Objective:To investigate the effect of metformin on the chemosensitivity of colon cancer cells to 5-fluorouracil (5-FU) and its possible mechanism.Methods:Colon cancer cell lines HCT-116 and HT-29 were cultured in vitro, and the CCK-8 method was used to determine cell viability after intervention with different concentrations (1.25, 2.5, 5, 10, 20, 40, 80 mmol/L) of metformin hydrochloride (MET-HCl) or different concentrations (1.25, 2.5, 5, 10, 20, 40 μmol/L) of 5-FU. The survival rate and 48-hour half maximal inhibitory concentration ( IC50) of the two drugs were calculated. The combined index (CI) of MET-HCl and 5-FU was calculated using the Chou-Talalay model, and the concentrations of MET-HCl and 5-FU in subsequent experiments were determined based on the concentration of the two drugs when the inhibition rate (Fa) was 0.5. The CCK-8 method and Annexin V-FITC/PI flow cytometry were used to detect the survival rate and apoptosis rate of HCT-116 cells or HT-29 cells treated with the two drugs alone and in combination at experimental concentrations, respectively. Cells treated with drug free culture flnid were used as the control group. CXCR4 specific small interfering RNA (siRNA) was transfected into HCT-116 cells and HT-29 cells, and the decreased ( P < 0.05) expression of CXCR4 protein detected by Western blotting indicated successful knockdown of CXCR4. CCK-8 method was used to detect the survival rate of cells with CXCR4 knockdown by two drugs alone and in combination, and the untransfected cells added drug free culture fluid were served as the si-control group. Western blotting was used to detect the expression of proteins related to the CXCR4/Akt signaling pathway in HCT-116 cells or HT-29 cells treated with two drugs alone or in combination. Results:CCK-8 assay showed that MET-HCl and 5-FU decreased the viability of HCT-116 and HT-29 cells in a concentration-time-dependent manner; at 48 h, IC50 of MET-HCl and 5-FU in HCT-116 cells were (13.0±5.8) mmol/L and (16.9±7.2) μmol/L, respectively, and IC50 in HT-29 cells were (8.6±2.8) mmol/L and (9.7±3.1) μmol/L, respectively. When the cell inhibition rates of HCT-116 cells or HT-29 cells detected by CCK-8 method were 50%, 75% and 90% after 48 h of treatment, the corresponding CI values of HCT-116 cells were 0.48, 0.37 and 0.25, and HT-29 cells were 0.57, 0.51 and 0.49, suggesting that the combination of the two drugs had a synergistic effect. Based on Fa = 0.5, the experimental concentrations of MET-HCl and 5-FU were determined to be 10 mmol/L and 5 μmol/L. CCK-8 method showed that after HCT-116 cells or HT-29 cells were treated with 10 mmol/L MET-HCl, 5 μmol/L 5-FU alone or in combination for 48 h, the cell viability of each drug group was lower than that of the control group (all P < 0.05), there was no statistically significant difference in cell viability between MET-HCl alone and 5-FU alone (both P > 0.05), and the cell viability of the combination of the two drugs was lower than that of the two drugs alone (both P < 0.01). After CXCR4 was knocked down, the cell viability of each drug group was lower than that of the si-control group (all P < 0.05), but there was no significant difference in cell viability between the two drugs alone and the combination of the two drugs (all P > 0.05). Flow cytometry showed that after 24 h of treatment, the apoptosis rate of HCT-116 cells or HT-29 cells treated with MET-HCl alone or the combination of the two drugs was higher than that of the control group (all P < 0.05), but there was no statistically significant difference in the apoptosis rate of cells treated with 5-FU alone compared to the control group (all P > 0.05); the apoptosis rate of HCT-116 cells treated with the combination of the two drugs was higher than that of the two drugs alone (both P < 0.05); the apoptosis rate of HT-29 cells treated with the combination of the two drugs was higher than that of 5-FU alone ( P < 0.05), but there was no significant difference with MET-HCl alone ( P > 0.05). Western blotting showed that the relative expression levels of CXCR4, p-Akt and XRCC1 proteins in HCT-116 or HT-29 cells treated with MET-HCl alone and in combination of two drugs were lower than those in the control group (all P < 0.05), but there was no statistically significant difference in the relative expression levels of the 3 proteins in the two cell lines treated with 5-FU alone compared to the control group (all P > 0.05); the relative expression levels of the 3 proteins in the two cell lines treated with MET-HCl alone and in combination of two drugs were lower than those treated with 5-FU alone (all P < 0.05), the relative expression levels of CXCR4 and p-Akt proteins in the two cell lines treated with the combination of two drugs were lower than those treated with MET-HCl alone (all P < 0.05), and the relative expression level of XRCC1 protein in HCT-116 cells treated with the combination of two drugs was lower than that in HCT-116 cells treated with MET-HCl alone ( P < 0.05), but there was no significant difference in the relative expression level of XRCC1 protein in HT-29 cells treated with the combination of two drugs and MET-HCl alone ( P > 0.05). Conclusions:MET-HCl may reduce the expression of XRCC1 through the CXCR4/Akt signaling pathway, thereby enhancing the sensitivity of colon cancer cells to 5-FU.

