， and excessive ROS accumulation. Compared with the model group， Sal F repaired the high glucose-induced injury in HK-2 cells （P<0.05）， lowered the levels of LDH and IL-1β （P<0.05， P<0.01）， and decreased the proportion of PI-positive cells （P<0.01）. In addition， Sal F down-regulated the protein levels of Bax， cytochrome C， Caspase-9， Caspase-3， and GSDME and up-regulated the protein level of Bcl-2 （P<0.05， P<0.01）， increased the mitochondrial membrane potential， and decreased the accumulation of ROS in HK-2 cells. ConclusionSal F can reduce the production of ROS， restore the balance of mitochondrial membrane potential， and inhibit pyroptosis via the Bax/Caspase-3/GSDME signaling pathway to repair the high glucose-induced injury in HK-2 cells.

Compromiso secundario de glándulas mamarias por linfoma folicular / Secondary involvement of breasts by follicular lymphoma

Resumen Las lesiones metastásicas que comprometen la glándula mamaria son excepcionales, ocupando las neoplasias hematolinfoides el segundo lugar en orden de frecuencia en series de casos reporta dos en la literatura con una prevalencia de 0.04% a 1.6% en relación a todos los tumores malignos de la mama, alcanzando una incidencia anual de 0.07%, los cuales corresponden principalmente a linfomas secundarios. El 80% de estos son linfomas B difusos de células grandes, seguido de linfoma folicular y linfoma de la zona marginal. Presentamos una mujer de 60 años con diagnóstico de linfoma folicular que comenzó con una masa perirrenal derecha y ganglios linfáticos ipsilaterales retroperitoneales e inguinales, quien, durante su tratamiento, presentó avance en el estadio clínico con compromiso secundario inusual de ambas glándulas mamarias por esta neoplasia hematolinfoide. Se evaluó el comportamiento biológico de esta enfermedad para comprender los mecanismos fisiopatológicos, mediante el análisis de factores clínicos, histológicos y pronósticos, permitiendo la estadificación definitiva, la cual fue determinante para la elección de la terapia individualizada acorde a las guías de práctica clínica basada en la evidencia científica, impactando positivamente en la evolución médica de la paciente.

Abstract Metastatic lesions involving the breast are exceptional; hematolymphoid neoplasias rank second as per their frequency in case series reported in the literature with a prevalence of 0.04% to 1.6% when consider ing all malignant breast tumors and reaching an annual incidence of 0.07%, mainly accounted for by secondary lymphomas. Eighty percent of them are diffuse, large B cells lymphomas (DLBCL), followed by follicular lymphoma and marginal zone lymphoma. This case is about a 60 year-old woman with a diagnosis of follicular lymphoma, who presented with a right perirenal mass and ipsilateral retroperitoneal and inguinal lymph nodes, whose clinical status progressed during the treatment with unusual secondary involvement of both breasts by hematolymphoid neoplasia. The biological behavior of the condition was evaluated to understand the pathophysiological mecha nisms; this was done analyzing clinical, histologic and prognostic factors that led to a definitive staging, which was key to select the individualized therapy following the clinical practice guidelines based on scientific evidence, with a positive impact on the patient's medical progress.

Secondary involvement of breasts by follicular lymphoma / Compromiso secundario de glándulas mamarias por linfoma folicular

Abstract Metastatic lesions involving the breast are exceptional; hematolymphoid neoplasias rank second as per their frequency in case series reported in the literature with a prevalence of 0.04% to 1.6% when considering all malignant breast tumors and reaching an annual incidence of 0.07%, mainly accounted for by secondary lymphomas. Eighty percent of them are diffuse, large B cells lymphomas (DLBCL), followed by follicular lymphoma and marginal zone lymphoma. This case is about a 60 year-old woman with a diagnosis of follicular lymphoma, who presented with a right perirenal mass and ipsilateral retroperitoneal and inguinal lymph nodes, whose clinical status progressed during the treatment with unusual secondary involvement of both breasts by hematolymphoid neoplasia. The biological behavior of the condition was evaluated to understand the pathophysiological mechanisms; this was done analyzing clinical, histologic and prognostic factors that led to a definitive staging, which was key to select the individualized therapy following the clinical practice guidelines based on scientific evidence, with a positive impact on the patient's medical progress.

Resumen Las lesiones metastásicas que comprometen la glándula mamaria son excepcionales, ocupando las neoplasias hematolinfoides el segundo lugar en orden de frecuencia en series de casos reportados en la literatura con una prevalencia de 0.04% a 1.6% en relación a todos los tumores malignos de la mama, alcanzando una incidencia anual de 0.07%, los cuales corresponden principalmente a linfomas secundarios. El 80% de estos son linfomas B difusos de células grandes, seguido de linfoma folicular y linfoma de la zona marginal. Presentamos una mujer de 60 años con diagnóstico de linfoma folicular que comenzó con una masa perirrenal derecha y ganglios linfáticos ipsilaterales retroperitoneales e inguinales, quien, durante su tratamiento, presentó avance en el estadio clínico con compromiso secundario inusual de ambas glándulas mamarias por esta neoplasia hematolinfoide. Se evaluó el comportamiento biológico de esta enfermedad para comprender los mecanismos fisiopatológicos, mediante el análisis de factores clínicos, histológicos y pronósticos, permitiendo la estadificación definitiva, la cual fue determinante para la elección de la terapia individualizada acorde a las guías de práctica clínica basada en la evidencia científica, impactando positivamente en la evolución médica de la paciente.

