Objective To investigate the clinical significance of CD44st expression in non-small cell lung carcinoma (NSCLC).Methods Specific primers for the CD44st gene (Gene Bank NO.FJ216964) which was found in multidrug resistance cells were designed,and reverse transcription-polymerase chain reaction (RT-PCR),gene sequencing and real-time PCR were used to validate the expression of the CD44st in the tissues and cancer adjacent tissues of 83 NSCLC patients (47 cases of adenocarcinoma,36 cases of squamous carcinoma).The clinicopathologic features were analyzed by statistical analysis method.Results The positive expressive rate of CD44st gene mRNA in NSCLC tissue was 63.9 ％ (53/83) and the negative expressive rate was 36.1％ (30/83),which were more higher than those of cancer adjacent tissues (0/30,0 ％) (P ＜ 0.001).DNA sequencing results showed that the sequence of CD44st mRNA was consistent with the CD44st mRNA found in MCF-7/Adr cells in our previous study.Expression of CD44st was closely associated with pathological pattern,lymphatic metastasis and TNM staging in NSCLC (P ＜ 0.05),but not with tumor size and differentiated degree (P ＞ 0.05).The rates of two years disease progression and stable condition in the group of lower CD44st expression were 45.2 ％ (19/42) and 54.8 ％ (23/42),respectively,and the rates in the group of higher CD44st expression were 68.3 ％ (28/41) and 31.7 ％ (13/41),respectively (P ＜ 0.05).Conclusions The expression level of CD44st is closely related to pathological pattern,the lymph node metastasis and the TNM-staging in NSCLC patients,which seriously influences the prognosis of the NSCLC patients.Therefore,the detection and the therapy to this special target may be a beneficial choice for the prognostic prediction of NSCLC.

Purpose: Isocitrate dehydrogenase 1 (IDH1) mutations are the most common genetic abnormalities in low-grade gliomas and secondary glioblastomas. Glioma patients with these mutations had better clinical outcomes. However, the effect of IDH1 mutation on drug sensitivity is still under debate. Materials and Methods: IDH1-R132H mutant cells were established by lentivirus. IDH1-R132H protein expression was confirmed by western blot. The expression of ataxia telangiectasia mutated (ATM) signaling pathway and apoptosis-related proteins were detected by immunofluorescence and western blot. Temozolomide (TMZ) induced cell apoptosis was detected by flow cytometry. Tumor cell proliferation was detected by Cell Counting Kit-8. In vivo nude mice were used to confirm the in vitro roles of IDH1 mutation. Results: We established glioma cell lines that expressed IDH1-R132H mutation stably. We found that TMZ inhibited glioma cells proliferation more significantly in IDH1 mutant cells compared to wild type. The IC50 of TMZ in IDH1-R132H mutant group was less than half that of wild-type group (p < 0.01). TMZ significantly induced more DNA damage (quantification of γH2AX expression in IDH1 mutation vs. wild type, p < 0.05) and apoptosis (quantification of AnnexinV+propidium iodide–cells in IDH1 mutation versus wild type, p < 0.01) in IDH1 mutant gliomas compared to wild-type gliomas. The ATM-associated DNA repair signal was impaired in IDH1 mutant cells. Inhibiting the ATM/checkpoint kinase 2DNA repair pathway further sensitized IDH1 mutant glioma cells to chemotherapy. We found that IDH1 mutation significantly inhibited tumor growth in vivo (the tumor size was analyzed statistically, p < 0.05). Moreover, we confirmed that gliomas with IDH1 mutation were more sensitive to TMZ in vivo compared to wild type significantly and the results were consistent with the in vitro experiment. Conclusion These results provide evidence that combination of TMZ and ATM inhibitor enhances the antitumor effect in IDH1 mutant gliomas.

