Aim To express and purify recombinant hCGH-CTP fusion protein in high-density suspension culture of Chinese hamster ovary cells (CHO-S), and to verify the lipid accumulation effect of rhCGH-CTP on 3T3-L1 mature adipocytes. Methods The recombinant protein expression vector (pcDNA3. 1-rhCGH-CTP) was constructed, achieved by fusing the human glycoprotein hormone beta 5/alpha 2 cDNA with CTP Linker. The expression plasmid was transiently transfected into the suspended CHO-S to express rhCGH-CTP protein and then purified, and the protein biological activity was verified. Intervention with 3T3-L1 mature adipocyte cells for 24 h was performed to detect the changes of intracellular triglyceride (TG) level. Results Western blot results showed that rhCGH-CTP protein was successfully expressed in CHO-S cells, and the yield was up to 715. 4 mg • L~ . The secreted protein was purified by AKTA pure system with higher purity that was up to 90% as identified by SDS-PAGE. In addition, the intracellular cAMP content of mature adipocytes with high expression of TSHR gene significantly increased after intervention with different concentrations of rhCGH-CTP protein by ELISA kit, indicating that rhCGH-CTP protein had biological activity. Oil red 0 staining showed that compared with the control group, the lipid content of mature adipocytes in the intervention groups with different concentrations of rhCGH-CTP protein significantly decreased (P < 0. 05) . Conclusions The rhCGH-CTP protein has been successfully expressed and purified with biological activity, and effectively reduce TG. This research provides an important theoretical basis for further revealing the physiological role of CGH protein and its potential application in clinical practice.

Aim To investigate the effect of recombi-nant glycoprotein hormone β5/α2(rCGH)on lipolysis in 3T3-L1 adipocytes,and explore the underlying mechanism.Methods 3T3-L1 preadipocytes were cultured and induced to differentiate into mature adipo-cytes,then treated with different concentrations of rCGH for 24 h in vitro.Cell viability of 3T3-L1 adipo-cytes was evaluated by CCK-8 assay,the levels of in-tracellular triglyceride(TG)and glycerol in the culture supernatant were measured by enzymatic method,and the changes of lipid droplets were observed by oil red O staining.The expression levels of HSL and ATGL lipo-lytic proteins in adipocytes were detected by Western blot.To carry out the intervention experiment with dif-ferent concentrations of rCGH with or without the PKA inhibitor,H89,on the mature 3T3-L1 adipocytes,the cultured cells were divided into the control group,H89 pre treatment group,1 μmol·L-1 rCGH group,and(1 μmol·L-1 rCGH+H89)combined intervention group.The contents of intracellular TG and free glycer-ol were measured by enzymatic method,and the ex-pression of CREB and lipolysis-related proteins was de-tected using Western blot.Results Different concen-trations of rCGH(0.25,0.5,1,and 2 μmol·L-1)had no significant effect on the cell viability of adipo-cytes(P＞0.05).Compared with the control group,the treatment with rCGH significantly decreased the size of lipid droplets and intracellular TG content,while significantly elevated glycerol concentration in cell supernatant.rCGH treatment also stimulated the protein expression of p-HSL,ATGL,and p-PKA.In addition,the addition of a PKA inhibitor,H89,atten-uated the effects of rCGH on free glycerol level,intra-cellular TG content,and the expression of p-HSL,p-PLIN1,and p-CREB.Conclusions rCGH enhances the lipolysis of 3T3-L1 adipocytes by up-regulating the activities of HSL,ATGL and PKA,promoting glycerol release,inhibiting TG synthesis and lipid accumula-tion,and its mechanism of action is related to the acti-vation of cAMP/PKA/CREB signaling pathway.