Objective:To investigate the effect of metformin on the chemosensitivity of colon cancer cells to 5-fluorouracil (5-FU) and its possible mechanism.Methods:Colon cancer cell lines HCT-116 and HT-29 were cultured in vitro, and the CCK-8 method was used to determine cell viability after intervention with different concentrations (1.25, 2.5, 5, 10, 20, 40, 80 mmol/L) of metformin hydrochloride (MET-HCl) or different concentrations (1.25, 2.5, 5, 10, 20, 40 μmol/L) of 5-FU. The survival rate and 48-hour half maximal inhibitory concentration ( IC50) of the two drugs were calculated. The combined index (CI) of MET-HCl and 5-FU was calculated using the Chou-Talalay model, and the concentrations of MET-HCl and 5-FU in subsequent experiments were determined based on the concentration of the two drugs when the inhibition rate (Fa) was 0.5. The CCK-8 method and Annexin V-FITC/PI flow cytometry were used to detect the survival rate and apoptosis rate of HCT-116 cells or HT-29 cells treated with the two drugs alone and in combination at experimental concentrations, respectively. Cells treated with drug free culture flnid were used as the control group. CXCR4 specific small interfering RNA (siRNA) was transfected into HCT-116 cells and HT-29 cells, and the decreased ( P < 0.05) expression of CXCR4 protein detected by Western blotting indicated successful knockdown of CXCR4. CCK-8 method was used to detect the survival rate of cells with CXCR4 knockdown by two drugs alone and in combination, and the untransfected cells added drug free culture fluid were served as the si-control group. Western blotting was used to detect the expression of proteins related to the CXCR4/Akt signaling pathway in HCT-116 cells or HT-29 cells treated with two drugs alone or in combination. Results:CCK-8 assay showed that MET-HCl and 5-FU decreased the viability of HCT-116 and HT-29 cells in a concentration-time-dependent manner; at 48 h, IC50 of MET-HCl and 5-FU in HCT-116 cells were (13.0±5.8) mmol/L and (16.9±7.2) μmol/L, respectively, and IC50 in HT-29 cells were (8.6±2.8) mmol/L and (9.7±3.1) μmol/L, respectively. When the cell inhibition rates of HCT-116 cells or HT-29 cells detected by CCK-8 method were 50%, 75% and 90% after 48 h of treatment, the corresponding CI values of HCT-116 cells were 0.48, 0.37 and 0.25, and HT-29 cells were 0.57, 0.51 and 0.49, suggesting that the combination of the two drugs had a synergistic effect. Based on Fa = 0.5, the experimental concentrations of MET-HCl and 5-FU were determined to be 10 mmol/L and 5 μmol/L. CCK-8 method showed that after HCT-116 cells or HT-29 cells were treated with 10 mmol/L MET-HCl, 5 μmol/L 5-FU alone or in combination for 48 h, the cell viability of each drug group was lower than that of the control group (all P < 0.05), there was no statistically significant difference in cell viability between MET-HCl alone and 5-FU alone (both P > 0.05), and the cell viability of the combination of the two drugs was lower than that of the two drugs alone (both P < 0.01). After CXCR4 was knocked down, the cell viability of each drug group was lower than that of the si-control group (all P < 0.05), but there was no significant difference in cell viability between the two drugs alone and the combination of the two drugs (all P > 0.05). Flow cytometry showed that after 24 h of treatment, the apoptosis rate of HCT-116 cells or HT-29 cells treated with MET-HCl alone or the combination of the two drugs was higher than that of the control group (all P < 0.05), but there was no statistically significant difference in the apoptosis rate of cells treated with 5-FU alone compared to the control group (all P > 0.05); the apoptosis rate of HCT-116 cells treated with the combination of the two drugs was higher than that of the two drugs alone (both P < 0.05); the apoptosis rate of HT-29 cells treated with the combination of the two drugs was higher than that of 5-FU alone ( P < 0.05), but there was no significant difference with MET-HCl alone ( P > 0.05). Western blotting showed that the relative expression levels of CXCR4, p-Akt and XRCC1 proteins in HCT-116 or HT-29 cells treated with MET-HCl alone and in combination of two drugs were lower than those in the control group (all P < 0.05), but there was no statistically significant difference in the relative expression levels of the 3 proteins in the two cell lines treated with 5-FU alone compared to the control group (all P > 0.05); the relative expression levels of the 3 proteins in the two cell lines treated with MET-HCl alone and in combination of two drugs were lower than those treated with 5-FU alone (all P < 0.05), the relative expression levels of CXCR4 and p-Akt proteins in the two cell lines treated with the combination of two drugs were lower than those treated with MET-HCl alone (all P < 0.05), and the relative expression level of XRCC1 protein in HCT-116 cells treated with the combination of two drugs was lower than that in HCT-116 cells treated with MET-HCl alone ( P < 0.05), but there was no significant difference in the relative expression level of XRCC1 protein in HT-29 cells treated with the combination of two drugs and MET-HCl alone ( P > 0.05). Conclusions:MET-HCl may reduce the expression of XRCC1 through the CXCR4/Akt signaling pathway, thereby enhancing the sensitivity of colon cancer cells to 5-FU.