Synergistic effect of sorafenib with Platycladus orientalis (L) leaf extract on cervical cancer

The extracts of Platycladus orientalis (L.) Franco leaves have shown promising anti-cancer, anti-oxidant and anti-inflammatory potency with the traditional knowledge of healing HPV associated warts. The purpose of this research is to assess the synergistic activity of sorafenib and Platycladus orientalis (L) leaf extraction on cervical cancer cells. The cytotoxicity efficiency of different concentrations of Sorafenib and ethanol extract of Platycladus orientalis (L.) leaves were tested on HeLa cells by MTT and Trypan blue assays. The synergistic effect of the IC50 concentrations of Sorafenib and Platycladus orientalis (L.) on HeLa cell by MTT assay, and mRNA expression levels of tumor suppressor tazarotene-induced gene 3 (TIG3), proliferating cell nuclear antigen (PCNA) gene and apoptosis modulator (Bcl-2) gene by RT-PCR were evaluated with individual treatments. Combination treatment showed a relatively more expression of TIG3 and less expression of Bcl-2 and PCNA was observed. Growth factor-induced MAPKP activation was arrested by compound combination treatment, which and suppression of proliferation-induced apoptosis of cervical cancer cells. Based on the our results, the combination of sorafenib and crude leaf extract from Platycladus orientalis (L.) can effectively suppress cervical cancer cell growth, thereby providing an interesting rationale for further clinical trials and in-vivo studies.

Expressions of STMN1, BubR1, bcl-2 and Bad in patients with esophageal squamous cell carcinoma and their relationship with chemotherapy effect of paclitaxel-containing regimen / 肿瘤研究与临床

Objective:To investigate the correlation between the expression levels of STMN1, BubR1, bcl-2 and Bad and the chemotherapy effect of paclitaxel-containing regimen in patients with esophageal squamous cell carcinoma (ESCC).Methods:The clinical data of ESCC patients who received paclitaxel-containing chemotherapy at Fenyang Hospital Affiliated to Shanxi Medical University from September 2016 to June 2021 were retrospectively analyzed. Among them, 59 cases received maintenance chemotherapy and 27 cases received surgery after 3 courses of neoadjuvant chemotherapy. The expression levels of STMN1, BubR1, bcl-2 and Bad in tumor tissues before chemotherapy were detected by immunohistochemistry. The imaging efficacy after 3 courses of chemotherapy and pathological efficacy after neoadjuvant chemotherapy were evaluated. The imaging efficacy, pathological efficacy and progression-free survival (PFS) were compared between the high expression group and the low expression group of each protein.Results:The proportion of patients with stage Ⅳ (46.3%, 19/41), the proportion of patients with low differentiation (22%, 9/41) and the incidence of lymph node metastasis (95.1%, 39/41) in STMN1 high expression group were higher than those in STMN1 low expression group (17.8%, 8/45; 4.4%, 2/45; 64.4%, 29/45), and the differences were statistically significant (all P < 0.05). The proportion of patients with stage Ⅳ in Bad high expression group was lower than that in Bad low expression group, and the difference was statistically significant ( P < 0.05). In the evaluation of imaging efficacy, the chemotherapy sensitivity rates in STMN1 and BubR1 high expression groups (29.3%, 12/41; 37.9%, 22/58) were lower than those in STMN1 and BubR1 low expression groups (75.6%, 34/45; 85.7%, 24/28), and the chemotherapy sensitivity rate of patients in Bad high expression group (65.9%, 27/41) was higher than that in Bad low expression group (42.2%, 19/45), and the difference was statistically significant (all P < 0.05). There was no statistical correlation between bcl-2 expression and chemotherapy sensitivity rate ( P > 0.05). In the evaluation of pathological efficacy, the proportion of patients with tumor regression grade (TRG) score 0-1 after neoadjuvant therapy in STMN1 high expression group (27.3%, 3/11) was lower than that in STMN1 low expression group (75.0%, 12/16), and the difference was statistically significant ( P = 0.022). There were no statistical differences in the proportions of patients with TRG score 0-1 after neoadjuvant therapy between high and low expression groups of BubR1, bcl-2 and Bad (all P > 0.05). The PFS rate was 15.2% (9/59) for patients received maintenance chemotherapy, and the median PFS time was 6 months. Kaplan-Meier analysis showed that PFS in STMN1 low expression group was better than that in STMN1 low expression group ( χ2 = 12.90, P < 0.001). PFS in BubR1 low expression group was better than that in BubR1 high expression ( χ2 =12.04, P < 0.001). PFS in Bad high expression group was better than that in Bad low expression group ( χ2 =9.69, P = 0.004). There was no statistical difference in PFS between high and low bcl-2 expression groups ( χ2 =1.43, P = 0.320). Conclusions:ESCC patients with low expression of STMN1, low expression of BubR1 and high expression of Bad have better chemotherapy effect after receiving paclitaxel-containing regimen, but there is no correlation between bcl-2 expression and chemotherapy efficacy.

