Table of Content

05 August 2023, Volume 43 Issue 8

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Original Articles

LncRNA FGD5-AS1 promotes osteogenic differentiation of human bone marrow mesenchymal stem cells by regulating miR-93-5p/BMP2 axis

ZHANG Liangliang, ZHAO Chengjin, ZHOU Yuhu, CAO Bo, DUAN Mingming, FENG Yangyang

2023, 43(8):  1179-1185.  doi:10.16352/j.issn.1001-6325.2023.08.1179

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Objective To investigate the impact of long non-coding RNA (lncRNA) FGD5-AS1 on the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) through regulation of the microRNA miR-93-5p/bone morphogenetic protein-2(BMP2) axis. Methods hBM-MSCs in logarithmic growth phase were taken, and the expression levels of lncRNA FGD5-AS1, miR-93-5p and BMP2 mRNA were detected before and after osteogenic differentiation; The cells were transfected or co-transfected with pcDNA FGD5-AS1, miR-93-5p inhibitor, miR-93-5p mimics and corresponding negative controls, respectively, then divided into pcDNA NC group, pcDNA FGD5-AS1 group, inhibitor NC group, miR-93-5p inhibitor group, pcDNA FGD5-AS1+mimics NC group, or pcDNA FGD5-AS1+miR-93-5p mimics group and non-transfected cells were taken as blank group. CCK-8 assay was applied to detect cell proliferation ability of each group; Alkaline phosphatase (ALP) kit was applied to detect its activity; Alizarin red staining was applied to identify cellular mineralized nodule formation; Western blot was applied to detect the levels of BMP2, osteogenesis-related markers-osteocalcin (OCN), osteopontin (OPN), and osterix(OSX); Dual-luciferase experiment was applied to verify the targeting relationship of miR-93-5p with FGD5-AS1 and BMP2, respectively. Results lncRNA FGD5-AS1 and BMP2 were found to be targets of miR-93-5p. After osteogenic differentiation, the expression of FGD5-AS1 and BMP2 was increased and the expression of miR-93-5p was decreased (P＜0.05). Compared with pcDNA NC group, the expression of FGD5-AS1 in pcDNA FGD5-AS1 group was significantly increased(P＜0.05), indicating successful transfection; Mineralized nodules, cell proliferation, ALP activity and the expression of BMP2, OCN, OPN and OSX were obviously higher (P＜0.05) in pcDNA FGD5-AS1 group. Compared with the inhibitor NC group, the expression of miR-93-5p in miR-93-5p inhibitor group was significantly decreased (P＜0.05), indicating successful transfection; Mineralized nodules, cell proliferation, ALP activity and the expression of BMP2, OCN, OPN and OSX were obviously improved (P＜0.05) in miR-93-5p inhibitor group. Over-expression of miR-93-5p inhibited the promoting effect of FGD5-AS1 on the osteogenic differentiation of hBM-MSCs(P＜0.05). Conclusions Up-regulation of FGD-AS1 promotes the osteogenic differentiation of hBM-MSCs, which might be related to the miR-93-5p/BMP2 axis.

Synergistic pro-apoptotic effect of triptolide combined with doxorubicin in human breast cancer cell line MCF-7

CHEN Ning, PENG Tianying, ZHANG Weimiao, GAO Yanuo, LI Xuan, YAN Caizhen, LI Junxia

2023, 43(8):  1186-1192.  doi:10.16352/j.issn.1001-6325.2023.08.1186

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Objective To study the effect of triptolide(TPL)combined with chemotherapy drug doxorubicin(DOX)on apoptosis of human breast cancer cell line MCF-7 and its molecular mechanism. Methods MCF-7 cells were treated with triptolide and different concentrations of doxorubicin alone or in combination. Cell viability was analyzed by CCK-8 method with the combination index calculated. Cell proliferation was detected by plate cloning formation assay. Cell apoptosis and cell cycle were detected by flow cytometry. The expressions of apoptosis-related proteins Bcl-2, Bax, cleaved caspase-3 and proteins in PI3K/AKT signaling pathway were analyzed by Western blot. Results Triptolide combined with different concentrations of doxorubicin significantly inhibited cell proliferation as compared with the doxorubicin group alone after incubation for 24, 48 and 72 hours(P＜0.05). The clone formation was restrained and the cell apoptosis rate was significantly higher(P＜0.01). The expressions of Bax and cleaved caspase-3 was up-regulated, and the expressions of Bcl-2, p-PI3K, p-AKT was down-regulated(P＜0.05). The cells were blocked in S phase after combination treatment of the two drugs(P＜0.05). Conclusions The combination of triptolide and doxorubicin significantly increases the sensitivity of MCF-7 cells to doxorubicin and promotes the apoptosis of cells, which might be related to the inhibition of PI3K/AKT signaling pathway.