Objective:To explore the effect of long non-coding RNA (lncRNA) C10orf25 on the proliferation and invasion ability of prostate cancer cells and the possible role of miRNA-671-5p (miR-671-5p).Methods:Data from the Gene expression omnibus (GEO) database (data updated in January 2023) were used to analyze the differences in the expression levels of C10orf25 in 137 cases of prostate cancer tissues and paracancerous tissues. Prostate cancer C4-2B, DU-145, 22Rv1, PC-3, LNCaP cell lines and immortalized prostate epithelial RWPE-1 cell lines were selected, and then real-time quantitative fluorescence polymerase chain reaction (qRT-PCR) was used to detect the relative expression levels of C10orf25 in cell lines. The 22Rv1 cells with the lowest relative expression level of C10orf25 were selected and divided into the control group (transfected with negative plasmid) and the C10orf25 group (transfected with C10orf25 plasmid); the CCK-8 method was used to detect the proliferation activity of 22Rv1 cells in both groups at day 1, 2, 3, 4, 5 (expressed as absorbance value); the Transwell method was used to detect the invasion ability of 22Rv1 cells. Linc2GO software was used to predict miR-671-5p with binding sites for C10orf25. Dual luciferase reporter gene assay was used to verify the targeting relationship between C10orf25 and miR-671-5p. qRT-PCR was used to detect the relative expression levels of C10orf25 and miR-671-5p. Western blot was used to detect the expression of proteins related to the NF-κB signaling pathway of 22Rv1 cells in the both groups.Results:In the GEO database, the relative expression level of C10orf25 in prostate cancer tissues was lower than that in paracancerous tissues ( P < 0.01). The relative expression levels of C10orf25 in immortalized prostate epithelial cell line RWPE-1 and prostate cancer cell lines C4-2B, DU-145, 22Rv1, PC-3, and LNCaP were 1.00±0.05, 0.63±0.04, 0.42±0.03, 0.18±0.04, 0.81±0.02, 0.50±0.07, and the difference was statistically significant ( F = 43.29, P < 0.05). The proliferation ability of 22Rv1 cells in C10orf25 group was lower than that in the control group from the second day, and the differences were statistically significant (all P < 0.05). The number of invasive cells in the control group and C10orf25 group were (97±11) and (36±9), respectively, and the difference was statistically significant ( t = 4.15, P < 0.01). Linc2GO software prediction results showed that C10orf25 had a binding site for miR-671-5p. The dual luciferase reporter gene assay showed that the relative luciferase activity of miR-671-5p and C10orf25 wild plasmid co-transfecting 22Rv1 cells was lower than that of miR-NC and C10orf25 wild plasmid co-transfecting 22Rv1 cells, and the difference was statistically significant ( P < 0.01); when miR-671-5p or miR-NC was co-transfected with C10orf25 mutant plasmid, the difference in the luciferase activity of 22Rv1 cells was not statistically significant ( P > 0.05). The relative expression levels of miR-671-5p in 22Rv1 cells were 7.33±0.99 and 0.98±0.16, respectively in the control group and C10orf25 group, and the difference was statistically significant ( t = 6.32, P < 0.01). The results of Western blot showed that the expression levels of NF-κB signaling pathway protein p50, matrix metalloproteinase 9, c-myc, and vascular endothelial growth factor protein in 22Rv1 cells in C10orf25 group were lower than those in the control group. Conclusions:The overexpression of C10orf25 may inhibit the proliferation and invasion of prostate cancer cells through the miR-671-5p-NF-κB axis.

Objective    To explore the predictive value of CT signs of mixed ground-glass nodules in the pathological subtype and differentiation of lung adenocarcinoma. Methods    The clinical data of 66 patients with mixed ground-glass nodules pathologically diagnosed as invasive adenocarcinoma (IAC) in the Second Department of Thoracic Surgery, the First Affiliated Hospital of Xiamen University from May to December 2021 were retrospectively analyzed, including 20 males and 46 females, aged 35-75 years. The CT findings were analyzed before operation, and the lesion profile was cut after operation to distinguish the ground-glass and solid components, and the pathological results of different positions were obtained. According to the postoperative pathological results, the patients were divided into a low-risk group (containing adherent type and no components of micropapillary subtype and solid subtype, n=16), a medium-risk group (containing niple or acinar type and no components of micropapillary subtype and solid subtype, n=38), and a high-risk group (containing micropapillary or solid subtype, n=12). The relationships between CT features and the pathological subtype and degree of differentiation were analyzed and compared. Results    In 66 patients with IAC, the infiltration degree of solid components was greater than that of ground-glass components. When the solid component ratio (CTR) was≥25% (sensitivity 90.2%, specificity 64.0%, P=0.005), and the average CT value was>−283.95 HU (sensitivity 82.9%, specificity 64.0%, P=0.000), the histological grade was more inclined to medium and low differentiation. The CTR, Ki-67, average CT value and histological grade of IAC in the medium- and high-risk groups were higher than those of nodules in the low-risk group. Conclusion    The infiltration degree of solid components is higher than that of ground-glass components in IAC mixed ground-glass nodules. The pathological subtype, Ki-67 expression and histological grade of lung adenocarcinoma can be predicted according to its CT characteristics, which has important clinical significance for determining the timing of surgery.