Study on mechanism of inhibition effect of N 6-methyladenosine methyltransferase-like 3 on vascular calcification in chronic kidney disease through Bax/Bcl-2 / 中华肾脏病杂志

Objective:To investigate the role and mechanism of N 6-methyladenosine (m 6A) methyltransferase-like 3 (METTL3) in vascular calcification (VC) of chronic kidney disease (CKD) through apoptosis-associated protein. Methods:(1) Real-time fluorescence quantitative PCR was used to test METTL3 mRNA in serum of maintenance hemodialysis (MHD) patients. (2) Western blotting was used to detect the expression of METTL3 protein in high-phosphorus stimulated vascular smooth muscle cells (VSMCs), and immunofluorescence double lable was used to observe the distribution of METTL3 and Runt-related transcription factor 2 (Runx2). The METTL3 overexpressed and knockdown plasmids were constructed and transfected into VSMCs. Alizarin red staining was used to detect calcification degree. Western blotting was used to detect the expressions of osteogenic markers [Runx2, bone morphogenetic protein-2(BMP-2), collagen Ⅰ] and apoptosis- related proteins Bax and Bcl-2. (3) SD rats were randomly divided into control group, CKD-VC group and S-adenosylhomocysteine (SAH) intervention group. The calcification of thoracic aorta was evaluated by von Kossa staining, and the protein expressions of Runx2, Bax and Bcl-2 were detected by immunohistochemistry and Western blotting.Results:(1) METTL3 mRNA expression in MHD patients with VC was significantly lower than that in non-VC patients ( P<0.05), and was negatively correlated with coronary artery calcium score ( r=-0.65, P<0.001). (2) The expression of METTL3 in VSMCs stimulated by high phosphorus was decreased and showed a time dependence. Immunofluorescence double label showed that METTL3 and Runx2 were co-expressed in the nucleus. METTL3 was overexpressed in high-phosphorus induced VSMCs, and the expressions of Runx2, collagen I and BMP-2 were significantly decreased, accompanied by the decrease of calcified nodules and Bax/Bcl-2 ratio (all P<0.05). Conversely, METTL3 knockdown aggravated VSMCs calcification by inducing apoptosis. (3) Furthermore, METTL3 inhibitor SAH was administered in vivo, and it was found that inhibition of METTL3 expression significantly increased the calcification of rat thoracic aorta, and the Bax/Bcl-2 ratio and Runx2 expression were up-regulated. Conclusions:Serum METTL3 level is reduced in MHD patients with VC. In vivo and in vitro studies demonstrate that METTL3 inhibits VC in CKD by mediating the apoptosis-related protein Bax/Bcl-2.

Regulatory effect of CCCTC binding factor on Bcl-2 gene in pterygium and its mechanism / 中华实验眼科杂志