Platelet-rich plasma promotes bone injury repairment in rats with diabetes mellitus

WU Zhigang, QUAN Dong, CHANG Xin, CHEN Xuexue, ZHANG Ziru

2023, 43(8):  1193-1200.  doi:10.16352/j.issn.1001-6325.2023.08.1193

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Objective To investigate whether platelet-rich plasma (PRP) can promote osteoblasts differentiation in diabetic mellitus(DM) adipose-derived stem cells(ADSCs) by M2 polarization of DM bone marrow derived macrophages(BMDMs). Methods PRP was extracted from the heart of rats according to the instructions of the kit. BMDMs and ADSCs of DM rats were isolated and cultured, then identified by flow cytometry. BMDMs were divided into control group (PRP 0%) and PRP group (1%, 5%, 10%); ADSCs were divided into: control group (control, ADSCs, 2×10⁵/mL, + same volume as PRP group without PRP plasma), ADSCs+BMDMs group (BMDMs group, 2×10⁵/mL, + plasma without PRP), ADSCs+10% PRP group (PRP group, medium with 10% PRP), and ADSCs+BMDMs+10% PRP group (BMDMs+PRP). Transwell was used to count ADSCs by polarized BMDMs in invasion experiment. The polarization of BMDMs and osteogenic differentiation of ADSCs were determined by RT-qPCR and Western blot. The expression of osteogenic protein Osterix was detected by immunofluorescence. The bone defect model of DM rats was constructed with 3 mm×5 mm at femoral epiphysis, and the rats were divided into: control group (DM rats), ADSCs+ clodronate liposomes (CLP)group (ADSCs group), ADSCs+BMDMs group (BMDMs group without CLP injection), ADSCs+CLP+PRP group (PRP group), ADSCs+PRP group (BMDMs+PRP group without CLP injection). CLP(5 mL/kg) was injected into tail vein of rat 48 hours before operation followed by injection twice a week after surgery for 8 weeks. ADSCs (1×10⁷/mL) and alginate gel mixture were injected into the bone defect, and PRP was treated once every 2 weeks with 500 μL PRP for 4 times. Micro-CT was used to reconstruct and quantitatively analyze the bone defect repair in DM rats. Results The purity of ADSCs positive markers Sca-1 and CD29 were 85.4% and 99.9% respectively and and the purity of BMDMs positive markers CD11b and F4/80 were 94% and 90%. Compared with the control group, BMDMs polarized into M2-type dependent on PRP concentration (P＜0.05). Compared with PRP group, the cell counting of ADSCs migrated in BMDMs+PRP group was significantly increased(P＜0.05). Compared with other groups, the osteogenic differentiation activity and the expressions of osteogenic proteins and genes were significantly increased in ADSCs with BMDMs+PRP treatment(P＜0.05). Bone volume/ tissue volume(BV/TV),trabecular number(Tb.N)and trabecular thickness(Tb.Th)were significantly increased in the BMDMs+PRP group compared with the other groups (P＜0.001). Conclusions PRP promotes ADSCs migration and osteogenic differentiation by stimulating the polarization of BMDMs towards M2 in vitro. BMDMs+PRP can significantly promote the differentiation of ADSCs into osteoblasts and accelerate bone formation in vivo, thus improving the poor healing of diabetic bone defects of rats.

Neuroprotective effects of exosomes derived from human umbilical cord mesenchymal stem cells in cerebral palsy mice

CHEN Xingxing, SAI Yipa, HU Xiaoxia, WANG Sanping, LUO Xuan, LIU Jing

2023, 43(8):  1201-1207.  doi:10.16352/j.issn.1001-6325.2023.08.1201

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Objective To investigate the neuroprotective effects of exosomes derived from human umbilical cord mesenchymal stem cells(HUCMSCs) in cerebral palsy (CP) mice. Methods The exosomes from HUCMSCs were verified by transmission electron microscopy (TEM), nanoparticle tracking analyzer(NTA) and Western blot. The animals were randomly divided as sham operation group (sham group, n=8), model group[CP group, n=8, hypoxic induction after unilateral common carotid artery ligation combined with lipopolysaccharide(LPS) infection] and model exosome group (CP-Exo group, n=8, 125 μg exosomes was injected intravenously). Neuromotor function, the ability of grasping and balance were evaluated by Longa score, grid test and pole climbing test. The pathological lesion in brain tissue was observed with hematoxylin-eosin (HE) staining, the synaptic structure of neurons was observed by TEM, the survival of neurons was detected by toluidine blue staining, and the expression levels of PSD-95 and synaptophysin (SYP) were detected by Western blot. Results The exosomes isolated were spherical and double-membrane vesicles with an average particle size of 123nm, which expressed TSG101 protein. In animal behavior tests, Longa score in CP group was significantly higher than sham group(P＜0.01) and CP-Exo group (P＜ 0.05). There was no plaque necrosis found in exosome group and the incidence of nerve fiber demyelination in the white matter area was significantly reduced than in the CP group (P＜0.01). The synaptic structure of the sham group was relatively normal, while the other two groups showed varying degrees of pathological changes. The general trend of pathological changes was CP group＞CP-Exo group＞ sham group. The number of Nissl bodies in hippocampal pyramidal cells in CP group was significantly less than that in sham group(P＜0.01), however, which was also less than that in CP-Exo group (P＜ 0.01). The expression of PSD-95 and SYP in CP group was significantly lower than that in sham group (P＜0.01), and the levels PSD-95 and SYP in CP-Exo group was significantly higher than those in CP group(P＜0.01). Conclusions HUCMSCs exosomes can promote the recovery of neural function of cerebral palsy mice, reduce synaptic damage and protect neuron cells by increasing the expression of PSD-95 and SYP.

