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Natural killer (NK) cells recognize stress-activated NK group 2, member D (NKG2D) ligands in tumors. In the present study, the expression levels of NKG2D ligands were examined in four lung cancer cell lines (A549, PLA801D, NCI-H157 and NCI-H520). In the A549 cells, the expression of MHC class I polypeptiderelated sequence (MIC)A/B and UL16 binding protein (ULBP)1 was weak, the expression of ULBP2 was typical, and neither ULBP3 nor ULBP4 were expressed. The mechanism underlying the regulatory effect of a cancer treatment agent on the expression of NKG2D ligands was investigated using the proteasome inhibitor MG132. Following treatment for 8 h with MG132, the transcription levels of MICB and ULBP1 were upregulated 10.62- and 11.09-fold, respectively, and the expression levels of MICB and ULBP1 were increased by 68.18 and 23.65%, respectively. Notably, MICB exhibited significant time-dependent change. MG132 increased the transcription of MICB by acting at a site in the 480-bp MICB upstream promoter. The activity of the MICB promoter was upregulated 1.77-fold following treatment with MG132. MG132 treatment improved the cytotoxicity of NK cells, which was partially blocked by an antibody targeting NKG2D, and more specifically the MICB molecule. The expression of MICB induced by MG132 was inhibited by KU-55933 [ataxia telangiectasia mutated (ATM) kinase inhibitor], wortmannin (phosphoinositide 3 kinase inhibitor) and caffeine (ATM/ATM-Rad3-related inhibitor). The phosphorylation of checkpoint kinase 2 (Chk2), an event associated with DNA damage, was observed following treatment with MG132. These results indicated that MG132 selectively upregulates the expression of MICB in A549 cells, and increases the NKG2D-mediated cytotoxicity of NK cells. The regulatory effect of MG132 may be associated with the activation of Chk2, an event associated with DNA damage. The combination of MG132 with NK cell immunotherapy may have a synergistic effect that improves the therapeutic effect of lung cancer treatment. [ABSTRACT FROM AUTHOR]

With the growing population, aging, extended lifespans and anti‑aging have become popular areas of research in the life and social sciences. With increasing age, the structure and function of the testes, the spermatogenetic and androgen‑producing organ in the male reproductive system, gradually declines. Ginsenoside Rg1 is an extract of Panax ginseng in traditional Chinese medicine. The extract facilitates anti‑aging through its anti‑inflammatory and antioxidant properties. However, it has not been reported whether ginsenoside Rg1 delays testicular aging. The present study established D‑galactose (D‑gal)‑induced aging mouse models to examine the protective effects of ginsenoside Rg1 on the structure and function of the testes, and the underlying mechanism. A total of 60 healthy specific pathogen‑free male C57BL/6 mice were randomly divided into four groups: Control group; Rg1 group; D‑gal + Rg1 group; and D‑gal group. The tissues of the mice were used for further experiments. The present study further investigated the effects of Rg1 on the volume of serum testosterone, the testicular index, testicular microscopic structures, the senescence of spermatogenetic cells, the apoptosis of spermatogenetic cells, the activity of the antioxidant enzymes, the levels of inflammatory cytokines, and the levels of S‑phase kinase‑associated protein (p19), cyclin‑dependent kinase inhibitor 1 (p21) and cellular tumor antigen p53 (p53) in D‑gal‑induced aging mice. In general, compared with the D‑gal group, the treatment of Rg1 increased the testis index, serum testosterone level and the active content of superoxide dismutase and the total antioxidant capacity. The percentage of senescence‑associated β‑galactosidase‑positive cells, the level of apoptosis and the volume of methane dicarboxylic aldehyde, tumor necrosis factor‑α, interleukin (IL)‑1β and IL‑6 in testicular tissues were significantly decreased, and the expression of p19, p53 and p21 was downregulated due to the treatment with Rg1. The results of the present study demonstrated that ginsenoside Rg1 was able to protect the testes against D‑gal‑induced aging in mice. In addition, the protective effect of Rg1 may be achieved via antioxidation and downregulation of the p19/p53/p21 signaling pathway. [ABSTRACT FROM AUTHOR]