Objective:To investigate the regulation of transcription factor CCCTC binding factor (CTCF) on the expression of B-cell lymphoma 2 ( Bcl-2) gene in pterygium and its molecular mechanism. Methods:Pterygium tissue samples from 22 primary pterygium patients who underwent pterygium excision combined with autologous limbal stem cell transplantation in The First Hospital of Changsha from June 2017 to February 2019 were collected during the operation as pterygium group.Normal conjunctival tissue from 20 patients with ocular trauma due to conjunctiva rupture, eyeball rupture or eyeball perforation in the same period were collected during the repair of ocular trauma as control group.Real-time PCR and Western blot were used to detect the expression levels of CTCF and Bcl-2 in the two groups.The DNA methylation level of the Bcl-2 promoter in the samples of the two groups was detected by bisulfite sequencing PCR (BSP). Pterygium fibroblasts were isolated and cultured.Fibroblasts were identified by immunohistochemistry using vimentin antibody.The cultured pterygium fibroblasts were divided into a CTCF interference group transfected with CTCF interference plasmid, and a control group transfected with control plasmid.The expression levels of CTCF and Bcl-2 in pterygium fibroblasts in CTCF interference and control groups were detected by real-time PCR and Western blot.The cell vitality was detected with cell counting kit-8 at 12, 24, and 48 hours after transfection.The DNA methylation level of the Bcl-2 promoter in the cells of the CTCF interference and control groups after transfection was determined by BSP.Differences of the indexes among groups were analyzed.Correlation between Bcl-2 mRNA and Bcl-2 gene promoter methylation level of CTCF protein in pterygium tissue was analyzed by Pearson linear correlation analysis.This study protocol was approved by the Ethics Committee of The First Hospital of Changsha (No.KL-2017021). Written informed consent was obtained from the patients from whom the specimens were collected.Results:The relative expression levels of CTCF mRNA and protein in pterygium group were 7.23±3.34 and 0.92±0.21, respectively, which were significantly higher than 1.10±0.44 and 0.28±0.07 in normal conjunctiva group ( t=-8.136, -13.025; both at P<0.01). The relative expression levels of Bcl-2 mRNA and protein in pterygium group were 10.27±4.64 and 0.95±0.27, which were higher than 1.10±0.41 and 0.32±0.14 in normal conjunctiva group, showing statistically significant differences ( t=-8.789, -10.782; both at P<0.01). The CTCF protein expression was significantly positively correlated with the Bcl-2 mRNA expression in pterygium group ( r=0.746, P<0.01). The DNA methylation level of the Bcl-2 promoter in pterygium group was 0.65±0.09, which was lower than 0.83±0.06 in normal conjunctiva group, with a statistically significant difference ( t=7.408, P<0.01). The DNA methylation level was significantly negatively correlated with the Bcl-2 mRNA expression in pterygium group ( r=-0.635, P<0.01). After the interference of CTCF expression in pterygium fibroblasts, the relative expression levels of CTCF and Bcl-2 mRNA in CTCF interference group were 0.37±0.03 and 0.53±0.06, which were significantly lower than 1.02±0.06 and 0.99±0.07 in control group ( t=20.035, 9.029; both at P<0.01). The relative expression levels of CTCF and Bcl-2 proteins in CTCF interference group were 0.23±0.06 and 0.56±0.07, which were lower than 0.52±0.05 and 0.92±0.12 in control group, showing statistically significant differences ( t=6.914, 4.719; both at P<0.01). The cell viability of pterygium fibroblasts in CTCF interference group was 0.10±0.01, 0.17±0.01, 0.38±0.04 at 12, 24, and 48 hours after interference, respectively, which were lower than 0.12±0.01, 0.29±0.01 and 0.85±0.06 in control group, and the differences were statistically significant ( t=3.718, 18.350, 15.621; all at P<0.01). The DNA methylation level of Bcl-2 promoter in CTCF interference group was 0.75±0.04, which was significantly higher than 0.61±0.03 in control group ( t=-4.472, P<0.05). Conclusions:CTCF is excessively expressed in pterygium, which may mediate the overexpression of Bcl-2 through down-regulating DNA methylation level.

Research Progress in Treatment of Chronic Lymphocytic Leukemia --Review / 中国实验血液学杂志

Chronic lymphocytic leukemia (CLL) is a low-grade lymphoproliferative tumor that occurs frequently in middle-aged and elderly people. Early and precise intervention can effectively improve the clinical prognosis of CLL patients. In the past, chemotherapy was the main treatment plan. With the development of molecular biology and the continuous advent of immune targeting drugs, targeted drugs targeting B cell receptor signaling pathway have shown high clinical application value in the diagnosis and treatment path of CLL. Cellular immunotherapies such as CAR-T also offer hope for patients with relapsed and refractory CLL. Allogeneic hematopoietic stem cell transplantation and multi-drug combination have also shown remarkable results in clinical practice. The purpose of this article is to review the latest research progress in the treatment of CLL.

Geju Hugan Tablets Ameliorate Alcohol-induced Liver Injury in Mice by Regulating NF-κB and Bcl-2/Bax Signaling Pathways / 中国实验方剂学杂志

ObjectiveTo investigate the protective effect of Geju Hugan tablets on the liver of mice with alcohol-induced liver injury， and explore the underlying mechanism based on nuclear factor-κB p65 （NF-κB p65） and B-cell lymphoma-2 （Bcl-2）/Bcl-2-associated X protein （Bax） signaling pathways. MethodAccording to the body weight， 60 SPF-grade male ICR mice were randomized into normal， model， Compound Yiganling tablets （0.16 g·kg-1）， and low-， medium-， and high-dose （0.2， 0.4， 0.8 g·kg-1， respectively） Geju Hugan tablets groups. The drugs were administrated at the corresponding doses by gavage， and the normal and model groups with equal volume of pure water once a day for 28 consecutive days. On day 29， the mice in other groups except the normal group were administrated with liquor （53% Vol） by gavage twice a day at the doses of 20， 10 mL·kg-1 and with the interval of 6 h. Samples were harvested on day 30. The histopathological changes in the liver were observed by hematoxylin-eosin （HE） staining， and the ultrastructural changes in hepatocytes were observed by transmission electron microscopy. The enzyme-linked immunosorbent assay was employed to measure the levels of malonaldehyde （MDA）， reduced glutathione （GSH）， and triglycerides （TG） in the liver tissue and the levels of alanine aminotransferase （ALT） and aspartate aminotransferase （AST） in the serum. Western blotting was employed to determine the protein levels of NF-κB p65， phosphorylated p-inhibitor kappa B alpha （p-IκBα）， Bcl-2， and Bax in the liver tissue. ResultCompared with the normal group， the model group showed increases in the ALT， AST， MDA， and TG levels， a decrease in the GSH level， and increases in the liver injury scores evaluated based on the HE， oil red O， and transmission electron microscopy （P<0.01）. Moreover， the model group showed up-regulated expression of NF-κB， p-IκBα， and Bax （P<0.05， P<0.01） and down-regulated expression of Bcl-2 （P<0.05） in the liver tissue. Compared with the model group， Geju Hugan tablets of all the doses lowered the ALT， AST， MDA， and TG levels and elevated the GSH level （P<0.01）. The liver injury scores assessed based on HE staining and transmission electron microscopy in the medium- and high-dose Geju Hugan tablets groups were lower than those in the model group （P<0.01）. Compared with the model group， medium- and high-dose Geju Hugan tablets down-regulated the protein levels of NF-κB， p-IκBα， and Bax （P<0.01） and all doses of Geju Hugan tablets up-regulated the protein level of Bcl-2 （P<0.01）. ConclusionGeju Hugan tablets protect mice from alcohol-induced liver injury by down-regulating NF-κB signaling pathway to alleviate inflammation in the liver tissue and down-regulating the expression of Bax and up-regulating the expression of Bcl-2 to inhibit hepatocyte apoptosis.