Short-chain fatty acid sodium acetate reduces hypoxia- reoxygenation induced injury of human renal tubular epithelial cell line HK2

JIANG Luojia, XU Haibo

2023, 43(8):  1208-1214.  doi:10.16352/j.issn.1001-6325.2023.08.1208

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Objective To study the protective effect and related mechanisms of short-chain fatty acid sodium acetate on hypoxia/reoxygenation (H/R)-induced injury of human renal tubular epithelial cell line HK2. Methods The H/R model was established, and HK2 cells were incubated with sodium acetate (SA) for 2 h. The survival rate of HK2 cells was detected by CCK8 assay; Enzyme activity kit was used for detection of inflammatory factors, cellular reactive oxygen species and ATP production; Flow cytometry was used to measure mitochondrial membrane potential (MMP) and mitochondrial oxidative stress products (mitoSOX) levels; Electron microscope was used to observe mitochondrial ultrastructural damage; Western blot was used to detected the expression of inflammatory signaling pathway IκB-α/NF-κB and mitochondrial energy disorder signaling pathway AMPK/PGC-1α. Results Compared with the control group, the survival rate of HK2 cells in the H/R group was significantly decreased(P＜0.05); the expression of intracellular ROS, mitoSOX and inflammatory factors was significantly increased(P＜0.05); the ultrastructure of mitochondria was severely damaged, and the content of ATP and MMP was significantly decreased(P＜0.05); it was further found that the protein expression of p-IκB-α and NF-κB-p-P65 was significantly increased while the protein expression of p-AMPK and PGC-1α was significantly decreased(P＜0.05). Compared with H/R group, SA significantly enhanced the survival rate of HK2 cells (P＜0.05); SA inhibited the release of intracellular ROS, mitoSOX and inflammatory factors; SA inhibited mitochondrial ultrastructural damage, decreased ATP and MMP (P＜0.05); SA promoted the expression of p-AMPK and PGC-1α and inhibited the expression of p-IκB-α and NF-κB-p-P65 (P＜0.05). Conclusions Sodium acetate plays a protective role with potential mechanisms of anti-inflammation, anti-oxidative stress, protection of mitochondrial structure and function of HK2 cells induced by H/R through inhibiting IκB-α/ NF-κB and activating AMPK/PGC-1α signal pathway.

miR-146a-5p inhibits proliferation and invasion of prostate cancer cell line PC-3 by targeting SMAD4

LIU Bide, LI Xun, WANG Shuheng, JIN Hongyong, ZHANG Xiao'an, LI Jiuzhi

2023, 43(8):  1215-1221.  doi:10.16352/j.issn.1001-6325.2023.08.1215

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Objective To explore the inhibition effect and mechanism of miR-146a-5p on proliferation and invasion of prostate cancer (PCa) cell line PC-3 by targeting SMAD4. Methods RT-qPCR was used to detect the expression of miR-146a-5p in PCa tissues and cell lines. The relevance of miR-146a-5p expression with Gleason score was also analyzed. MTT, BrdU experiment, cell colony formation experiment, scratch experiment, Transwell assay and nude mouse xenograft model experiment were conducted to detect the effect of miR-146a-5p on cell proliferation, tumorigenicity, migration and invasion. The expression of SMAD4 in PCa tissues was detected by RT-qPCR, and the targeting relationship of SMAD4 and miR-146a-5p was confirmed by double luciferase reporter gene assay and rescue experiment. Western blot was used to detect the expression of SMAD2/SMAD3 complex in nucleus affected by miR-146a-5p and SMAD4. Finally, double luciferase reporter gene assay and ChIP experiment were performed to examine the targeting regulation of TIM3 by miR-146a-5p/SMAD4/SMAD2/SMAD3 signaling axis. Results miR-146a-5p was low expressed in PCa tissues and cell lines; its expression was negatively correlated to Gleason score and had the lowest expression in PC-3 cells. miR-146a-5p inhibited the proliferation and invasion of PC-3 cells by targeting SMAD4. SMAD2/SMAD3/TIM3 axis seemed to be the downstream mechanism of miR-146a-5p/SMAD4 signaling pathway. Conclusions miR-146a-5p can inhibit the proliferation and invasion of PC-3 cells by targeting SMAD4, and the downstream mechanism might be related to the SMAD2/SMAD3/TIM3 signaling pathway.

Platycodon D attenuates renal ischemia-reperfusion injury in rats

WANG Qiong, DONG Qianlan, ZHU Yanting, JIN Gang, ZHANG Linping

2023, 43(8):  1222-1228.  doi:10.16352/j.issn.1001-6325.2023.08.1222

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Objective To investigate the protective effect of Platycodin D (PD) on renal ischemia-reperfusion injury in rats. Methods The rats were divided into sham group, model group, low, middle and high-dose groups with 10 animals in each. The rat model of ischemia reperfusion injury was established by clamping the bilateral renal pedicles in model group, low, middle and high-dose group. The rats in sham group underwent the same modeling operation but did not clamp the renal pedicles. Before 5 minutes of modeling, the rats of low, middle and high-dose group were intra-peritoneally injected with 12.5, 25 and 50 mg/kg PD, respectively. After 24 h of modeling, the serum creatinine (Cr) and blood urea nitrogen (BUN) levels, as well as antioxidant markers[superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA)] levels in kidney tissues were measured. HE staining was used to evaluate renal tissue lesions. Immuno-histochemical staining was used to detect the expression of caspase-3 in renal tissue, the apoptosis index of renal tubular epithelial cells was evaluated by the counting of caspase-3 positive cells. RT-qPCR was used to detect the expression of interleukin-1β (IL-1β), IL-6 and IL-10 mRNA in renal tissues. Western blot was used to detect the expression of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), p65, p-p65, inhibitor of nuclear factor kappa B (NF-κB) (IκB) and p-IκB. Results Compared with sham group, the kidney lesions, serum Cr and BUN levels, apoptosis index of renal tubular epithelial cells in model group were all increased (P＜0.05). Compared with model group, the kidney lesions, serum Cr and BUN levels, apoptosis index of renal tubular epithelial cells in low, middle and high-dose group were decreased (P＜0.05). Compared with sham group, the level of MDA and mRNA of IL-1β and IL-6, the protein expression of TLR4/MyD88/NF-κB signaling pathway in kidney tissue in model group were all significantly increased while the level of SOD and GSH-Px and mRNA of IL-10 in rat kidney tissue were decreased(P＜0.05). Compared with model group, the level of MDA, mRNA of IL-1β and IL-6, the protein expression of TLR4/MyD88/NF-κB signaling pathway in kidney tissue in low, middle and high-dose group were all decreased, SOD and GSH-Px and IL-10 mRNA in rat kidney tissue were increased (P＜0.05). Conclusions Platycodin D has a protective effect on renal ischemia-reperfusion injury in rats, and the underlying mechanism may be related to the improvement of antioxidant capacity and inhibition of TLR4/MyD88/NF-κB signaling pathway.

