Objective:To investigate the efficacy and safety of a combination of programmed death receptor 1 (PD-1) inhibitor and multi-target tyrosine kinase inhibitor anlotinib in second-line treatment of non-small cell lung cancer (NSCLC).Methods:A prospective randomized controlled study was conducted. Using the random number table method, 118 NSCLC patients who were admitted to Shaanxi Provincial Cancer Hospital from June 2021 to June 2023 were randomly divided into the control group and the observation group, with 59 patients in each group. The observation group was treated with PD-1 inhibitor combined with anlotinib, while the control group was treated with PD-1 inhibitor. There were 36 males and 23 females in the observation group, with an age of (56±5) years; there were 34 males and 25 females in the control group, with an age of (56±5) years. There was no statistically significant difference in general clinicopathological data between the two groups (all P > 0.05). The short-term clinical efficacy [objective response rate (ORR) and disease control rate (DCR)], tumor-related factor levels [vascular endothelial growth factor (VEGF), matrix metalloproteinase 2 (MMP-2), tissue inhibitor of matrix metalloproteinase (TIMP), and tumor necrosis factor β1 (TNF-β1)], inflammatory status [plasma fibrinogen-to-albumin ratio (FAR)], lung function [forced vital capacity (FVC) and peak expiratory flow (PEF)] before and after treatment, the European Organization for Research and Treatment on Cancer (EORTC) core questionnaire for quality of life assessment (QLQ-C30) score, and occurrence of adverse reactions were compared between two groups of patients. Results:The ORR and DCR of the observation group were 33.90% (20/59) and 69.49% (41/59), respectively; the ORR and DCR of the control group were 10.17% (6/59) and 44.07% (26/59), respectively; the comparison of ORR and DCR between the two groups showed statistically significant differences ( χ2 values were 9.67 and 7.77, both P < 0.05). There was no statistically significant difference in the levels of tumor-related factors between the observation group and the control group before treatment (all P > 0.05); after 4 cycles of treatment, the levels of VEGF and MMP-2 in the observation group were lower than those in the control group, while the levels of TIMP and TNF-β1 were higher than those in the control group, and the differences were statistically significant (all P < 0.001). The FAR of the observation group and the control group before treatment were (0.15±0.06) g/L and (0.16±0.06) g/L, respectively, with no statistically significant difference ( t = 0.90, P = 0.367); after 4 cycles of treatment, the FAR were (0.07±0.01) g/L and (0.11±0.04) g/L, respectively, with statistically significant difference ( t = 7.45, P < 0.001). Before treatment, there was no statistically significant difference in FVC and PEF between the observation group and the control group (both P > 0.05); after 4 cycles of treatment, the FVC and PEF in the observation group were higher than those in the control group, and the differences were statistically significant (both P < 0.001). There were no statistically significant differences in the EORTC QLQ-C30 scores of functional dimension, symptom dimension and global health status/quality of life dimension between the observation group and the control group before treatment (all P > 0.05); after 4 cycles of treatment, the scores of functional dimension and global health status/quality of life dimension in the observation group were higher than those in the control group, while the symptom dimension score was lower than that in the control group, and the differences were statistically significant (all P < 0.001). The incidence of adverse reactions in the observation group was 6.78% (4/59), while in the control group it was 10.17% (6/59), and the difference was not statistically significant ( P = 0.741). Conclusions:The combination of PD-1 inhibitor and anlotinib in second-line treatment of NSCLC has good clinical efficacy, it can reduce the inflammatory response, improve the lung function and quality of life, and has good safety.

To prepare a recombinant adeno-associated virus(rAAV9-ZsGreen1-EsASP)capable of expressing the aspartic protease protein of Eimeria stiedai(E.stiedai),and explore its in vitro ac-tivity.The EsASP gene was amplified by PCR,and recombinant adeno-associated viral vector pAAV-IRES-ZsGreen1 was combined with the EsASP gene using homologous recombination to construct the pAAV-ZsGreen1-EsASP expression plasmid;pAAV-ZsGreen1-EsASP expression plasmid,pHelper,and pAAV-RC9 plasmids were cotransfected into HEK-293T cells by liposomal transfection to package and produce recombinant adeno-associated virus rAAV9-ZsGreen1-EsASP capable of expressing EsASP protein.rAAV9-ZsGreen1-EsASP was purified using chloroform treatment-PEG/NaCl precipitation-chloroform extraction method,the purity of the virus was iden-tified by silver staining,the virus morphology was observed by TEM,and virus titer was detected by qRT-PCR;the purified recombinant virus was further infected into HEK-293T cells,and EsASP expression was detected by observing green fluorescent protein ZsGreen1 and Western blot method.The results indicated that double enzyme digestion and DNA sequencing confirmed that the EsASP gene had been successfully constructed into the pAAV-IRES-ZsGreen1 expression plas-mid.The expression of green fluorescent protein in HEK-293T cells suggested that co-transfection was successful.Western blot results of cell protein preparation showed that EsASP protein was successfully expressed;The purified recombinant viral capsid proteins show three distinct bands(VP1-3).The purified rAAV9-ZsGreen1-EsASP was uniform in size,around 20 nm,and had a titer higher than 1.0 × 1012 vg/mL;Green fluorescent protein expression was observed after infection of HEK-293T cells with the recombinant virus,and EsASP expression was detected by Western blot.The results suggest that rAAV9-ZsGreen1-EsASP with in vitro infectious activity was suc-cessfully obtained,providing a material basis for the development of a novel vaccine against Eimer-ia stiedai.