Objective:To summarize the clinical manifestations, gene variations,and treatment of cases with SPTAN1 gene variations characterized by global developmental delay or epileptic encephalopathy. Methods:Three patients with SPTAN1 gene mutations which caused developmental epileptic encephalopathy type 5 admitted to the Department of Pediatrics, Xiangya Hospital, Central South University from August 2019 to September 2021 were collected. The studies till December 2021 were searched with keywords of " SPTAN1" and "developmental and epileptic encephalopathy 5" in both English and Chinese databases of China National Knowledge Infrastructure, Wanfang, Online Mendelian Inheritance in Man, and PubMed. The clinical manifestations, genetic variations, treatments and prognosis of patients with SPTAN1 gene variations were summarized. Results:All 3 patients presented with global developmental delay, infant onset. Patient 1 showed early-onset epileptic encephalopathies and microcephaly. Patient 2 had an atrial septal defect. Cranial magnetic resonance imaging (MRI) of patient 3 showed cerebellar hypoplasia.Antiepileptic seizure therapy was partially effective, but failed to control the spasm. Development was slightly improved after rehabilitation training and other treatments, but still lagged behind the children of the same age. The SPTAN1 gene mutations of the 3 cases were heterozygous mutations, c.6923_6928dup, c.6619_6621delGAG and c.6749T>C, respectively. c.6749T>C was not reported in the previous literature. Thirteen case reports, including 69 patients, were collected. Sixty-seven patients had heterozygous mutations, inherited in an autosomal dominant fashion, including 35 missense mutations, 12 deletion mutations, 11 repetition mutations, 9 nonsense mutations, and the rest 2 patients had compound heterozygous missense mutations. A total of 38 different variation sites were reported. The phenotypes of 69 patients from the previous studies mainly included intellectual impairment (32/69), seizures (30/69), developmental delay (28/69), progressive microcephaly (27/69), hypotonia (23/69), poor visual attention (15/69), spastic quadriplegia (9/69), and gastrointestinal abnormalities (7/69). The primary type of seizures was epileptic spasm. Cranial MRI abnormalities mainly included cerebellar and brainstem atrophy, corpus callosum dysplasia, myelin dysplasia, and brain atrophy. Previous reports showed that a variety of anti-seizure drugs were effective for epileptic seizures. The prognosis varied greatly. Severe cases could be fatal, and mild cases only manifested as mild mental retardation or movement disorders. Conclusions:SPTAN1 gene mutation leads to developmental epileptic encephalopathy type 5, the phenotypes of which include intellectual impairment, global developmental delay, infantile spasms, and head deformity.Antiepileptic drugs and functional training can improve the symptoms, but the prognosis is still poor. This study expands the SPTAN1 gene variant spectrum, enriches the mutant spectrum of SPTAN1 gene associated with developmental epileptic encephalopathy type 5.

