ObjectiveTo establish an allele-specific polymerase chain reaction （PCR） method for identifying Scolopendra dispensing granules， so as to ensure the quality and therapeutic effects of Scolopendra and its preparations. MethodThe primer interval suitable for the PCR was selected based on the cytochrome c oxidase subunit 3（COX-3） gene sequence of Scolopendra， and the single nucleotide polymorphism （SNP） loci of Scolopendra and its adulterants were mined from the interval for the design of specific primers. The samples of Scolopendra and its adulterants were collected. The PCR system was established and optimized regarding the annealing temperature， cycles， Taq enzymes， DNA template amount， PCR instruments， and primer concentrations， and the specificity and applicability of this method were evaluated. ResultThe PCR system was composed of 12.5 μL 2×M5 PCR Mix， 0.4 μL forward primer （10 μmol·L^-1）， 0.4 μL reverse primer （10 μmol·L^-1）， 2.5 μL DNA template， and 9.2 μL sterile double distilled water. PCR parameters： Pre-denaturation at 94 ℃ for 3 min， 30 cycles （94 ℃ for 20 s， 62 ℃ for 20 s， 72 ℃ for 45 s）， and extension at 72 ℃ for 5 min. After PCR amplification with the system and parameters above， the electrophoresis revealed a bright band at about 135 bp for Scolopendra and no band for the adulterants. ConclusionThe established allele-specific PCR method can accurately identify the medicinal materials， decoction pieces， and standard decoction freeze-dried powder of Scolopendra， as well as the intermediates and final products of Scolopendra dispensing granules， which is of great significance for ensuring the quality and clinical efficacy of Scolopendra and its preparations.

AIM:To investigate the effect of Ganoderma leucocontextum extract(GLE)on mice with cisplatin-induced acute kidney injury(AKI)and lipopolysaccharide(LPS)-induced cellular inflammation.METHODS:Eight-week-old male C57BL/6 mice were randomly divided into control group,AKI group,low-dose(100 mg/kg)GLE group,high-dose(300 mg/kg)GLE group,and quercetin(100 mg/kg)group,with 6 mice in each group.The AKI model was es-tablished by intraperitoneal injection of a 20 mg/kg cisplatin solution.After GLE intervention for 3 d,serum creatinine(SCr)and blood urea nitrogen(BUN)levels were measured.Renal pathology was observed using HE and PAS staining.The expression of β-catenin and Axin2 protein in each group was detected by immunohistochemistry.The expression lev-els of interleukin-1β(IL-1β),IL-6,β-catenin and Axin2 in each group were detected by Western blot and RT-qPCR.The TCMK1 cells were stimulated with 2 mg/L LPS to simulate cellular inflammatory injury.After GLE treatment(0.2,0.4,0.6 and 0.8 g/L)for 24 h,the expressions of IL-1β,IL-6,β-catenin and Axin2 in each group were detected.Fur-ther overexpression of Axin2 was used to verify the changes in the above-mentioned indices.RESULTS:High doses of GLE significantly reduced SCr(P<0.01)and BUN(P<0.05)levels compared with the AKI mice.AKI mice showed re-nal tubule dilatation,tubular epithelial cell necrosis,vacuolation,and other pathological manifestations,which were im-proved after GLE intervention.Immunohistochemistry showed increased expression of Axin2 and β-catenin protein in the kidneys of AKI mice,which was reduced by GLE intervention.Western blot and RT-qPCR results in vitro and in vivo showed that GLE intervention significantly inhibited the expression and mRNA levels of IL-1β,IL-6,Axin2 and β-catenin(P<0.05).Overexpression of Axin2 antagonized the effect of GLE on IL-1β,IL-6,Axin2 and β-catenin,resulting in sig-nificantly up-regulated expressions of these proteins and mRNAs(P<0.01).CONCLUSION:GLE significantly allevi-ates the inflammatory response in AKI mice and LPS-induced cells,and protects against cisplatin-induced kidney injury in mice by inhibiting the Axin2/β-catenin signaling pathway.