Effect of Bushen Huoxuetang on Expression of Bax/Bcl-2 and Cleaved Caspase-3 in Nude Mice with Bone Metastasis of Breast Cancer / 中国实验方剂学杂志

ObjectiveTo study the effect of Bushen Huoxuetang on the apoptosis and the expression of B-cell lymphoma （Bcl-2）-associated X protein （Bax）/ Bcl-2 and cleaved cysteine-containing aspartate proteolytic enzyme-3 （cleaved Caspase-3） in the nude mouse model of bone metastasis of breast cancer， and explore the mechanism of Bushen Huoxuetang in inhibiting bone destruction. MethodThirty BALB/c female nude mice were randomly assigned into blank group （n=6） and model group （n=24）. The suspension of 4T1 breast cancer cells was injected into the tibia of mouse right lower limb to establish model of bone metastasis of breast cancer. The successfully modeled nude mice were randomly assigned into model group， Bushen Huoxuetang group， zoledronic acid group， and combined drug group， with 6 mice in each group. Bushen Huoxuetang was administrated at a dose of 36.67 g·kg-1， once a day， and zoledronic acid was administrated by subcutaneous injection at a dose of 100 μg·kg-1， twice a week. The combined drug group was administrated with the same doses of Bushen Huoxuetang group by gavage and zoledronic acid by subcutaneous injection. The mice in the blank group and the model group were administrated with the same volume of distilled water by gavage for 14 days. On the next day at the end of drug administration， the mice were sacrificed by cervical dislocation. The general situation and weight changes of the mice were examined. The right lower limb was collected， and X-ray scanning and hematoxylin-eosin （HE） staining methods were used for observation of pathological changes in the bone. The terminal deoxynucleotidyl transferase dUTP nick-end labeling （TUNEL） was employed to detect the apoptosis of bone tissue in nude mice， and Western blot to determine the expression of Bax/Bcl-2 and cleaved Caspase-3 in the bone tissue. ResultCompared with the blank group， the modeling reduced the body weight （P<0.01） and increased the right lower limb weight of the nude mice （P<0.01）. Compared with the model group， Bushen Huoxuetang， zoledronic acid， and their combination increased the body weight （P<0.01） and decreased the right lower limb weight （P<0.01）. Compared with the blank group， the other groups showed obvious tumor cell atypia， deep nuclear staining， and clear bone metastasis， and the model group showed obvious osteolytic damage in right lower limb and loss of proximal tibia and knee joint. Compared with the model group， Bushen Huoxuetang， zoledronic acid， and their combination reduced the osteolytic lesions in the right lower limb and recovered part of the bone structure， demonstrating an inhibitory effect on bone destruction. The TUNEL assay showed that the model group had lower apoptosis rate of bone metastatic tumor cells than the blank group， Bushen Huoxuetang group， zoledronic acid group， and combined drug group （P<0.01）. Compared with the blank group， the modeling down-regulated the expression of Bax and cleaved Caspase-3 （P<0.01） and up-regulated the expression of Bcl-2 （P<0.01）. Compared with the model group， Bushen Huoxuetang， zoledronic acid， and their combination up-regulated the expression of Bax （P<0.01） and cleaved Caspase-3 （P<0.05， P<0.01） and down-regulated the expression of Bcl-2 （P<0.05， P<0.01）. ConclusionBushen Huoxuetang may inhibit bone destruction in the nude mouse model of bone metastasis of breast cancer by up-regulating the expression of Bax， down-regulating the expression of Bcl-2， activating cleaved Caspase-3， and further inducing apoptosis.