This study explores the effect of apigenin(APG), oxymatrine(OMT), and APG+OMT on the proliferation of non-small cell lung cancer cell lines and the underlying mechanisms. Cell counting kit-8(CCK-8) assay was used to detect the vitality of A549 and NCI-H1975 cells, and colony formation assay to evaluate the colony formation ability of the cells. EdU assay was employed to examine the proliferation of NCI-H1975 cells. RT-qPCR and Western blot were performed to detect the mRNA and protein expression of PLOD2. Molecular docking was carried out to explore the direct action ability and action sites between APG/OMT and PLOD2/EGFR. Western blot was used to study the expression of related proteins in EGFR pathway. The viability of A549 and NCI-H1975 cells was inhibited by APG and APG+OMT at 20, 40, and 80 μmol·L~(-1) in a dose-dependent manner. The colony formation ability of NCI-H1975 cells was significantly suppressed by APG and APG+OMT. The mRNA and protein expression of PLOD2 was significantly inhibited by APG and APG+OMT. In addition, APG and OMT had strong binding activity with PLOD2 and EGFR. In APG and APG+OMT groups, the expression of EGFR and proteins in its downstream signaling pathways was significantly down-regulated. It is concluded that APG in combination with OMT could inhibit non-small lung cancer, and the mechanism may be related to EGFR and its downstream signaling pathways. This study lays a new theoretical basis for the clinical treatment of non-small cell lung cancer with APG in combination with OMT and provides a reference for further research on the anti-tumor mechanism of APG in combination with OMT.

The present study explored the effects and its underlying mechanisms of four active fractions of Camellia nitidissima(leaf polyphenols, leaf saponins, flower polyphenols, and flower saponins in C. nitidissima) in inhibiting the proliferation and migration of non-small cell lung cancer(NSCLC) by suppressing the epidermal growth factor receptor(EGFR). MTT assay was used to detect the effect of four active fractions on the proliferation of NCI-H1975 and HCC827 cells. Wound healing assay and Transwell assay were adopted to evaluate the effect of four active fractions on the migration of NSCLC. The effect of four active fractions on the enzyme activity of EGFR was detected. Molecular docking was carried out to explore the direct action capacity and action sites between representative components of the four active fractions and EGPR. Western blot assay was employed to investigate the effect of four active fractions on the protein expression in EGFR downstream signaling pathways. The results of the MTT assay indicated that the cell viability of NCI-H1975 and HCC827 cells was significantly inhibited by four active fractions at 50, 100, 150, and 200 μg·mL~(-1) in a dose-dependent manner. Wound healing assay and Transwell assay revealed that the migration of NCI-H1975 and HCC827 cells was significantly suppressed by four active fractions. In addition, the results of the protein activity assay showed that the enzyme activity of EGFR was significantly inhibited by four active fractions. The molecular docking results confirmed that various components in four active fractions possessed strong binding activity to EGFR enzymes. Western blot assay revealed that four active fractions down-regulated the protein expression of EGFR and its downstream signaling pathways. It is concluded that the four active fractions of C. nitidissima can inhibit NSCLC. The mechanism may be related to EGFR and its downstream signaling pathways. This study provides a new scientific basis for the clinical treatment of NSCLC with active fractions of C. nitidissima, which is of reference significance for further research on the anti-tumor mechanism of C. nitidissima.