Transcription factor CREB regulates the expression of miR-30a in mouse CD4⁺ lymphocytes

HAN Jingjing, WANG Xuanruo, ZHANG Xinyi, GAO Han, CHENG Xiumei, YANG Liucai, QU Xuebin

2023, 43(8):  1229-1233.  doi:10.16352/j.issn.1001-6325.2023.08.1229

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Objective To study the regulatory effect of the transcription factor cAMP response element binding protein (CREB) on the expression of miR-30a in CD4⁺ lymphocytes. Methods The promoter region of miR-30a gene was determined by UCSC and Ensembl website. The methylation of DNA sequence in the promoter region was calculated on EMBOSS Cpgplot website. The transcription factor binding region and its conservation were analyzed via ECR Browser website. The special binding transcription factors were predicted with support from JASPAR database. Chromatin immunoprecipitation assay was used to confirm the binding of CREB in the promoter region of miR-30a gene in mouse CD4⁺ lymphocytes. According to whether CREB inhibitor KG-501 was added to the culture medium, mouse CD4⁺ lymphocytes were divided into three groups, control, vehicle and KG-501 treated group. The effect of KG-501 on the expression of miR-30a in the cells was detected by RT-qPCR. Results None methylated CpG islands were found in the promoter region of miR-30a gene. The promoter region contained highly conserved transcription factor binding sequences, including several CREB binding sites. Transcription factor CREB bound to the upstream of miR-30a gene promoter region. KG-501 significantly down-regulated the expression of miR-30a(P＜0.05). Conclusions Transcription factor CREB binds to specific sites in the promoter region of miR-30a gene to regulate the expression of miR-30a in mouse CD4⁺ lymphocytes.

DNA damage response activated by high calcium and phosphorus induces premature aging of human aortic smooth muscle cells

FAN Zhijuan, WU Yujing, TIAN Yaqiong, LIU Shuang, ZHANG Die, LIU Shuye

2023, 43(8):  1234-1240.  doi:10.16352/j.issn.1001-6325.2023.08.1234

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Objective To investigate the mechanism of DNA damage response(DDR) pathway regulating calcification in human aortic smooth muscle cells(HASMCs). Methods The HASMCs were divided into the control group, model group, ATM treatment group, and PARP treatment group, and they were cultured for 12 days. Cell calcification was measured by Alizarin red staining and σ-Cresolphthalein; phosphorylation levels of histone γH2AX, protein levels of p16 and p21, and phosphorylation levels of ATM on Ser1981 were tested by Western blot, premature cell senescence by β-galactosidase staining; and p16 and p21 mRNA by qPCR. The level of oxidative stress was measured by 8-hydroxy-2′-deoxyguanosine (8-OHDG), and the level of IL-6 and IL-8 was measured by ELISA kit. Results The calcification was evident in the model group as compared with that in control group. There were significant changes in 8-OHDG, histone γH2AX phosphorylation, β -galactosidase staining, mRNA and protein of p16, p21 mRNA, release of IL 6 and IL 8 and ATM phosphorylation(P＜0.05).The changes in the model group alleviated by ATM and PARP treatment. Conclusions High calcium and phosphorus environment stimulates HASMCs to produce sustained DNA damage, triggers ATM phosphorylation, activates p16 protein expression, and induces premature cell senescence causing cell death and resulted in calcification.

Rapamycin alleviates hepatocellular carcinoma progressing in transgenic mice

LIN Yingxue, CUI Haipeng, WANG Aiguo, YAO Yinhui, ZHAO Juan

2023, 43(8):  1241-1246.  doi:10.16352/j.issn.1001-6325.2023.08.1241

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Objective Exploring the influence of rapamycin on the progressing of hepatocellular carcinoma(HCC) in H-ras 12V transgenic mice. Methods The expression level of miRNA in liver tissue and peritumor tissue of transgenic mice was detected by high-throughput sequencing of miRNAs. Treatment of transgenic mice with rapamycin and the changes of tumor morphology were detected. The miR-183 expression levels were detected by RT-qPCR. The protein expression levels of mTOR, ERK and PDCD4, the downstream target gene of miR-183, were detected by Western blot. Results Compared with the peritumor tissue, the miR-183 expression in hepatocellular carcinoma tissue was increased significantly (P＜0.01). The protein expression levels of PDCD4 were decreased significantly(P＜0.05). Compared with the control group, after intervention with rapamycin, the counting of liver tumors decreased significantly(P＜0.05). The liver co-efficient decreased significantly(P＜0.05). The ERK phosphorylation increased significantly (P＜0.05). The miR-183 expression was decreased significantly (P＜0.05). The protein level of PDCD4 increased significantly (P＜0.05). Conclusions Rapamycin can alleviate hepatocellular carcinoma progression in H-ras 12V transgenic mice.