Research progress of BCL-2 family apoptotic regulation and its mediated drug resistance after antitumor drug therapy / 中国胸心血管外科临床杂志

@#Apoptosis is an important means to regulate cell proliferation and maintain homeostasis. Recent researches have shown that the B-cell lymphoma-2 (BCL-2) family not only plays a dominant role in the regulation of normal cell apoptosis, but also plays a crucial role in the formation of tumor genesis, progression and subsequent drug resistance mediated by the escape mode of apoptosis. The phenomenon that BCL-2 family antagonized the apoptosis induced by antitumor drugs and then acquired drug resistance has been reported in the clinical treatment of hematologic lymphatic system tumors, breast cancer, lung cancer, gastric cancer and other diseases. Thus, specific inhibitors targeting anti-apoptotic members of the BCL-2 family have emerged with the development of research. In this paper, we systematically reviewed the regulation of apoptosis mediated by BCL-2 family and the drug resistance mediated by BCL-2 family. Meanwhile, we summarized the research advances of BCL-2 family specific inhibitors to provide new strategy for solving the problems on tumor therapeutic resistance and for finding new therapeutic targets in the future.

Doxorubicin-conjugated siRNA lipid nanoparticles for combination cancer therapy

Evasion of apoptosis is a hallmark of cancer, attributed in part to overexpression of the anti-apoptotic protein B-cell lymphoma 2 (Bcl-2). In a variety of cancer types, including lymphoma, Bcl-2 is overexpressed. Therapeutic targeting of Bcl-2 has demonstrated efficacy in the clinic and is the subject of extensive clinical testing in combination with chemotherapy. Therefore, the development of co-delivery systems for Bcl-2 targeting agents, such as small interfering RNA (siRNA), and chemotherapeutics, such as doxorubicin (DOX), holds promise for enabling combination cancer therapies. Lipid nanoparticles (LNPs) are a clinically advanced nucleic acid delivery system with a compact structure suitable for siRNA encapsulation and delivery. Inspired by ongoing clinical trials of albumin-hitchhiking doxorubicin prodrugs, here we developed a DOX-siRNA co-delivery strategy via conjugation of doxorubicin to the surface of siRNA-loaded LNPs. Our optimized LNPs enabled potent knockdown of Bcl-2 and efficient delivery of DOX into the nucleus of Burkitts' lymphoma (Raji) cells, leading to effective inhibition of tumor growth in a mouse model of lymphoma. Based on these results, our LNPs may provide a platform for the co-delivery of various nucleic acids and DOX for the development of new combination cancer therapies.

Effect of SMAC Gene on Sensitivity of Lung Adenocarcinoma Cells to Paclitaxel and Cell Viability Based on caspase-3/Bcl-2/Bax Signaling Pathway / 肿瘤防治研究

Objective To investigate the effect of the SMAC gene on paclitaxel sensitivity and cellular activity in lung adenocarcinoma cells based on the caspase-3/Bcl-2/Bax signaling pathway. Methods A paclitaxel-resistant cell line A549/Taxol was established for lung adenocarcinoma, and the cells were divided into four following groups: pcDNA-NC (transfected with pcDNA-NC blank vector), pcDNA-SMAC (transfected with pcDNA-SMAC vector), siRNA-NC (transfected with siRNA-NC empty virus vector), and siRNA-SMAC groups (transfected with siRNA-SMAC lentiviral vector). The SMAC mRNA expression in cells was detected by qRT-PCR; cell sensitivity was detected by MTT; cell proliferation ability was detected by cloning assay; cell invasion ability was detected by Transwell; apoptosis ability was detected by flow cytometry assay; and caspase-3, Bcl-2 and Bax protein expression in cells were detected by Western blot analysis. Results The SMAC mRNA expression was significantly lower in A549 cells compared with BEAS-2B cells (P < 0.05). The SMAC mRNA expression was significantly higher in the pcDNA-SMAC group than that in the pcDNA-NC group cells (P < 0.05). The SMAC mRNA expression was significantly lower in the cells of the siRNA-SMAC group (P < 0.05) than that in the siRNA-NC group. The SMAC mRNA expression was significantly lower in the cells of the siRNA-SMAC group (P < 0.05) than in the siRNA-NC group. Compared with the pcDNA-NC group, the cell IC50, cell clone number, cell invasion ability, and Bcl-2 protein and Bcl-2/Bax ratio were significantly lower in the pcDNA-SMAC group, the cell resistance index reversal was 2.51-fold, and the apoptosis ability and caspase-3, as well as Bax protein expression, were significantly higher (P < 0.05). Compared with the siRNA-NC group, cell IC50, cell clone number, cell invasion ability, and Bcl-2 protein and Bcl-2/Bax ratio were significantly higher in the siRNA-SMAC group, and apoptosis ability and caspase-3 and Bax protein expression were significantly lower (P < 0.05). Conclusion High expression of SMAC increases paclitaxel sensitivity, inhibits cell growth and invasion, promotes apoptosis in lung adenocarcinoma cells, and has a regulatory effect on the caspase-3/Bcl-2/Bax signaling pathway.