As a novel and promising antitumor target, AXL plays an important role in tumor growth, metastasis, immunosuppression and drug resistance of various malignancies, which has attracted extensive research interest in recent years. In this study, by employing the structure-based drug design and bioisosterism strategies, we designed and synthesized in total 54 novel AXL inhibitors featuring a fused-pyrazolone carboxamide scaffold, of which up to 20 compounds exhibited excellent AXL kinase and BaF3/TEL-AXL cell viability inhibitions. Notably, compound 59 showed a desirable AXL kinase inhibitory activity (IC₅₀: 3.5 nmol/L) as well as good kinase selectivity, and it effectively blocked the cellular AXL signaling. In turn, compound 59 could potently inhibit BaF3/TEL-AXL cell viability (IC₅₀: 1.5 nmol/L) and significantly suppress GAS6/AXL-mediated cancer cell invasion, migration and wound healing at the nanomolar level. More importantly, compound 59 oral administration showed good pharmacokinetic profile and in vivo antitumor efficiency, in which we observed significant AXL phosphorylation suppression, and its antitumor efficacy at 20 mg/kg (qd) was comparable to that of BGB324 at 50 mg/kg (bid), the most advanced AXL inhibitor. Taken together, this work provided a valuable lead compound as a potential AXL inhibitor for the further antitumor drug development.

This study aimed to investigate the mechanism of Psoraleae Fructus in improving the learning and memory ability of APP/PS1 mice by serum metabolomics, screen the differential metabolites of Psoraleae Fructus on APP/PS1 mice, and reveal its influence on the metabolic pathway of APP/PS1 mice. Thirty 3-month-old APP/PS1 mice were randomly divided into a model group and a Psoraleae Fructus extract group, and another 15 C57BL/6 mice of the same age were assigned to the blank group. The learning and memory ability of mice was evaluated by the Morris water maze and novel object recognition tests, and metabolomics was used to analyze the metabolites in mouse serum. The results of the Morris water maze test showed that Psoraleae Fructus shortened the escape latency of APP/PS1 mice(P<0.01), and increased the number of platform crossing and residence time in the target quadrant(P<0.01). The results of the novel object recognition test showed that Psoraleae Fructus could improve the novel object recognition index of APP/PS1 mice(P<0.01). Eighteen differential metabolites in serum were screened out by metabolomics, among which the levels of arachidonic acid, tryptophan, and glycerophospholipid decreased after drug administration, while the levels of glutamyltyrosine increased after drug administration. The metabolic pathways involved included arachidonic acid metabolism, glycerophospholipid metabolism, tryptophan metabolism, linoleic acid metabolism, α-linolenic acid metabolism, and glycerolipid metabolism. Therefore, Psoraleae Fructus can improve the learning and memory ability of APP/PS1 mice, and its mechanism may be related to the effects in promoting energy metabolism, reducing oxidative damage, protecting central nervous system, reducing neuroinflammation, and reducing Aβ deposition. This study is expected to provide references for Psoraleae Fructus in the treatment of Alzheimer's disease(AD) and further explain the mechanism of Psoraleae Fructus in the treatment of AD.
Mice ; Animals ; Amyloid beta-Protein Precursor/genetics* ; Mice, Transgenic ; Arachidonic Acid ; Tryptophan ; Mice, Inbred C57BL ; Alzheimer Disease/genetics* ; Maze Learning ; Glycerophospholipids ; Disease Models, Animal ; Amyloid beta-Peptides/metabolism*

Objective:To explore the mechanism of osteoclast stimulatory transmembrane protein (OC-STAMP) overexpression on epithelial-mesenchymal transition (EMT) .Methods:In April 2021, mice alveolar type Ⅱ epithelial cells MLE-12 were divided into five groups: overexpression control group (NC group), Ocstamp overexpression group (over- Ocstamp group), Fasudil intervention group (over- Ocstamp+Fasudil group), silence control group (si-NC group), Ocstamp silence group (si- Ocstamp group). The protein expressions of OC-STAMP, epithelial marker protein-E-cadherin (E-cad), interstitial marker protein-α-smooth muscle actin (α-SMA), Ras homolog gene family member A (RhoA), Rho GDP dissociation inhibitor α (Rho GDIα), Rho-associated protein kinase (ROCK), phosphate myosin phosphatase (p-MYPT) were examined by Western blotting and Immunocytochemical staining. The filamentous actin (F-actin) was detected by Phalloidin method. t test was used to compare the relative expression of each protein between the two groups. Results:Western blotting and Immunocytochemical staining showed that compared with the NC group, the expression level of E-cad was down-regulated, while the expression levels of α-SMA, Rho GDIα, RhoA, ROCK, p-MYPT were increased, and F-actin expression was enhanced in the over- Ocstamp group. The differences were statistically significant ( P<0.05). There were no significant differences in E-cad and α-SMA protein expression in si- Ocstamp group compared with si-NC group ( P>0.05). Compared with over- Ocstamp group, the expression level of E-cad protein in over- Ocstamp+Fasudil group was up-regulated, the expression levels of α-SMA, Rho GDIα, RhoA, ROCK and p-MYPT protein were decreased, and F-actin expression was weakened, with statistical significance ( P<0.05) . Conclusion:OC-STAMP overexpression in alveolar type Ⅱ epithelial cells may induce actin cytoskeleton remodeling through activation of Rho GDIα/RhoA/ROCK signaling pathway, thus promoting EMT.