Purpose: Isocitrate dehydrogenase 1 (IDH1) mutations are the most common genetic abnormalities in low-grade gliomas and secondary glioblastomas. Glioma patients with these mutations had better clinical outcomes. However, the effect of IDH1 mutation on drug sensitivity is still under debate. Materials and Methods: IDH1-R132H mutant cells were established by lentivirus. IDH1-R132H protein expression was confirmed by western blot. The expression of ataxia telangiectasia mutated (ATM) signaling pathway and apoptosis-related proteins were detected by immunofluorescence and western blot. Temozolomide (TMZ) induced cell apoptosis was detected by flow cytometry. Tumor cell proliferation was detected by Cell Counting Kit-8. In vivo nude mice were used to confirm the in vitro roles of IDH1 mutation. Results: We established glioma cell lines that expressed IDH1-R132H mutation stably. We found that TMZ inhibited glioma cells proliferation more significantly in IDH1 mutant cells compared to wild type. The IC50 of TMZ in IDH1-R132H mutant group was less than half that of wild-type group (p < 0.01). TMZ significantly induced more DNA damage (quantification of γH2AX expression in IDH1 mutation vs. wild type, p < 0.05) and apoptosis (quantification of AnnexinV+propidium iodide–cells in IDH1 mutation versus wild type, p < 0.01) in IDH1 mutant gliomas compared to wild-type gliomas. The ATM-associated DNA repair signal was impaired in IDH1 mutant cells. Inhibiting the ATM/checkpoint kinase 2DNA repair pathway further sensitized IDH1 mutant glioma cells to chemotherapy. We found that IDH1 mutation significantly inhibited tumor growth in vivo (the tumor size was analyzed statistically, p < 0.05). Moreover, we confirmed that gliomas with IDH1 mutation were more sensitive to TMZ in vivo compared to wild type significantly and the results were consistent with the in vitro experiment. Conclusion These results provide evidence that combination of TMZ and ATM inhibitor enhances the antitumor effect in IDH1 mutant gliomas.

OBJECTIVE: To explore the effect of interleukin-17A (IL-17A) on liver and kidney injury and prognosis in septic mice. METHODS: A total of 84 SPF male C57BL/6 mice were randomly divided into sham operation group (Sham group), cecal ligation and puncture (CLP) induced sepsis model group (CLP group), and IL-17A intervention group. IL-17A intervention group were then divided into five subgroups according to the dose of IL-17A (0.25, 0.5, 1, 2, 4 μg). Mice in the IL-17A intervention group were intraperitoneally injected with the corresponding dose of IL-17A 100 μL immediately after surgery. The other groups were intraperitoneally injected with 100 μL phosphate buffer solution (PBS). The survival rate of mice was observed at 7 days, and peripheral blood and liver, kidney and spleen tissues were collected. According to the 7-day survival, another 18 mice were randomly divided into Sham group, CLP group, and 1 μg IL-17A intervention group. Peripheral blood samples were collected at 12 hours and 24 hours after CLP, and the mice were sacrificed to obtain liver, kidney, and spleen tissues. The behavior and abdominal cavity of each group were observed. The levels of peripheral blood liver and kidney function indexes and inflammatory factors were detected. The histopathological changes of liver and kidney were observed under light microscope. The peripheral blood and spleen tissues were inoculated in the medium, the number of bacterial colonies was calculated, and the bacterial migration of each group was evaluated in vitro. RESULTS: Except for the Sham group, the 7-day survival rate of mice in the 1 μg IL-17A intervention group was the highest (75.0%), so this condition was selected as the intervention condition for the subsequent study. Compared with Sham group, the liver and kidney functions of CLP group were significantly damaged at each time point after operation. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and serum creatinine (SCr) reached the peak at 24 hours after operation, and the liver and kidney pathological scores reached the peak at 7 days after operation, the levels of inflammatory cytokines interleukin (IL-17A, IL-6, IL-10) reached the peak at 12 hours after operation, and tumor necrosis factor-α (TNF-α) reached the peak at 24 hours after operation. In addition, a large number of bacteria proliferated in the peripheral blood and spleen, which reached the peak on day 7. Compared with the CLP group, exogenous administration of 1 μg IL-17A significantly delayed the rising trend of each index in the early stage of sepsis [24-hour ALT (U/L): 166.95±5.20 vs. 271.30±6.11, 24-hour AST (U/L): 599.42±7.25 vs. 1 013.27±3.37, 24-hour BUN (mg/L): 815.4±26.3 vs. 1 191.2±39.4, 24-hour SCr (μmol/L): 29.34±0.87 vs. 60.75±3.83, 7-day liver pathological score: 2.50 (2.00, 3.00) vs. 9.00 (8.50, 9.00), 7-day kidney pathological score: 1.00 (1.00, 2.00) vs. 5.00 (4.50, 5.00), 12-hour IL-17A (ng/L): 105.21±0.31 vs. 111.28±1.37, 12-hour IL-6 (ng/L): 83.22±1.01 vs. 108.88±0.99, 12-hour IL-10 (ng/L): 731.54±3.04 vs. 790.25±2.54, 24-hour TNF-α (μg/L): 454.67±0.66 vs. 576.18±0.76, 7-day peripheral blood colony count (CFU/mL): 600 (400, 600) vs. 4 200 (4 200, 4 300), 7-day spleen tissue colony count (CFU/g): 4 600 (4 400, 4 600) vs. 23 400 (23 200, 23 500), all P < 0.05]. CONCLUSIONS Appropriate dose (1 μg) of exogenous IL-17A can reduce the lethal inflammatory response induced by CLP and improve the ability of bacterial clearance, thereby alleviating liver and kidney injury and improving the 7-day survival rate of septic mice.
Animals ; Male ; Mice ; Interleukin-10 ; Interleukin-17/pharmacology* ; Interleukin-6 ; Kidney/physiopathology* ; Liver/physiopathology* ; Mice, Inbred C57BL ; Prognosis ; Sepsis ; Tumor Necrosis Factor-alpha

OBJECTIVE To analyze the serum chemical composition of rats after intragastric administration of water extract of crude Anemarrhenae Rhizoma-crude Phellodendri Chinensis and salted Anemarrhenae Rhizoma-salted Phellodendri Chinensis based on liquid chromatography-mass spectrometry(LC-MS)technology,predict the effect of salt processing on the treatment of type 2 dia-betes in Anemarrhenae Rhizoma-Pheellodendri Chinensis combined with network pharmacology,and preliminarily verify it through in vitro experiments.METHODS Rats were continuously intragastrically administered with crude Anemarrhenae Rhizoma-crude Phello-dendri Chinensis drug pair and salted Anemarrhenae Rhizoma-salted Phellodendri Chinensis drug pair water extract twice,with an in-terval of 1 h.After 60 min of the last administration,the blood was taken from the abdominal aorta,and the protein was precipitated by methanol.After dissolution,the chromatographic column was Shim-pack GIST C18(4.6 mm×150 mm,5 μm);the mobile phase A was 0.1%formic acid water,and the mobile phase B was 0.1%formic acid-acetonitrile;gradient elution,positive and negative ion full scan mode,mass scan range 100-1 500 m/z.Combined with the secondary spectrum of the database and literature,the blood compo-nents of crude Anemarrhenae Rhizoma-crude Phellodendri Chinensis drug pair and salted Anemarrhenae Rhizoma-salted Phellodendri Chinensis drug pair were analyzed and identified.The disease targets of type 2 diabetes were retrieved,and the protein interaction net-work analysis,GO and KEGG pathway enrichment analysis were performed on the intersection targets of blood components and diseases.The"blood components-targets"network diagram was constructed,and the selected core components and core targets were verified by molecular docking using AutoDock software.In the verification experiment,HepG2 cells were used as the experimental ob-ject,and the insulin resistance model was induced by high insulin and high glucose.CCK8 method was used to test the effect of Rhizo-ma Anemarrhenae-Phellodendri Chinensis on cell proliferation before and after salt processing.Western blot was used to detect the ex-pression of PI3K-AKT signaling pathway-related proteins.RESULTS 15 prototype components and 1 mangiferin metabolic compo-nent were identified in the serum of rats.17 prototype components and 1 mangiferin metabolite were identified