To prepare a recombinant adeno-associated virus(rAAV9-ZsGreen1-EsASP)capable of expressing the aspartic protease protein of Eimeria stiedai(E.stiedai),and explore its in vitro ac-tivity.The EsASP gene was amplified by PCR,and recombinant adeno-associated viral vector pAAV-IRES-ZsGreen1 was combined with the EsASP gene using homologous recombination to construct the pAAV-ZsGreen1-EsASP expression plasmid;pAAV-ZsGreen1-EsASP expression plasmid,pHelper,and pAAV-RC9 plasmids were cotransfected into HEK-293T cells by liposomal transfection to package and produce recombinant adeno-associated virus rAAV9-ZsGreen1-EsASP capable of expressing EsASP protein.rAAV9-ZsGreen1-EsASP was purified using chloroform treatment-PEG/NaCl precipitation-chloroform extraction method,the purity of the virus was iden-tified by silver staining,the virus morphology was observed by TEM,and virus titer was detected by qRT-PCR;the purified recombinant virus was further infected into HEK-293T cells,and EsASP expression was detected by observing green fluorescent protein ZsGreen1 and Western blot method.The results indicated that double enzyme digestion and DNA sequencing confirmed that the EsASP gene had been successfully constructed into the pAAV-IRES-ZsGreen1 expression plas-mid.The expression of green fluorescent protein in HEK-293T cells suggested that co-transfection was successful.Western blot results of cell protein preparation showed that EsASP protein was successfully expressed;The purified recombinant viral capsid proteins show three distinct bands(VP1-3).The purified rAAV9-ZsGreen1-EsASP was uniform in size,around 20 nm,and had a titer higher than 1.0 × 1012 vg/mL;Green fluorescent protein expression was observed after infection of HEK-293T cells with the recombinant virus,and EsASP expression was detected by Western blot.The results suggest that rAAV9-ZsGreen1-EsASP with in vitro infectious activity was suc-cessfully obtained,providing a material basis for the development of a novel vaccine against Eimer-ia stiedai.

Objective:To investigate the efficacy and safety of a combination of programmed death receptor 1 (PD-1) inhibitor and multi-target tyrosine kinase inhibitor anlotinib in second-line treatment of non-small cell lung cancer (NSCLC).Methods:A prospective randomized controlled study was conducted. Using the random number table method, 118 NSCLC patients who were admitted to Shaanxi Provincial Cancer Hospital from June 2021 to June 2023 were randomly divided into the control group and the observation group, with 59 patients in each group. The observation group was treated with PD-1 inhibitor combined with anlotinib, while the control group was treated with PD-1 inhibitor. There were 36 males and 23 females in the observation group, with an age of (56±5) years; there were 34 males and 25 females in the control group, with an age of (56±5) years. There was no statistically significant difference in general clinicopathological data between the two groups (all P > 0.05). The short-term clinical efficacy [objective response rate (ORR) and disease control rate (DCR)], tumor-related factor levels [vascular endothelial growth factor (VEGF), matrix metalloproteinase 2 (MMP-2), tissue inhibitor of matrix metalloproteinase (TIMP), and tumor necrosis factor β1 (TNF-β1)], inflammatory status [plasma fibrinogen-to-albumin ratio (FAR)], lung function [forced vital capacity (FVC) and peak expiratory flow (PEF)] before and after treatment, the European Organization for Research and Treatment on Cancer (EORTC) core questionnaire for quality of life assessment (QLQ-C30) score, and occurrence of adverse reactions were compared between two groups of patients. Results:The ORR and DCR of the observation group were 33.90% (20/59) and 69.49% (41/59), respectively; the ORR and DCR of the control group were 10.17% (6/59) and 44.07% (26/59), respectively; the comparison of ORR and DCR between the two groups showed statistically significant differences ( χ2 values were 9.67 and 7.77, both P < 0.05). There was no statistically significant difference in the levels of tumor-related factors between the observation group and the control group before treatment (all P > 0.05); after 4 cycles of treatment, the levels of VEGF and MMP-2 in the observation group were lower than those in the control group, while the levels of TIMP and TNF-β1 were higher than those in the control group, and the differences were statistically significant (all P < 0.001). The FAR of the observation group and the control group before treatment were (0.15±0.06) g/L and (0.16±0.06) g/L, respectively, with no statistically significant difference ( t = 0.90, P = 0.367); after 4 cycles of treatment, the FAR were (0.07±0.01) g/L and (0.11±0.04) g/L, respectively, with statistically significant difference ( t = 7.45, P < 0.001). Before treatment, there was no statistically significant difference in FVC and PEF between the observation group and the control group (both P > 0.05); after 4 cycles of treatment, the FVC and PEF in the observation group were higher than those in the control group, and the differences were statistically significant (both P < 0.001). There were no statistically significant differences in the EORTC QLQ-C30 scores of functional dimension, symptom dimension and global health status/quality of life dimension between the observation group and the control group before treatment (all P > 0.05); after 4 cycles of treatment, the scores of functional dimension and global health status/quality of life dimension in the observation group were higher than those in the control group, while the symptom dimension score was lower than that in the control group, and the differences were statistically significant (all P < 0.001). The incidence of adverse reactions in the observation group was 6.78% (4/59), while in the control group it was 10.17% (6/59), and the difference was not statistically significant ( P = 0.741). Conclusions:The combination of PD-1 inhibitor and anlotinib in second-line treatment of NSCLC has good clinical efficacy, it can reduce the inflammatory response, improve the lung function and quality of life, and has good safety.