Background: and Purpose Intravenous tenecteplase (TNK) efficacy has not been well demonstrated in acute ischemic stroke (AIS) beyond 4.5 hours after onset. This study aimed to determine the effect of intravenous TNK for AIS within 4.5 to 24 hours of onset. Methods: In this pilot trial, eligible AIS patients with diffusion-weighted imaging (DWI)-fluid attenuated inversion recovery (FLAIR) mismatch were randomly allocated to intravenous TNK (0.25 mg/kg) or standard care within 4.5–24 hours of onset. The primary endpoint was excellent functional outcome at 90 days (modified Rankin Scale [mRS] score of 0–1). The primary safety endpoint was symptomatic intracranial hemorrhage (sICH). Results: Of the randomly assigned 80 patients, the primary endpoint occurred in 52.5% (21/40) of TNK group and 50.0% (20/40) of control group, with no significant difference (unadjusted odds ratio, 1.11; 95% confidence interval 0.46–2.66; P=0.82). More early neurological improvement occurred in TNK group than in control group (11 vs. 3, P=0.03), but no significant differences were found in other secondary endpoints, such as mRS 0–2 at 90 days, shift analysis of mRS at 90 days, and change in National Institutes of Health Stroke Scale score at 24 hours and 7 days. There were no cases of sICH in this trial; however, asymptomatic intracranial hemorrhage occurred in 3 of the 40 patients (7.5%) in the TNK group. Conclusion This phase 2, randomized, multicenter study suggests that intravenous TNK within 4.5–24 hours of onset may be safe and feasible in AIS patients with a DWI-FLAIR mismatch.

Objective:To explore the mechanism of osteoclast stimulatory transmembrane protein (OC-STAMP) overexpression on epithelial-mesenchymal transition (EMT) .Methods:In April 2021, mice alveolar type Ⅱ epithelial cells MLE-12 were divided into five groups: overexpression control group (NC group), Ocstamp overexpression group (over- Ocstamp group), Fasudil intervention group (over- Ocstamp+Fasudil group), silence control group (si-NC group), Ocstamp silence group (si- Ocstamp group). The protein expressions of OC-STAMP, epithelial marker protein-E-cadherin (E-cad), interstitial marker protein-α-smooth muscle actin (α-SMA), Ras homolog gene family member A (RhoA), Rho GDP dissociation inhibitor α (Rho GDIα), Rho-associated protein kinase (ROCK), phosphate myosin phosphatase (p-MYPT) were examined by Western blotting and Immunocytochemical staining. The filamentous actin (F-actin) was detected by Phalloidin method. t test was used to compare the relative expression of each protein between the two groups. Results:Western blotting and Immunocytochemical staining showed that compared with the NC group, the expression level of E-cad was down-regulated, while the expression levels of α-SMA, Rho GDIα, RhoA, ROCK, p-MYPT were increased, and F-actin expression was enhanced in the over- Ocstamp group. The differences were statistically significant ( P<0.05). There were no significant differences in E-cad and α-SMA protein expression in si- Ocstamp group compared with si-NC group ( P>0.05). Compared with over- Ocstamp group, the expression level of E-cad protein in over- Ocstamp+Fasudil group was up-regulated, the expression levels of α-SMA, Rho GDIα, RhoA, ROCK and p-MYPT protein were decreased, and F-actin expression was weakened, with statistical significance ( P<0.05) . Conclusion:OC-STAMP overexpression in alveolar type Ⅱ epithelial cells may induce actin cytoskeleton remodeling through activation of Rho GDIα/RhoA/ROCK signaling pathway, thus promoting EMT.

Animals ; Cell Cycle Proteins/metabolism* ; Insulin Resistance/genetics* ; Liver/physiopathology* ; Mice ; Sirtuin 1/metabolism*