Expression of DDX5 and DDX17 in ovarian cancer tissues and their effects on proliferation and drug resistance of human ovarian cancer cell line SKOV3

HUANG Jie, LI Runbo, GUO Jianxin, HAN Jian

2023, 43(8):  1247-1253.  doi:10.16352/j.issn.1001-6325.2023.08.1247

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Objective To analyze the expression of DDX5 and DDX17 in ovarian cancer and their relationship with patient survival, and to study the effects of DDX5 and DDX17 on proliferation and drug resistance of ovarian cancer cell line SKOV3. Methods The expression of DDX5 and DDX17 was detected by RT-qPCR in tissue samples from 40 cases of ovarian carcinoma and 27 controls. TCGA database was used to analyze the relationship between DDX15/DDX17 and survival prognosis of ovarian cancer patients. The expression of DDX5 and DDX17 in SKOV3 was down-regulated by lentivirus-mediated DDX5-shRNA and DDX17-shRNA. Western blot was used to verify the knockdown effect, and CCK8 was used to detect cell proliferation and resistance to paclitaxel and carboplatin. Results Compared with normal ovarian epithelium, the expression of DDX5 in samples from ovarian carcinoma was increased, while that of DDX17 was decreased significantly(P＜0.05); Ovarian cancer patients with high DDX5 expression had shorter progression-free survival and overall survival time, while those with high DDX17 expression had longer progression-free survival and overall survival time. Down-regulated DDX5 expression inhibited the SKOV3 cells proliferation, while down-regulated DDX17 expression promoted SKOV3 cells proliferation. The drug sensitivity of SKOV3 cells to paclitaxel and carboplatin was significantly increased after the down-regulation of DDX5 expression, while the drug sensitivity of SKOV3 cells to paclitaxel and carboplatin was not significantly changed after the down-regulation of DDX17 expression. Conclusions DDX5 and DDX17 regulate proliferation and drug resistance of ovarian cancer cells, and are potential regulatory factors functioning in ovarian cancer.

Expression of death receptor apoptosis pathway- correlated factors in pancreatic tissue of mouse diabetic model

MIAO Miao, LIU Yan, ZHANG Huan, ZHAO Huichao, LI Zhuofan, ZHANG Haolan, YUAN Panpan

2023, 43(8):  1254-1258.  doi:10.16352/j.issn.1001-6325.2023.08.1254

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Objective To study the expression of apoptosis related factors in the apoptosis pathway of death receptor in the pancreas of mice diabetic models. Methods The mice were divided into control group and model group. They were injected alloxan intraperitoneally twice (120 mg/kg,80 mg/kg). Blood glucose was measured on the third and seventh days after the model was established; Pancreatic tissue was taken for HE observation, TUNEL was used to detect the apoptosis rate, and RT-qPCR was used to detect the mRNA expression of tumor necrosis factor receptor 1 (TNFR1), Fas-related death domain (FADD), caspase-8 and caspase-3. Results Compared with the control group, pancreatic acinar cells in the model group showed granular degeneration, capillary congestion, decreased islet volume and the number of cells in islets. Compared with the control group, the apoptosis rate of pancreatic cells in the model group was significantly increased (P＜0.01). In addition, compared with the control group, the expression of Tnfr1, Fadd, caspase-8 and caspase-3 mRNA in the pancreas of the model group increased significantly or highly significantly on the third and seventh days (P＜0.05 or P＜0.01). Conclusions Alloxan could promote the over-expression of Tnfr1, Fadd, caspase-8 and caspase-3 mRNA in pancreatic tissue of diabetes model mice, and then induce apoptosis of pancreatic cells.

TARBP2 promotes human breast cancer cell metastasis by degrading AKAP12 transcript

WANG Jihui, CAO Jilie, ZHANG Lijun, ZHANG Jun, LU Wenbao

2023, 43(8):  1259-1264.  doi:10.16352/j.issn.1001-6325.2023.08.1259

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Objective To study the role and mechanism of trans-activation response RNA-binding protein 2 (TARBP2) in post-transcriptional regulation of the expression of AKAP12, a metastasis suppressor gene, in breast cancer cells. Methods The expression of TARBP2 in breast tumor tissues and potential relationship between TARBP2 expression and tumor metastasis were analyzed by bioinformatics methods. Bioinformatics methods were used to analyze the correlation between TARBP2 and AKAP12 expression in different breast cancer datasets. MDA-MB-231 cells were transfected with GFP-tagged TARBP2 plasmids by Lipofectamine 2000 and screening with G418. Lung metastasis in tumor-bearing nude mice was detected by HE staining. RT-qPCR was used to test the expression of AKAP12 and its half-life. Luciferase assay was carried out to determine whether the transcript decay was mediated through targeting the 3′UTR. EMSA assay was performed to detect the physically binding of TARBP2 with stem-loop structure of AKAP12. Results The high expression of TARBP2 in human breast tumor tissue was closely related to lymph node metastasis. TARBP2 over-expression in MDA-MB-231 cells significantly promoted tumor metastasis in vitro(P＜0.05). TARBP2 inhibited the expression of AKAP12(P＜0.01) and reduced its mRNA half-life(P＜0.05). TARBP2 significantly suppressed luciferase activity of AKAP12 3′UTR reporter(P＜0.05) and bound to the stem-loop structure. There was a significant negative correlation between TARBP2 and AKAP12 expression in human breast cancer samples(P＜0.001). Conclusions TARBP2-mediated inhibition of AKAP12 gene expression might be one of the mechanisms of TARBP2 promoting breast cancer metastasis.