This is a study on the relationship between the protective effect of ginsenoside Rg1 on senescent neural stem cells and Wnt- β /catenin signaling pathway. Background . Recent studies have shown that overactivation of the Wnt/ β -catenin signaling pathway is closely related to stem cell senescence. Whether Rg1 delays the senescence of NSCs is related to the regulation of this signaling pathway. Methods . The whole brain of Nestin-GFP transgenic newborn rat was extracted, and NSCs were extracted and cultured to P3 generation. The following indicators were detected: (1) NSC culture identification, (2) the effect of LiCl on the proliferation and survival rate of NSCs, (3) the effect of ginsenoside Rg1 on the proliferation and survival of NSCs, (4) the growth of NSCs in each group observed by an optical microscope, (5) the cell cycle of each group detected by flow cytometry, (6) the proliferative ability of each group detected by BrdU, (7) the fluorescence intensity of Nestin and Sox2 of NSCs in each group observed by a fluorescence microscope, (8) the positive rate of senescence staining analyzed by SA- β -Gal staining, (9) the localization of β -catenin in NSCs observed by laser confocal microscopy, and (10) the changes of the Wnt/ β -catenin pathway-related proteins in each group detected by Western blotting. Results . LiCl activates the Wnt/ β -catenin pathway and promotes mouse neural stem cell senescence. Ginsenoside Rg1 promotes proliferation of neural stem cells and inhibits Wnt/ β -catenin pathway activation. Conclusions . LiCl can activate the Wnt/ β -catenin signaling pathway of NSCs, and ginsenoside Rg1 can antagonize the senescence of NSCs caused by activation of the Wnt/ β -catenin signaling pathway and delay brain aging., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2019 Yue Xiang et al.)

Objective: To construct an expression vector of anti-MM scFv-tP fusion protein and test its expression efficiency and function., Methods: The truncated protamine (tP) gene sequence was added to the gene of single chain antibody against the specific antigen on the surface of malignant melanoma tumor cells using PCR. A GST-fusion expression vector was constructed and the soluable protein was expressed in the E.coli system. After cleavage and purification, the purified fusion protein was obtained. The binding activity of Anti-MM scFv-tP and siRNA was detected by EMSA. Flow cytometry and confocal microscopy were used to detect the cell surface antigen binding activity of the fusion protein., Results: The expression vector of Anti-MM scFv-tP fusion protein was successfully constructed. The soluable protein could be expressed in the E.coli system, and the purified fusion protein was obtained. The anti-MM scFv-tP fusion protein retained siRNA binding ability and could directly target malignant melanoma (MM) LiBr cells., Conclusion: The recombinant GST- Anti-MM-scFv-tp expression vector was successfully constructed. The fusion protein retains siRNA binding ability and can directly target LiBr cells to provide a reliable tool for further study.

This study was purpose to investigate the effects of CD147 on the invasiveness of leukemia cells U937. The experiments were divided into 4 groups: control group, LPS group, CD147mAb group and LPS+CD147 mAb group. Cells were treated by lipopolysaccharide (LPS) or anti-CD147 monoclonal antibody, and the expression of CD147 and MMP-2, -9, the invasive potential of the cells in vitro and ex vivo, as well as the invasion of the implanted tumors in SCID mice were analysed by RT-PCR, FCM, gel zymography and invasion test in vitro respectively. The results showed that the expression of CD147 was elevated by the induction of LPS, and the enhanced expression of CD147 on U937 cells increased the production and secretion of MMP-2 and MMP-9 as measured by reverse transcription-PCR and gel zymography. An increased number of LPS-induced cells invading through a reconstituted basement membrane were observed by invasion assays. These responses were down-regulated after blocking CD147 with anti-CD147 antibody. At 30 days after intravenous injection of LPS pretreated U937 cells to SCID mice human U937 cells were found in the bone marrow and lung of the mice, indicating the invasion of the tumor cells. And overexpressions of CD147, MMP-2 and MMP-9 were found in the lung tissue of the mice injected with LPS-treated but not anti-CD147 antibody treated tumor cells. It is concluded that overexpression of CD147 on U937 cells may increase the secretion and activation of MMP-2 and MMP-9 and thus promote the invasiveness of the tumor cells.