in the rat serum of the water extract of Anemarrhenae Rhizoma-Phellodendri Chinensis after salt processing.The contents of mangiferin,berberine and 3-isobutylglutaric acid in the blood components after salt-processing were higher than those in the raw products.According to the results of KEGG and GO,the treatment of type 2 diabetes may be related to the transcriptional regulation of RNA polymerase,inflammatory re-sponse,AGE-RAGE,PI3K-AKT pathway and insulin resistance.Cell experiments showed that the ratio of p-PI3K/PI3K,p-AKT/AKT and GLUT4 protein expression could be up-regulated before and after salt processing,and the effect of salt processing group was better than that of the crude group.CONCLUSION This experiment preliminarily revealed the components of Anemarrhenae Rhizo-ma-Phellodendri Chinensis drug pair entering the blood before and after salt exposure,and suggested that ferulic acid,berberine,ber-berrubine,mangiferin,mTOR,SIRT1,EGFR and PPARA may be the main components and targets of Rhizoma Anemarrhenae-Phel-lodendri Chinensis after salt processing to enhance the therapeutic effect of type 2 diabetes.The mechanism may be to enhance the role of PI3K-AKT and other related signaling pathways,providing an important reference for the pharmacodynamic material basis and clini-cal application of Anemarrhenae Rhizoma-Phellodendri Chinensis before and after salt processing.

ObjectiveTo investigate the key compounds affecting the irritation and to clarify the effect of heating and the addition of ginger juice as the auxiliary material during the processing on the irritation of Magnoliae Officinalis Cortex（MOC） by comparing the irritation and composition of volatile oil in MOC and its different processed products. MethodVolatile oil in raw products， water-processed products， ginger-dried products， ginger-fried products（the amounts of ginger were 10%， 50%， respectively） of MOC were extracted by steam distillation and subjected to rabbit eye irritation experiment， and the volatile components of each sample were detected by gas chromatography-mass spectrometry（GC-MS）. Principal component analysis（PCA）and orthogonal partial least squares-discriminant analysis（OPLS-DA） were used to analyze the data of each sample by SIMCA 14.1. The relative contents of different processed products were compared two by two with those of and raw products or ginger-fried products， and the markers that might be related to the irritation were sorted out according to the principles of variable importance in the projection（VIP） value >1 and P<0.05， and the factors influencing the differences in irritation were analyzed. ResultCompared with the blank group， the administration groups all had irritation to the eyes of rabbits， and the degree of irritation was in the order of raw products>water-processed products>ginger-dried products>ginger-fried products（10%）>ginger-fried products（50%）. The results of PCA and OPLS-DA showed that there were differences in the volatile oil from raw products and different processed products. According to VIP value>1 and P<0.05， and combined with the results of eye irritation experiment， ten volatile compounds related to irritation changes were screened out. Among them， cis-cinnamaldehyde was only detected in raw products， the relative contents of β-caryophyllene， （+）-delta-cadinene， α-humulene， γ-muurolene， （-）-isoledene and citral all increased to different degrees， the contents of p-cymene， 1（10）-4-cadinadien-15-ol and β-eudesmol all decreased to different degrees. ConclusionThe irritation of MOC is reduced after heating and processing with ginger juice， and the synergistic effect of both is more effective for reducing irritation. Among the differential markers associated with changes in irritation， the increase in the relative content of citral is closely related to the addition of ginger juice， while the decrease in the relative contents of cis-cinnamaldehyde， p-cymene， 1（10）-4-cadinadien-15-ol is related to heating， and the changes of other components may be related to the synergistic effect of heating and ginger juice.