Objective To investigate the effect of estrogen on the expression of matrix metallopro-teinase protein 9 and 2(MMP-9,MMP-2)and tight junction protein Occludin in mouse brain mi-crovascular endothelium cell line bEnd.3 injured by homocysteine(Hcy),and explore the protec-tive effects and underlying mechanisms of estrogen on small cerebral vascular diseases.Methods bEnd.3 cells were treated with Hcy andβ-estradiol respectively,and the optimal concentration was determined for cell proliferation and toxicity with enhanced cell counting kit-8.The cells were divided into control group,Hcy group(200 μmol/L),E2β group(200 nmol/L)and combined treatment group(200 nmol/L E2β treatment for 30 min followed by 200 μmol/L Hcy).After 6 h corresponding treatment,Western blotting was used to detect the protein levels of MMP-9,MMP-2 and Occludin in each group of cells.Results There were no significant differences in the protein levels of MMP-9,MMP-2 and Occludin in above groups after treatment for 24 and 48 h(P＞0.05).Compared with the control group,the expression levels of MMP-9 and MMP-2 was signifi-cantly increased in the Hcy Group after treatment of 72 h(1.45±0.14 vs 1.00±0.00,1.35±0.15 vs 1.00±0.00,P＜0.05),while the level of Occludin was obviously decreased(0.64±0.05 vs 1.00±0.00,P＜0.05).Compared in the Hcy Group,the MMP-9 and MMP-2 levels were obviously decreased(0.84±0.19 vs 1.45±0.14,1.01±0.78 vs 1.35±0.15,P＜0.05),and that of Occludin was notably elevated in the combined treatment group(0.91±0.10 vs 0.64±0.05,P＜0.05).Con-clusion E2β can ameliorate blood-brain barrier damage in Hcy-treated bEnd.3 cells,resulting in decreased MMP-9 and MMP-2 expressions and increased Occludin expression.