Objective: To observe the characteristics of changes in hemorheology at the early stage of irreversible hemorrhagic shock in a rodent model., Methods: Rodent model of irreversible hemorrhagic shock was reproduced. Animals were randomized into 4 groups. In the first group, survival rate and mean arterial pressure (MAP) in 180 minutes were observed after hemorrhagic shock (S group). In the second group, animals were sacrificed soon after hemorrhagic shock (S0 group). In the third group, animals were sacrificed 60 minutes after hemorrhagic shock (S1 group). In the fourth group, animals were sacrificed 120 minutes after hemorrhagic shock (S2 group). Blood samples of animals of S0, S1 and S2 were all obtained before hemorrhagic shock. Blood lactate, hemorheological parameters, red blood cell (RBC) deformability and RBC aggregation index were determined., Results: Mean blood loss of S group was (22.9+/-3.8) ml/kg, constituting about (38.1+/-6.3)% of total blood volume. At 60, 120 and 180 minutes after hemorrhagic shock, survival rates were 100%, 72% and 64%, respectively. Compared with baseline, 0, 60 and 120 minutes after hemorrhagic shock, blood lactate increased significantly (all P<0.01), but 120 minutes after hemorrhagic shock, it decreased significantly compared with 0 minute after hemorrhagic shock (P<0.05). Compared with baseline, 0 minute and 60 minutes after hemorrhagic shock, blood viscosity was found to be decreased at shear rate of 10 s(-1), 60 s(-1) and 100 s(-1) (all P<0.01); 120 minutes after hemorrhagic shock, at shear rate of 10 s-1 and 60 s(-1), blood viscosity decreased significantly (both P<0.01); 0, 60 and 120 minutes after hemorrhagic shock, plasma viscosity, RBC deformability and RBC aggregation index at shear rates of 600 s(-1), 800 s(-1) and 1 000 s(-1) decreased significantly (all P<0.01)., Conclusion: At the early stage of irreversible hemorrhagic shock, blood lactate increased significantly, and decreased afterwards. These indicate reversal of deterioration of metabolism. At different time after the early stage of irreversible hemorrhagic shock, blood and plasma viscosity, RBC deformability and aggregation index lowered significantly and did not improve. Changes in viscosity and RBC aggregation are different from the changes in late stage, and this indicates that hemorheological disorders should be corrected in the treatment at the early stage after hemorrhagic shock.

Aim: To study the protective effect of sodium pyruvate on ischemia/reperfusion injury following hemorrhagic shock., Methods: Rat models of hemorrhagic shock were built up. When the shed blood was infused, the rats were also randomly provided by one of normal saline, glutathione and sodium pyruvate. Rats were killed 3 hours after the reperfusion, the activity of plasma lactate dehydrogenase (LDH) and glutamic-oxaloacetic transaminase (GOT), the level of tissue malondialdehyde (MDA) and the activity of tissue myeloperoxidase (MPO) were detected. Biopsy specimens were obtained to investigate morphological changes of the myocardial, hepatic, lung and renal tissue., Results: The activity of plasma LDH and GOT, the level of MDA of hepatic, lung and renal tissue and the activity of MPO of myocardial, lung and renal tissue decreased remarkably in group given sodium pyruvate compared with group given normal saline, and the effect of group given sodium pyruvate was more remarkable than group given glutathione., Conclusion: These data support the view that sodium pyruvate shows protective effect on ischemia/reperfusion injury following hemorrhagic shock. It is possibly relevant to scavenging of oxygen free radicals, reduction of neutrophil, and anti-inflammatory response.

Aim: To express fusion protein of GST and vWf binding domain(GP302) of platelet GPIbalpha in E.coli and its preparation of rabbit anti-serum., Methods: GP302 gene was inserted into pGEX-4T-1. The recombinant vector was identificated by restriction endonuclease digestion analysis. Fusion protein GST-GP302 was expressed in E.coli via IPTG induction. The rabbit antibody against GST-GP302 was prepared by using renatured GST-GP302 as immuneogen and the specificity of polyclonal antibody was identified by Western blot., Results: The restriction endonuclease digestion analysis of recombinant plasmid demonstrated that the GP302 gene had been exactly inserted into pGEX-4T-1. SDS-PAGE analysis showed that the ralative molecular mass(M(r)) of the fusion protein was about 59 000. ELISA analysis proved that the titer of rabbit serum against GST-GP302 was 10(-5). The polyclonal antibody specifically bound to purified platelet GPIbalpha., Conclusion: The preparation of polyclonal antibody against GP302 peptide provides an usefal reagent for the detection of platelet GPIbalpha.