Role of Mitophagy in Myocardial Ischemia/Reperfusion Injury and Chinese Medicine Treatment / 中国结合医学杂志

Mitophagy is one of the important targets for the prevention and treatment of myocardial ischemia/reperfusion injury (MIRI). Moderate mitophagy can remove damaged mitochondria, inhibit excessive reactive oxygen species accumulation, and protect mitochondria from damage. However, excessive enhancement of mitophagy greatly reduces adenosine triphosphate production and energy supply for cell survival, and aggravates cell death. How dysfunctional mitochondria are selectively recognized and engulfed is related to the interaction of adaptors on the mitochondrial membrane, which mainly include phosphatase and tensin homolog deleted on chromosome ten (PTEN)-induced kinase 1/Parkin, hypoxia-inducible factor-1 α/Bcl-2 and adenovirus e1b19k Da interacting protein 3, FUN-14 domain containing protein 1 receptor-mediated mitophagy pathway and so on. In this review, the authors briefly summarize the main pathways currently studied on mitophagy and the relationship between mitophagy and MIRI, and incorporate and analyze research data on prevention and treatment of MIRI with Chinese medicine, thereby provide relevant theoretical basis and treatment ideas for clinical prevention of MIRI.

Mechanism of Bcl-2 BH4 selective inhibitor BDA-366 on NK/T cell lymphoma cells / 国际肿瘤学杂志

Objective:To investigate the inhibitory effect and killing mechanism of Bcl-2 BH4 selective inhibitor BDA-366 on NK/T cell lymphoma (NK/TCL) .Methods:Human NK cell leukemia cell line YT and human NK/TCL cell line NK92 cells were treated with 0, 0.05, 0.10, 0.20, 0.30, 0.40, 0.50 μmol/L BDA-366. CCK-8 assay was used to calculate the half inhibitory concentration (IC 50) value of BDA-366 on these cells. The apoptosis levels of cells in control group and IC 50 BDA-366 treated group were detected by flow cytometry. Western blotting was used to detect the expression levels of apoptosis-related proteins in cells of control group and 1/2 IC 50, IC 50, 2× IC 50 BDA-366 treated groups. TMRE and Fluo-3 fluorescent probe were used to detect mitochondrial membrane potential of control group and IC 50 BDA-366 treated group, and the intracellular Ca 2+ concentration of control group, IC 50, 2× IC 50 BDA-366 treated groups. NOD-SCID mice in control group and 10 mg/kg BDA-366 intraperitoneal injection group were weighed and HE staining was performed to evaluate the toxicity of BDA-366 in vivo. Results:The IC 50 of BDA-366 for YT and NK92 cells were 0.065 and 0.086 μmol/L respectively. The apoptosis rates of YT cells in the control group and 0.065 μmol/L BDA-366 group were (6.62±1.59) % and (34.60±3.06) % respectively. The apoptosis rates of NK92 cells in the control group and 0.086 μmol/L BDA-366 group were (5.57±0.88) % and (29.18±0.90) % respectively, both with statistically significant differences ( t=14.05, P<0.001; t=32.58, P<0.001). The relative expression of Bax in NK92 cells of the control group, 0.043, 0.086 and 0.172 μmol/L BDA-366 groups were 0.85±0.00, 1.26±0.04, 1.51±0.18, 1.15±0.10 ( F=20.70, P<0.001), the relative expression of Bax in BDA-366 groups were higher than that in the control group (all P<0.05). The fluorescence intensity of TMRE of YT cells in the control group and 0.065 μmol/L BDA-366 group were 8 372.00±330.47 and 6 419.67±311.34, and that of NK92 cells in the control group and 0.086 μmol/L BDA-366 group were 9 169.00±535.72 and 7 311.67±295.52 respectively, and there were statistically significant differences ( t=7.45, P=0.002; t=5.26, P=0.006). In YT cells, the intracellular Ca 2+ concentrations of 0.065 and 0.130 μmol/L BDA-366 groups were significantly higher than that of the control group (5 791.67±220.45, 6 729.33±585.39, 4 874.67±112.61, F=19.16, P=0.003) ( P=0.039; P=0.002). In NK92 cells, the intracellular Ca 2+ concentrations of 0.086 and 0.172 μmol/L BDA-366 groups were significantly higher than that of the control group (4 553.67±17.62, 4 740.33±254.50, 4 185.67±17.67, F=10.96, P=0.010) ( P=0.039; P=0.007). There was no statistically significant difference in body weight change on day 12 compared with day 0 of NOD-SCID mice between BDA-366 group and control group [ (3.18±0.01) g vs. (2.73±0.58) g, t=0.60, P=0.570], and HE staining showed no abnormal morphology of heart, liver, spleen, lung and kidney in BDA-366 group. Conclusion:BDA-366 promotes NK/TCL cells apoptosis in vitro, but does not cause weight loss and morphological changes of organs by HE staining in vivo. The inhibitory effect of BDA-366 on NK/TCL cells may be achieved by increasing Bax expression, inducing Ca 2+ release and reducing mitochondrial membrane potential.