Objective To investigate the causal association between bronchial asthma and bone mineral density at different sites using a two-sample Mendelian randomization (MR) approach. Methods Summary data for exposure factors and outcome were obtained from different genome-wide association studies.Single nucleotide polymorphisms strongly associated with bronchial asthma were selected as instrumental variables,and those in linkage disequilibrium were excluded.The inverse-variance weighted (IVW) method was used as the primary method for MR analysis,complemented by weighted median,simple mode,weighted mode,and MR-Egger regression methods.Sensitivity analyses were conducted to assess the stability of the results. Results The random-effects model of IVW analysis showed that heel bone mineral density (OR=0.986;95% CI,0.974 to 0.998;P=0.023) as the outcome dataset had a reverse causal effect with bronchial asthma,while lumbar spine bone mineral density (OR=1.031;95% CI,0.984 to 1.081;P=0.195),femoral neck bone mineral density (OR=1.014;95% CI,0.973 to 1.057;P=0.505),and forearm bone mineral density (OR=1.011;95% CI,0.935 to 1.094;P=0.775) as outcome datasets showed no causal effect with bronchial asthma.The MR-Egger intercept test results indicated that the P-values for the intercepts of lumbar bone mineral density,femoral neck bone mineral density,forearm bone mineral density,and calcaneal bone mineral density were all over 0.05,suggesting no horizontal pleiotropy and relatively stable results. Conclusion MR analysis reveals a reverse causal effect between bronchial asthma and heel bone mineral density,suggesting that clinicians should strengthen the monitoring of heel bone mineral density in patients with bronchial asthma to timely detect and intervene osteoporosis.

Using chemoproteomic techniques, we first identified EIF2AK2, eEF1A1, PRDX3 and VPS4B as direct targets of berberine (BBR) for its synergistically anti-inflammatory effects. Of them, BBR has the strongest affinity with EIF2AK2 via two ionic bonds, and regulates several key inflammatory pathways through EIF2AK2, indicating the dominant role of EIF2AK2. Also, BBR could subtly inhibit the dimerization of EIF2AK2, rather than its enzyme activity, to selectively modulate its downstream pathways including JNK, NF-κB, AKT and NLRP3, with an advantage of good safety profile. In EIF2AK2 gene knockdown mice, the inhibitory IL-1β, IL-6, IL-18 and TNF-α secretion of BBR was obviously attenuated, confirming an EIF2AK2-dependent anti-inflammatory efficacy. The results highlight the BBR's network mechanism on anti-inflammatory effects in which EIF2AK2 is a key target, and inhibition of EIF2AK2 dimerization has a potential to be a therapeutic strategy against inflammation-related disorders.

A series of new monobactam sulfonates is continuously synthesized and evaluated for their antimicrobial efficacies against Gram-negative bacteria. Compound 33a (IMBZ18G) is highly effective in vitro and in vivo against clinically intractable multi-drug-resistant (MDR) Gram-negative strains, with a highly druglike nature. The checkerboard assay reveals its significant synergistic effect with β-lactamase inhibitor avibactam, and the MIC values against MDR enterobacteria were reduced up to 4-512 folds. X-ray co-crystal and chemoproteomic assays indicate that the anti-MDR bacteria effect of 33a results from the dual inhibition of the common PBP3 and some class A and C β-lactamases. Accordingly, preclinical studies of 33a alone and 33a‒avibactam combination as potential innovative candidates are actively going on, in the treatment of β-lactamase-producing MDR Gram-negative bacterial infections.