Silencing SF3B1 promotes apoptosis and inhibits proliferation and invasion of human lung cancer cell line A549

ZHANG Xiaowan, KANG Xia, YAO Xiaoying, XIE Fang, LI Ying

2023, 43(8):  1265-1270.  doi:10.16352/j.issn.1001-6325.2023.08.1265

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Objective To explore the effect of splicing factor 3B subunit (SF3B1) on apoptosis, proliferation and invasion of human lung cancer cells. Methods Non-small cell lung cancer(NSCLC) patients in the General Hospital of Western Theater Command PLA from June 2017 to June 2020 were selected as the research objects to detect the level of SF3B1 in cancer and adjacent tissues, and to analyze the relationship between SF3B1 and the pathological characteristics, survival and prognosis of patients. In addition, A549 cells were cultured and divided into control group, transfected si-NC group and si-SF3B1 group. Cell apoptosis was detected by flow cytometry, cell proliferation was detected by CCK-8, cell invasion was detected by Transwell, and the expression of Ras/Raf/ERK signal protein was detected by Western blot. Results The level of SF3B1 mRNA in cancer tissues was significantly higher than that in adjacent tissues(P＜0.05), and its level in A549 cells was significantly higher than that in normal lung epithelial cell line BEAS-2B(P＜0.05). The high level of SF3B1 was related to lymph node metastasis, tumor size and differentiation(P＜0.05). The median overall survival(OS) and progression free survival(PFS) of patients with low expression of SF3B1 were significantly higher than those with high expression of SF3B1(P＜0.05). After silencing SF3B1, the apoptosis of A549 cells was significantly increased but the proliferation and invasion were significantly decreased. The silence of SF3B1 suppressed the expression of Ras, Raf and p-ERK in A549 cells. Conclusions SF3B1 is highly expressed in NSCLC. Silencing SF3B1 can promote lung cancer cell apoptosis, inhibit cell proliferation and invasion.

Clinical Sciences

Immunoglobulin is valuable in the differential diagnosis of primary biliary cholangitis, hepatitis E and cirrhosis

XING Xuemei, YANG Jianrui, LI Zhijun, CHEN Yajuan, YAO Fengxia, LIU Zheng

2023, 43(8):  1271-1274.  doi:10.16352/j.issn.1001-6325.2023.08.1271

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Objective To explore the value of immunoglobulin in differential diagnosis of primary biliary cholangitis (PBC), hepatitis E and liver cirrhosis. Methods Blood samples were collected from 121 patients with PBC and 107 healthy people. The serum r-glutamyltransferase (r-GT), alkaline phosphatase (ALP), immunoglobulin IgG and IgM, total cholesterol (TCH) and lipoprotein (a)[Lp(a)] were detected by biochemical routine tests; Blood routine counted white blood cells (WBC) and platelets (PC); The IgG and IgM antibodies of HEV were detected by enzyme-linked immunosorbent assay (ELISA). Results r-GT, ALP, IgG and IgM in PBC patients increased significantly (P＜0.05) accompanied by significant decrease in WBC and PC values (P＜0.05). In patients with PBC cirrhosis, IgG increased significantly (P＜0.05), while WBC and PC values decreased significantly(P＜0.05). IgG and IgM in patients with PBC+cirrhosis+HEV antibody positive increased significantly(P＜0.05), while WBC and PC values decreased significantly (P＜0.05). Conclusions The increase of IgG and IgM might be used as diagnostic markers for the development of cirrhosis in PBC patients with HEV infection.

Comparison of airway anatomical indices in magnetic resonance imaging between growth hormone pituitary adenoma and nonfunctioning pituitary adenoma

JING Longnian, YAO Jingxin, HAN Ruquan

2023, 43(8):  1275-1279.  doi:10.16352/j.issn.1001-6325.2023.08.1275

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Objective To evaluate the application of airway anatomical indices in magnetic resonance imaging (MRI) to predict difficult airways in patients with growth hormone pituitary adenoma. Methods This is a retrospective analysis for patients with pituitary adenoma excision under general anesthesia at Beijing Tiantan Hospital, Capital Medical University, from December 2018 to December 2022. Patients with growth hormone pituitary adenoma (GH group, n=81) and patients with nonfunctioning pituitary adenoma (NF group, n=81) were included. Linear distance between upper lip and uvula (ULUD), tongue thickness on the plane of cervical vertebra 2/3 (TT_C2/3), linear distance of supraglottic airway cervical vertebra 2/3(SAD_C2/3) and linear distance between underlip and posterior pharyngeal wall (ULPD) were measured in the median sagittal plane of head MRI. The data in the two groups were compared. Results ULUD, TT_C2/3, and ULPD in the GH group were significantly longer than those in the NF group (P＜0.05). SAD_C2/3 was not significantly different (P＞0.05). GH was confirmed to be correlated with TT_C2/3 and ULPD. Conclusions ULUD, TT_C2/3, ULPD found by head MRI and GH can be used as predictors of difficult airway in patients with growth hormone pituitary adenoma.