Mechanism of Ethyl Acetate Extract of Gentianopsis paludosa in Treatment of Ulcerative Colitis Based on Bcl-2/Bax Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of ethyl acetate extract of Tibetan medicine dampness bud Gentianopsis paludosa in the prevention and treatment of recurrent ulcerative colitis （UC） in rats with dampness-heat in large intestine syndrome based on the apoptotic pathway mediated by the B-cell lymphoma-2 （Bcl-2） and Bcl-2-associated X protein （Bax）. MethodUsing the disease-syndrome combination method， a recurrent UC model of dampness-heat in large intestine syndrome was constructed in rats. Seventy SPF-grade male SD rats were randomly divided into control group， model group， high-， medium-， and low-dose ethyl acetate of G.paludosa groups （150， 75， 37.5 mg·kg-1）， and mesalazine group （135 mg·kg-1）. The rats were orally administered with respective drugs for 14 days. The general conditions of the rats were recorded， and colon length and mucosal damage were observed. The colon wet weight index and organ coefficients of the liver， spleen， and thymus were calculated. Enzyme-linked immunosorbent assay （ELISA） was used to measure the levels of interleukin-6 （IL-6） and interleukin-1β （IL-1β） in the serum of each group. Hematoxylin-eosin （HE） staining was performed to observe pathological changes in the colon. Terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） was used to detect apoptosis in colonic epithelial cells. Western blot was used to measure the expression levels of Bcl-2， Bax， Caspase-3， Caspase-9， Zona Occludens-1 （ZO-1）， Claudin3， and Occludin in colonic tissue. Immunohistochemistry （IHC） was used to observe the expression of Bax and Caspase-3 in colonic epithelial cells. ResultCompared with the control group， the model group showed significant increases in the disease activity index （DAI） score， colonic mucosal damage index （CMDI）， intestinal epithelial apoptosis， liver and spleen indexes， and levels of inflammatory factors IL-1β and IL-6 in the serum （P<0.01）， decreased expression of intestinal mucosal protective proteins ZO-1， Claudin3， and Occluding （P<0.01）， increased expression of pro-apoptotic proteins Bax， Caspase-3， and Caspase-9 （P<0.01）， and decreased expression of anti-apoptotic protein Bcl-2 （P<0.01）. Compared with the model group， the high-， medium-， and low-dose ethyl acetate of G.paludosa groups all significantly improved the general condition of the rats， reduced colonic lesions， decreased intestinal epithelial cell apoptosis， reduced liver and spleen indexes， upregulated the expression of ZO-1， Claudin3， Occludin， and Bcl-2 proteins， and downregulated the expression of Bax， Caspase-3， and Caspase-9 proteins， with the high- and medium-dose ethyl acetate of G.paludosa groups showing the superior effects （P<0.05， P<0.01）. ConclusionEthyl acetate of G.paludosa can alleviate colonic mucosal damage and exert a therapeutic effect on UC by regulating the Bcl-2/Bax signaling pathway.

Multicenter Prospective Study of Different Induction Regimens of Azacytidine in Treatment of Elderly Patients with Acute Myeloid Leukemia / 中国实验血液学杂志

OBJECTIVE@#To observe the efficacy and safety of different induction regimens of same total dosage of azacitidine (Aza), including standard dose (standard dose group) and low-dose long-term (adjusted dose group), in the treatment of elderly acute myeloid leukemia (AML).@*METHODS@#A total of 103 elderly patients with AML (non-acute promyelocytic leukemia) from January 2020 to June 2021 were enrolled. Aza was administered at the standard dose of 75 mg/(m2·d) for 7 days in the standard dose group (50 cases), while at 100 mg/d for 7-12 days in the adjusted dose group (53 cases). The administration days in adjusted dose group was calculated based on the total standard dose of the patient's single course of treatment. The efficacy and safety between standard dose group and adjusted dose group were compared. Subgroup analysis were performed in the two groups for Aza alone, Aza combined with BCL-2 inhibitor, and Aza combined with low-dose chemotherapy for efficacy and safety.@*RESULTS@#There were no significant differences in overall response rate (ORR), incidence of adverse reaction, and 1-year overall survival (OS) rate between standard dose group and adjusted dose group (P >0.05). The ORR of combination was higher than that of Aza alone (P < 0.05), while there was no significant difference in ORR between Aza combined with BCL-2 inhibitor and Aza combined with low-dose chemotherapy (P >0.05). The combination of BCL-2 inhibitor did not increase the incidence of adverse reactions compared wtih Aza alone. There was a higher risk of myelosuppression and pulmonary infection with a combination of low-dose chemotherapy than with a combination of BCL-2 inhibitor and Aza alone (P <0.05). No significant difference was observed in 1-year OS between Aza alone, Aza combined with BCL-2 inhibitor, and Aza combined with low-dose chemotherapy (P >0.05).@*CONCLUSIONS@#Both two induction regimens can be used in elderly AML patients who cannot tolerate intensive chemotherapy with similar overall effectiveness and safety. Aza combined with low-dose chemotherapy may result in increased ORR and an increased incidence of serious adverse reactions, and may not result in longer survival compared with Aza alone. Aza combined with BCL-2 inhibitor not only has similar effect in complete remission, objective response rate, and OS compared with Aza combined with low-dose chemotherapy, but also has higher safety.