Objective:To investigate the expression of promyelocytic leukemia zinc finger (PLZF) in human peripheral blood with asthma and its clinical significance.Methods:Forty patients with stable asthma from May 2021 to October 2021 in the Department of Respiratory Medicine of the Shanghai Fifth People's Hospital were enrolled, and forty healthy controls were recruited in the study. The levels of cytokines in serum were measured by enzyme-linked immunosorbent assay (ELISA). Quantitative real-time PCR (qPCR) was used to detect the expression of PLZF mRNA in plasma. The level and distribution of PLZF+ cells in PBMCs were detected by flow cytometry after isolating peripheral blood mononuclear cells (PBMCs). Independent sample t test, Mann-Whitney U test, χ 2 test, ROC curve and Logistic regression were used to analyze the results with SPSS 26.0 and Graphpad Prism 7.0. A P<0.05 was considered statistically significant. Results:The levels of cytokines IFN-γ, IL-2, IL-4, TNF-α and IL-17 in human peripheral blood from the asthma group were obviously higher than those in the control group ( P<0.05), whereas there was no significant difference in the level of cytokine IL-10 between the two groups. The level of PLZF mRNA in PBMCs from the asthma group was significantly up-regulated compared to that in the control group [(3.40%±2.52%) vs. (1.23%±0.78%), P<0.05]. CD8+PLZF+ and Vβ11+PLZF+T cells in the asthma group were significantly outnumbered than those in the control group ( P<0.05). Logistic regression and ROC curve analysis showed that PLZF expression in PBMC was a risk factor for the development of asthma ( OR =3.67, AUC=0.87, P<0.05). Conclusions:The high expression of PLZF in peripheral blood may play an important role in the development of asthma, which needs to be further confirmed by large sample studies.

Objective:To investigate the effects of subanesthetic concentration of sevoflurane on the plasticity of dendritic spines in the prefrontal cortex neurons of juvenile rats.Methods:Thirty-six clean-grade male Sprague-Dawley rats, aged 24 days, weighing 50-60 g, were divided into control group (group C) and sevoflurane anesthesia group (group S) using a random number table method, with 18 rats in each group.Group S inhaled 1.2% sevoflurane and 50% oxygen (flow rate 1 L/min) for 3 h, while group C inhaled 50% oxygen (flow rate 1 L/min) for 3 h. Open-field test and Morris water maze test were performed at 3 days after anesthesia.Animals were sacrificed, and brain samples were then taken for determination of the number of apoptotic neurons in layer Ⅱ-Ⅲ of the prefrontal cortex, density of dendritic spines, and expression of postsynaptic density protein 95 and gephyrin by TUNEL staining, Golgi staining or Western blot.Results:Compared with group C, no significant change was found in total distance or time of staying at the central region in the open-field test or the average swimming velocity, escape latency or the number of apoptotic neurons in the Morris water maze test ( P>0.05), and the density of dendritic spines was significantly increased, and the expression of postsynaptic density protein 95 and gephyrin was up-regulated in group S ( P<0.05). Conclusion:Subanesthetic concentration of sevoflurane can enhance the plasticity of dendritic spines in the prefrontal cortex neurons of juvenile rats.

Objective:To investigate the expressions and correlation of bcl-2, programmed death-1 (PD-1) and programmed death ligand-1(PD-L1) in diffuse large B-cell lymphoma (DLBCL) tissue specimens, and the relationship between chemotherapy efficacy and prognosis of DLBCL patients.Methods:The expressions of bcl-2, PD-1 and PD-L1 in 82 patients with DLBCL who were admitted to Chenzhou First People's Hospital of Hunan Province from May 2011 to April 2014 were detected by using immunohistochemistry, and the correlation of the expressions of bcl-2, PD-1 and PD-L1 with clinicopathological characteristics and prognosis was analyzed.Results:The positive rate of bcl-2, PD-L1 and PD-1 in cancer tissues of DLBCL patients was 53.7% (44/82), 56.1% (46/82) and 32.9% (27/82), respectively. There was a correlation between bcl-2 and PD-L1 expression ( r = 0.306, P = 0.005). Bcl-2 was highly expressed in patients with international prognosis index (IPI) score 3-4 points, non-germinal center B-cell-like (non-GCB) subtype and B symptoms (all P < 0.05); PD-1 was highly expressed in patients with IPI score 3-4 points ( P < 0.05); PD-L1 was highly expressed in patients with IPI score 3-4 points, tumor stage Ⅲ-Ⅳ, B symptoms, ≥60 years old, and non-GCB (all P < 0.05). The overall survival (OS) and progression-free survival (PFS) in bcl-2-negative group were better than those in the bcl-2-positive group, and the differences were statistically significant (both P < 0.05); OS and PFS in PD-L1-negative group were better than those in PD-L1-positive group, and the differences were statistically significant(both P < 0.01). There were no statistical differences in OS and PFS between PD-1-positive group and PD-1-negative group (both P > 0.05). OS and PFS in bcl-2 and PD-L1 co-expression group were worse than those in both negative or any negative group (all P < 0.01), and PFS in bcl-2 and PD-1 co-expression group was worse than those in both negative or any negative group ( P = 0.044). Cox multivariate analysis showed IPI score 3-4 points and B symptoms were the independent influencing factors of OS in DLBCL patients (both P < 0.01); IPI score 3-4 points, B symptoms and PD-L1-positive were the independent influencing factors of poor PFS in DLBCL patients (all P < 0.05). Conclusion:The positive expressions of bcl-2 and PD-L1 are the independent factors for the poor prognosis of DLBCL patients, which may become new targets for the treatment of DLBCL.