Objective:To summarize the clinical manifestations, gene variations, diagnosis and treatment of 3 cases with SLC35A2 variations characterized by congenital glycosylation disorder Ⅱm (CDG Ⅱm). Methods:A total of 3 patients admitted to the Department of Pediatrics of Xiangya Hospital of Central South University in China from 2018 to 2020 were examined in detail. The studies till January 2022 were searched with key words of "congenital disorders of glycosylation Ⅱm", " SLC35A2" and "CDG Ⅱm" in both English and Chinese in the databases of China National Knowledge Infrast Ructure (CNKI), Wanfang, Online Mendelian Inheritance in Man and PubMed, and the clinical manifestations, genetic variation, treatments and prognosis of patients with SLC35A2 mutation were summarized. Results:The patients all presented with intractable infantile spasm and global developmental delay, onset in infancy. A variety of antiepileptic treatments had temporary and partial efficacy. Otherwise, proband 2 and 3 presented with abnormal glutamic-pyruvic transaminase and increased platelets. Funduscopy showed dysplasia of the retinal pigment epithelium in both eyes, and they both received D-galactose treatment. A total of 22 relevant case reports, including 99 patients, were collected. The 99 patients all were heterozygous mutations, and a total of 75 different variation sites were reported. The clinical manifestations were characterized by global developmental delay or mental retardation ( n=89), epileptic seizure ( n=75), hypotonia ( n=57), facial deformity ( n=57), skeletal abnormality ( n=50), visual impairment ( n=42), elevated glutamic-pyruvic transaminase ( n=31), gastrointestinal symptoms ( n=28), skin deformity ( n=26), microcephaly ( n=23) and congenital heart disease ( n=12). Craniocerebral magnetic resonance imaging may be normal in the early stage. With age, magnetic resonance imaging may show abnormal white matter signals, brain atrophy, dysplasia of corpus callosum, delayed myelination, enlargement of lateral ventricle, brain stem atrophy and so on. Studies have shown that galactose treatment may be effective. Conclusions:SLC35A2 variants lead to CDG Ⅱm, whose clinical manifestations mainly include epileptic encephalopathy and global developmental delay. Multiple antiepileptic therapies can temporarily or partially control seizures, while oral galactose may improve the clinical symptoms, showing its prospect as a dietary therapy.

Objective To study the preventive effect ofω-6 soybean oil fatty emulsion on gastric ulcer caused by acetic acid in rat model, and investigate its mechanisms. Methods Thirty healthy rats were randomly and equally assigned to the following 3 groups:sham operation,gastric ulcer,andω-6 Soybean oil fatty emulsion group.The model was induced by acetic acid. Five days after the model was established successfully,rats in ω-6 soybean oil group received the treatment by tail intravenous injection with the dose of 10 mL.kg-1 .d-1 ,the sham operation group and gastric ulcer group were given the same dose of 0.9%sodium chloride solution.The rats were sacrificed at 10th day after the treatment.The pathological changes of rat gastric ulcer tissue were observed by HE staining, and the concentration of gastric acid was detected by acid-base neutralization method,as well as the activity of pepsin was detected by colorimetry.Serum NO concentration was detected with nitrate reductive enzymatic method, and the expression of EGFR in gastric mucosal was detected with immunohistochemical method. Results Gastric ulcer area inω-6 soybean oil fatty emulsion group (5.67±2.32 mm2) was significantly lower than that in gastric ulcer group(8.68±1.98 mm2). The concentration of gastric acid (1.70±0.53 mmol.L-1), activity of pepsin(23.12±6.97 U) and NO level (64.62±13.86μmol.L-1 ) inω-6 soybean oil fatty emulsion group were much lower than those in the model control group.While the expression of EGFR in gastric ulcer tissue was increased after treatment withω-6 soybean oil fatty emulsion. Conclusion ω-6 soybean oil fatty emulsion exerts significant promotion effect on the healing of gastric ulcer,and its mechanism might be related to inhibiting the level of gastric acid, pepsin and NO, while improving the protective effect of EGFR on gastric mucosa.

Objective to investigate the protective effect of omega-3 fish oil fat emulsion on cyclophosphamide-induced gastric mucosal injury in mice. Methods Forty-five kunming mice were randomly divided into three groups as control,model,and omega-3 fish oil fat emulsion group(with 15 mice in each group). Mice of the two experiment groups were administrated with cyclophosphamide i.p. for 2 days to establish the damage model. then mice in omega-3 fish oil fat emulsion group received omega-3 fish oil fat emulsion at a dose of 15 mL/kg daily for 14 days. Meanwhile,the ani-mals in control group and model group were intravenously administered with the same volume of saline. the weight and food intake of the mice in each group were assessed daily. Five mice in each group were respectively sacrificed at day 1,day 7,day 14 after intravenous injection. Morphology of gastric mucosa was observed by HE staining and the activities of SOD and MAO in gastric mucosa were measured respectively by xanthine oxida-tion and ultraviolet spectrophotometry methods. Results Compared with the model group,the general status,nutritional status and the injury in stomach mucosa in omega-3 fish oil fat emulsion group were significantly improved. After 14 day′s treatment,the activities of SOD and MAO in gas-tric mucosa of mice in omega-3 fish oil fat emulsion group were significantly increased(P < 0.05)compared with model group. Conclusion omega-3 fish oil fat emulsion has a significant protective effect on the cyclophosphamide induced injury in gastric mucosa of mice,which may be related to the upregulation of MAO and SOD.