Objective:To investigate the correlation between serum anti-phospholipase A2 receptor antibody (SAb) combined with glomerular complement C3 (GC3) deposition and clinicopathologic features and prognosis in patients with idiopathic membranous nephropathy (IMN).Methods:It was a retrospective cohort study. The patients diagnosed with IMN in Affiliated Hospital of Qingdao University from July 1, 2019 to April 30, 2022 were enrolled, and the clinical and pathological data were collected and analyzed. The patients were divided into negative SAb and negative GC3 (SAb -/GC3 -) group, negative SAb and positive GC3 (SAb -/GC3 +) group, positive SAb and negative GC3 (SAb +/GC3 -) group and positive SAb and positive GC3 (SAb +/GC3 +) group according to the status of SAb titer and GC3 deposition. Clinical and pathological characteristics among the groups were compared. Kaplan-Meier survival curve was used to compare the cumulative renal remission rates of different groups. Cox regression analysis model was used to analyze the related factors of renal remission. Results:A total of 143 IMN patients aged (53.35±12.34) years old were included in the study, including 94 males (65.7%). There were 17 patients (11.9%) in the SAb -/GC3 - group, 30 patients (21.0%) in the SAb -/GC3 + group, 19 patients (13.3%) in the SAb +/GC3 - group, and 77 patients (53.8%) in the SAb +/GC3 + group. Compared with SAb -/GC3 - group, the level of serum albumin was lower in the SAb +/GC3 + group, and the level of 24 h urine protein, SAb titer, and the proportions of glomerular anti-phospholipase A2 receptor antigen and renal tubule atrophy were higher in the SAb +/GC3 + group (all P<0.05). After 26.0 (19.0, 36.0) months of follow-up, a total of 96 patients (67.1%) attained remission. The proportion of patients receiving immunosuppressive therapy in the SAb +/GC3 + group was higher than that in the SAb -/GC3 - group [93.5% (72/77) vs. 70.6% (12/17), fisher value=8.974, P=0.016] and the proportion of renal remission rate in the SAb +/GC3 + group was lower than that in the SAb -/GC3 - group [49.4% (38/77) vs. 100% (17/17), χ2=25.438, P<0.001]. Kaplan-Meier survival curve result showed that the cumulative renal remission rate in the SAb +/GC3 + group was significantly lower than that in the SAb -/GC3 - group (Log-rank χ2=31.538, P<0.01). Multivariate Cox regression analysis result showed that 24 h urine protein level ( HR=0.891, 95% CI 0.803-0.988, P=0.029), SAb titer ( HR=0.996, 95% CI 0.992-1.000, P=0.042) and SAb +/GC3 + (with SAb -/GC3 - group as reference, HR=0.414, 95% CI 0.204-0.827, P=0.013) were independent related factors for renal remission in patients with IMN. Conclusions:IMN patients with positive SAb and GC3 deposition have more severe clinical and pathological changes, lower renal cumulative remission rates, and are more likely to have poor prognosis. The combined assessment of SAb and GC3 deposition may be helpful for evaluating prognosis and guiding treatment in IMN patients.

Objective This study aims to explore the clinical characteristics of common mutation sites in the SOD1 gene and provide assistance for the early identification, diagnosis, and course evaluation of amyotrophic lateral sclerosis (ALS). Methods The clinical data and genetic testing results of a patient with ALS caused by the c.131A>G:p.H44R mutation in the second exon of the SOD1 gene were retrospectively analyzed and discussed in conjunction with the literature. Results The patient presented with pain and weakness in the right lower limb accompanied by muscle atrophy. No positive signs were observed in the sensory system. The electromyogram revealed subclinical neurogenic changes in the unaffected limbs. Whole-exome sequencing identified a rare mutation in exon c.131A>G:p.H44R of the SOD1 gene. Conclusion Early diagnosis of ALS is challenging, and the clinical manifestations vary depending on the gene site mutations. Genetic testing can assist in diagnosis and has significant identification value in the early stages of the disease.

Objective:To investigate the differences in the expression of microRNA (miR)-216a and its target gene SerpinB5 at the tissue level, and the effects of miR-216a on the proliferation of different liver cancer cells by regulating the expression of SerpinB5.Methods:Through bioinformatics prediction and selection of miR-216a that regulated SerpinB5. the expressions in liver cancer and normal tissues were detected by real time polymerase chain reaction (PCR). The miR-216a simulacrum and inhibitor, si-Serpinb5 and pcdna3.1-Serpinb5 to HepG2 and MHCC97H (97H) were transfected with liposomes, respectively. Real time PCR and Wester-Blot were used to detect the expression of miR-216a and SerpinB5 before and after transfection, and CCK8 was used to detect the influence of both on the proliferation of liver cancer cells.Results:The expression of miR-216a in human liver cancer tissues was higher than that in adjacent tissues, and the difference was statistically significant ( P < 0.01). The expression of SerpinB5 in human liver cancer tissues was lower than that adjacent tissues, and the difference was statistically significant ( P < 0.01). In HepG2 and 97H, miR-216a inhibitor and SerpinB5 overexpression group showed down-regulated miR-216a expression, which was statistically different from the control group ( P < 0.01). The proliferation of miR-216a inhibitor and pcdna3.1-serpinb5 group was lower than the control group, with statistically significant differences ( P < 0.01). Conclusions:The high expression of SerpinB5 can inhibit the proliferation of liver cancer cells, suggesting that SerpinB5 may have an anti-oncogene effect. MiR-216a may negatively regulate the expression of SerpinB5 and affect the proliferation of HCC cells.

Regarding radioactive iodine-refractory failure or advanced differentiated thyroid cancer,multiple multikinase inhibitors including sorafenib and lenvatinib,which target platelet derived growth factor receptor,vascular endothelial growth factor pathway,and rearranged during transfection (RET) pathway were proved to have obvious antitumor activity.Moreover,selective BRAF inhibitor,promoting drug uptake of radioactive iodine also showed a certain therapeutic effect.These molecular targets which are relevant with differentiated thyroid cancer occurrence,development,invasion and metastasis have become of its moment,and,selective inhibitors and re-differentiation agents were shown to be promising.In the future,individual genetic testing would provide more specific information in directing individualized molecular-targeted therapy.