The aim of this study is to investigate the effect of the chimeric intron in different directions on the expression of the nerve growth factor (NGF) in recombinant Chinese hamster ovary (CHO) cells. The chimeric intron that contained the splice sequence of the first intron of the human β-globin and the human immunoglobulin heavy chain variable region intron was used. NGF gene was cloned into the expression vectors containing the chimeric intron in the forward or reverse direction, followed by transfecting into CHO cells, and screened under G418 to produce the stable transfected CHO cells. Fluorescence quantitative PCR, ELISA, and Western blotting were performed to detect the recombinant NGF gene expression in CHO cells. The results showed that the chimeric introns could significantly enhance the expression of NGF in recombinant CHO cells. Moreover, the enhancing effect on NGF expression level by the intron in the forward direction showed stronger than that of the reverse direction both at mRNA and protein level. In conclusion, the chimeric intron could increase NGF expression in stably transfected CHO cells and the effect is associated with the direction of the intron insertion.
Animals ; Animals, Genetically Modified ; CHO Cells ; Cricetinae ; Cricetulus ; Gene Expression ; Humans ; Introns ; Transfection

Objective To investigate the CT and MR imaging feature of osseous metastasis of hepatic alveolar hydatida.Methods CT and MR imaging features of osseous metastasis of hepatic alveolar hydatid were retrospectively analyzed in 14 cases confirmed by clinic and radiology. Results Of the 10 vertebral metastasises,three vertebral bodies were involved in 6 cases,two adjacent vertebral bodies were involved in 2 cases, single vertebral body was involved in 1 case, corresponding vertebral accessory was involved in 9 cases,irregular mass was formed in paravertebral muscles with ill-defined margins in 9 cases, adjacent ribs were involved in 6 cases. On CT scans, the lesions of vertebral body showed osteolytic and geographical bone destruction with bone sclerosis. The lesions of vertebral accessory showed swelling and osteolytic bone destruction, small cystic density and calcification of cystic wall and irregular calcification were found in 7 cases.The lesions of scapula,costal cartilage,ribs and pelvis showed swelling and osteolytic bone destruction,and bone sclerosis,and revealed cystic density with calcified speckles.On MR scans,the lesions of vertebral body and paravertebral muscles showed high and low mixed signal,with hypointensity on T1WI and T2WI, vertebral accessory and adjacent ribs were involved in 6 cases. Small cyst with T2 hyperintensity was found in 4 cases. The lesions of ribs and pelvis showed swelling and osteolytic bone destruction and adjacent medullary appeared hypointensity on T1WI and T2WI, cystic signal of T2 hyperintensity was identified. There were 5 cases of pulmonary metastasis and 6 cases of retroperitoneal metastasis. Conclusion Imaging features of osteolytic,geographical bone destruction with bone sclerosis, small cystic density or cystic signal and calcified speckles or arc calcification in mass are helpful for the diagnosis of osseous metastasis of hepatic alveolar hydatid.