Monitoring effect of Aspergillus fumigatus specific IgG for recurrence of allergic bronchopulmonary aspergillosis

TANG Rui, LEI Shubin, SUN Jinlyu

2023, 43(8):  1280-1283.  doi:10.16352/j.issn.1001-6325.2023.08.1280

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Objective To investigate serological monitoring indicator of the recurrence of allergic broncho-pulmonary aspergillosis (ABPA). Methods The follow-up records of 18 outpatients in Peking Union Medical College Hospital from June 2001 to September 2021 were collected and analyzed statistically. Results In total, 68 records of 18 patients had been analyzed. Three models were conducted, model 1 included serological Aspergillus fumigatus specific IgG(Gm3) level as a single variant, model 2 added gender and age based on model 1, and model 3 added Aspergillus fumigatus specific IgE level and the count of white blood cells based on model 1. Serological Gm3 level was an independent risk factor in all 3 models. Conclusions Serological Gm3 level is potential serological marker used in monitoring the recurrence of ABPA.

Clinical characteristics and risk factors of macrovascular diseases in patients with different subtypes of type 1 diabetes mellitus

WANG Liting, XU Lingling, LI Wei, PING Fan, ZHANG Huabing, MA Yahong, LI Yuxiu

2023, 43(8):  1284-1288.  doi:10.16352/j.issn.1001-6325.2023.08.1284

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Objective To investigate the clinical characteristics of different subtypes of type 1 diabetes mellitus(T1DM) patients and to analyze the risk of macrovascular diseases in different subtypes of T1DM patients. Methods The clinical data among 79 T1DM patients who treated in the Endocrinology Department of Beijing Puren Hospital from March 2002 to July 2021 were retrospectively analyzed. There were 21 patients with idiopathic T1DM and 58 patients with autoimmune T1DM. Excluding 14 patients because of data missing, 65 patients were divided into macrovascular disease group (n=13) and non-macrovascular disease group (n=52). Results 1)Patients with idiopathic T1DM manifested a longer diabetic course,a higher high density lipoprotein cholesterol(HDL-c)level, a lower body mass index(BMI), hemoglobin A1c(HbA1c) and fasting C peptide(FC-P)level. 2)Compared with the group non-macrovascular disease, the macrovascular disease group was elder with higher prevalence of idiopathic T1DM and lower estimated glomerular filtration rate(eGFR) level and incidence of diabetic ketoacidosis(DKA) at the beginning of disease. 3)Macrovascular lesions were positively correlated with age(r=0.432) and diabetic course (r=0.245)(P＜0.05), were negatively correlated with eGFR(r=-0.392), positive islet autoantibody (r=-0.268) and DKA incidence at the beginning of disease(r=-0.313)(P＜0.05). 4)Multivariate regression analysis showed that negative auto-antibody finding was a risk factor for macrovascular disease (OR=0.03, 95% CI:0.001-0.858, P＜0.05) after age, diabetic course, BMI, eGFR, HDL-c and incidence of DKA at onset were adjusted. Conclusions Patients with idiopathic T1DM show a higher risk of macrovascular disease risk than those with autoimmune T1DM.

Evaluation of EEG, EMG and clinical semiology in the lateral location of bilateral extremities tonic seizures of focal epilepsy

SUN Li, LI Fang, WANG Xiaomei, LIU Xingzhou

2023, 43(8):  1289-1293.  doi:10.16352/j.issn.1001-6325.2023.08.1289

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Objective To evaluate the lateralizing value of electrical and clinical semiology in patients with bilateral extremities tonic seizure (BETS). Methods Ninety-two times of BETS were retrospectively observed from 26 patients with focal epilepsy, based on the surgery and follow-up results. The ictal electroencephalography(EEG),synchronous surface electromyography(EMG) and clinical symptoms were collected. Results The EEGs were correctly lateralizing in 59.8%(55/92)seizures.The accompanying lateral signs were correctly lateralized in 64.1%(59/92)seizures. The tonic joint movement lateralization was correct in 81.5%(75/92)seizures. The surface EMGs were 85.9%(79/92)correctly lateralized. The positivity rate of the latter two was higher than that of the first two, which was statistically significant. Conclusions Both the tonic joint movement and surface EMG have better lateralization value and guiding significance for preoperative evaluation of patients with epilepsy.

Mini Reviews

Research progress on the interaction between autophagy and ferroptosis in non-alcoholic fatty liver disease

LI Xiang, LI Ye

2023, 43(8):  1294-1298.  doi:10.16352/j.issn.1001-6325.2023.08.1294

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In hepatocytes, autophagy and ferroptosis, as important biological processes in cells, play important roles in maintaining lipid accumulation homeostasis, oxidative stress and lipid peroxidation balance. Here we reviewed the impact of autophagy on the abnormal lipids accumulation in the process of liver lipid metabolism, and its relationship with ferroptosis, which was marked by oxidative stress and lipid peroxidation. The impact of the interaction between autophagy and ferroptosis on the course of non-alcoholic fatty liver disease (NAFLD) might provide some new ideas for the treatment of NAFLD.

Research advances of Ghrelin in metabolic diseases

WANG Xinrui, QIN Yan

2023, 43(8):  1299-1303.  doi:10.16352/j.issn.1001-6325.2023.08.1299

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Ghrelin, a newly discovered gastric peptide, involves in the regulation of various biological processes and plays important role in the occurrence and development of metabolic diseases such as non-alcoholic fatty liver disease, diabetes and obesity by effectively regulating glucose and lipid metabolism and anti-oxidation.

Research progress on the role of TLR2 in the development of gastrointestinal cancer

ZOU Yuxiang, TANG Hui

2023, 43(8):  1304-1308.  doi:10.16352/j.issn.1001-6325.2023.08.1304

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Toll-like receptor 2 (TLR2) is a pattern recognition receptor (PRR) that mediates immune and inflammatory responses, angiogenesis, and other physiological processes using the TLR2-MyD88 pathway. It is expressed on the cell surface, activated by exogenous PAMPs and endogenous DAMPs, and mediates the development of gastrointestinal cancer by regulating nuclear RNA expression through classical and non-classical signaling pathways such as MyD88, PI3K/Akt, Wnt/β-catenin, and MAPK.

Research progress on the role of canonical transient receptor potential channels in skeletal muscle diseases

XIE Dongge, LI Junhao, HAN Han, LI Shoutian

2023, 43(8):  1309-1312.  doi:10.16352/j.issn.1001-6325.2023.08.1309

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Canonical transient receptor potential(TRPC) is a kind of non-selective cation channel located in the cell membrane, which plays an important role in electrophysiological activity of cells. Under pathological conditions, TRPC channels are more selective for Ca²⁺, which will break the Ca²⁺ homeostasis in skeletal muscle cells and lead to calcium overload and subsequent occurrence of a variety of skeletal muscle diseases. To study the role and mechanism of TRPC channels in skeletal muscle diseases is helpful to provide new ideas for disease treatment.

Progress in CRISPR/Cas9 for CAR-T cell therapy of tumors

WU Qi, WANG Shaobo

2023, 43(8):  1313-1316.  doi:10.16352/j.issn.1001-6325.2023.08.1313

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CRISPR/Cas9 gene editing technology can effectively solve the problem of insufficient source of T cells in chimerical antigen receptor-T (CAR-T) therapy and enhance their anti-tumor abilities by knocking out the genes related to the suppression of T cells activity. The technology can also be used to construct T cells with dual-targeting effect to stop tumor immune escape and inhibit recurrence and drug resistance development during chemotherapy. Compared with the other genetic engineering technologies that have complex processes, multiple genome editing by CRISPR/Cas9 is a technology of efficient and convenient to operate.So it is believed to be valuable and potential target molecules for CAR-T therapy.

Research progress of drugs targeting ferroptosis for the treatment of non-alcoholic fatty liver disease

HAO Dandan, ZHANG Lei, BAI Chunying

2023, 43(8):  1317-1321.  doi:10.16352/j.issn.1001-6325.2023.08.1317

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Emerging evidence indicates that ferroptosis, an iron-dependent form of regulated cell death, plays a critical role in the pathological progression of non-alcoholic fatty liver disease (NAFLD). Ferroptosis involves in the pathogenesis of NAFLD and drugs targeting at ferroptosis by its inhibitors can alleviate the progression of NAFLD both in vitro and in vivo. Pharmacological inhibition of ferroptosis might be a potential strategy of NAFLD treatment.

Research progress on the role of transcription factor T-bet in the pathogenesis of chronic obstructive pulmonary disease

CHEN Yuting, HUANG Ling, XIA Junjie, QIU Yu, YI Ke, WANG Jincheng

2023, 43(8):  1322-1325.  doi:10.16352/j.issn.1001-6325.2023.08.1322

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Chronic obstructive pulmonary disease (COPD) is a chronic and progressive respiratory disease. Transcription factor T-bet is involved in the inflammatory response and lung function changes of COPD, which is expected to become a new target for the prevention and treatment of COPD. It might provide a new idea for the targeted research of COPD.

Medical Education

Positive influence of setting up examination checkpoints and contents on the effect of medical training

WANG Hanbi, CHEN Jie, ZHANG Zhiyuan, ZHAO Chunxia, WU Tong, SUN Aijun

2023, 43(8):  1326-1329.  doi:10.16352/j.issn.1001-6325.2023.08.1326

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Objective To explore the influence of reasonable examination contents and checkpoints on the training outcomes of continuing medical education. Methods From September 2020 to February 2021, we carried out online and offline continuing medical education training for clinicians in maternal and child health centers in 16 districts of Beijing. The effect was assessed after the training. The assessment contents were assigned following the characteristics of continuing medical education, and examination checkpoints were set up according to the teaching content and arrangement to evaluate the learning effect. Results A total of three sections of online training programs were completed with 10 courses in each section and one examination was conducted at the end of each section. A total of 48 examinations were completed in 16 district maternal and child health care hospitals. The offline training was undertaken by the maternal and child health care hospitals in 16 districts of Beijing. Four to six training courses were completed in each hospital and one offline training examination was completed at the end of the course. The average score of test after three times of online training in 16 district maternal and child health care hospitals was 88.56±4.76, 93.39±3.22 and 90.71±5.18, respectively. Offline test average score was 88.65±4.35. Most students chose ‘excellent’ and ‘good’ for offline training satisfaction. Conclusions Reasonable establishment of examination node and examination content in medical continuing education may have positive influence on training outcomes.

Application of artificial intelligence in respiratory medicine teaching

ZHANG Xiaotong, XU Kaifeng

2023, 43(8):  1330-1333.  doi:10.16352/j.issn.1001-6325.2023.08.1331

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The development of artificial intelligence has received much attention recently and has been widely applied in the field of medicine. Development of clinical teaching technology based on artificial intelligence is important in improving the medical education. This article explores the current status and future prospects of the application of artificial intelligence in the training of respiratory medicine.