ObjectiveTo study the effect of Tangeretin on non-small cell lung cancer (NSCLC) and the tumor stemness, and to find the molecular mechanism of its effect. MethodsWe used cell counting and cell cloning experiments to study the effect of Tangeretin on the proliferation of NSCLC cells in vitro. The effect of Tangeretin on the invasion of NSCLC cells was detected by transwell assay. We detected the effect of Tangeretin on the proliferation of NSCLC cells in vivo by nude mouse tumor-bearing experiment. The effect of Tangeretin on tumor stemness of NSCLC cells was detected by self-renew assay, and CD133 and Nanog protein expressions. The expressions of PI3K/AKT/mTOR signaling pathway-related proteins were detected by Western blotting (WB). ResultsTangeretin had a good inhibitory effect on the proliferation of NSCLC cells in vivo and in vitro. Cell counting experiment, clonal formation experiment and nude mouse tumor-bearing experiment showed that Tangeretin could inhibit the proliferation activity, clonal formation ability, and tumor size of NSCLC cells in vivo. Self-renew experiments showed that Tangeretin could inhibit the self-renew ability of NSCLC cells. WB experiments showed that Tangeretin inhibited the expressions of tumor stemness markers CD133 and Nanog in NSCLC cells. Tangeretin could inhibit the activation of PI3K/AKT/mTOR signaling pathway-related proteins in NSCLC cells, and the activation of PI3K/AKT/mTOR signaling pathway could partially remit the inhibitory effect of Tangeretin on tumor stemness of NSCLC cells. ConclusionTangeretin can inhibit the tumor stemness of NSCLC cells, which may be related to the regulation of PI3K/AKT/mTOR signaling pathway.
ObjectiveTo investigate the effect of overexpressing the Indianhedgehog (IHH) gene on the chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells (BMSCs) in a simulated microgravity environment. MethodsThe 2nd generation BMSCs from rabbit were divided into 2 groups: the rotary cell culture system (RCCS) group and conventional group. Each group was further divided into the IHH gene transfection group (RCCS 1 group and conventional 1 group), green fluorescent protein transfection group (RCCS 2 group and conventional 2 group), and blank control group (RCCS 3 group and conventional 3 group). RCCS group cells were induced to differentiate into chondrocytes under simulated microgravity environment; the conventional group cells were given routine culture and chondrogenic induction in 6 well plates. During differentiation induction, the ELISA method was used to detect IHH protein expression and alkaline phosphatase (ALP) activity, and quantitative real-time PCR to detect cartilage and cartilage hypertrophy related gene expressions, and Western blot to detect collagen typeⅡ, agreecan (ANCN) protein expression; and methylene blue staining and Annexin V-cy3 immunofluorescence staining were used to observe cell slide. ResultsAfter transfection, obvious green fluorescence was observed in BMSCs under fluorescence microscopy in RCCS groups 1 and 2, the transfection efficiency was about 95%. The IHH protein levels of RCCS 1 group and conventional 1 group were significantly higher than those of RCCS 2, 3 groups and conventional 2, 3 groups (P < 0.05); at each time point, ALP activity of conventional 1 group was significantly higher than that of conventional 2, 3 groups (P < 0.05); ALP activity of RCCS 1 group was significantly higher than that of RCCS 2 and 3 groups only at 3 and 7 days (P < 0.05). Conventional 1 group expressed high levels of cartilage-related genes, such as collagen typeⅡand ANCN at the early stage of differentiation induction, and expressed high levels of cartilage hypertrophy-related genes, such as collagen type X, ALP, and Annexin V at the late stage (P < 0.05). RCCS 1 group expressed high levels of cartilage-related genes and low levels of cartilage hypertrophy-related genes at all stages. The expression of collagen typeⅡprotein in conventional 1 group was significantly lower than that of conventional 2 and 3 groups at 21 days after induction (P < 0.05); RCCS 1 group expressed high levels of collagen typeⅡand ANCN proteins at all stages (P < 0.05). Methylene blue staining indicated conventional 1 group was stained lighter than conventional 2 and 3 groups at 21 days after induction; while at each time point RCCS 1 group was significantly deeper than RCCS 2 and 3 groups. Annexin V-cy3 immunofluorescence staining indicated the red fluorescence of conventional 1 group was stronger than that of conventional 2 and 3 groups at each time point. The expression of red fluorescence in each RCCS subgroup was weak and there was no significant difference between the subgroups. ConclusionUnder the simulated microgravity environment, transfection of IHH gene into BMSCs can effectively promote the generation of cartilage and inhibit cartilage aging and osteogenesis. Therefore, this technique is suitable for cartilage tissue engineering.
ObjectiveTo investigate the effect of PI3K/Akt/mTOR signaling pathway on liver injury induced by severe acute pancreatitis (SAP). MethodsForty healthy adult male Sprague-Dawley (SD) rats were randomly divided into 4 groups: Sham operation group (SO group), SAP group, PI3K inhibitor LY294002 group (LY294002 group), and mTOR kinase inhibitor rapamycin group (rapamycin group). The rat model with SAP was made by injection with 5% sodium deoxycholate through retrogradely bilio pancreatic duct. Serum levels of amylase (AMY), alanine aminotransferase (ALT), and aspartate transaminase (AST) were detected through the inferior vena at 6 h after modeling. Pathologic change of the liver was observed under the light microscope. TUNEL analysis was used to detect apoptotic index (AI) of the heptocyte. Expressions of Akt, phosphated-Akt (p-Akt), mTOR, phosphated-mTOR (p-mTOR) protein were evaluated by Western blot. Results①Compared with the SO group, the serum levels of AMY, ALT, AST, and the hepatocyte AI were significantly increased among the other three groups (P < 0.05). Compared with the SAP group, the serum levels of AMY, ALT, AST, and the hepatocyte AI were significantly decreased in the LY294002 group and rapamycin group (P < 0.05).②Compared with the SO group, the damages of the liver tissues were aggravated among the other three groups. The pathologies of the liver tissues were ameliorated in the LY294002 group and rapamycin group as compared with the SAP group.③Compared with the SO group, the levels of p-Akt/Akt, p-mTOR/mTOR were significantly increased among the other three groups (P < 0.05). Compared with the SAP group, the levels of p-Akt/Akt, p-mTOR/mTOR were significantly decreased in the LY294002 group (P < 0.05), but in the rapamycin group, only the p-mTOR/mTOR level was significantly decreased (P < 0.05). ConclusionThe activation of PI3K/Akt/mTOR signaling pathway might be one of the reasons for the liver injury induced by SAP and blocking this signaling pathway might be a potential target of preventing progress of SAP and alleviating liver injury induced by SAP.
Objective To investigate the effect of resveratrol (RES) on inflammation-induced cartilage endplate (CEP) degeneration, and its regulatory mechanism on high mobility group box-1 protein (HMGB1) signaling pathway. Methods The intervertebral CEP cells of Sprague Dawley (SD) rats aged 3 weeks were extracted and identified by toluidine blue staining and immunofluorescence staining of rabbit anti-rat collagen type Ⅱ. The cell counting kit 8 (CCK-8) method was used to screen the optimal concentration of RES on intervertebral CEP cells. Gene chip analysis was used to determine the target of RES on intervertebral CEP cells. Interleukin 1β (IL-1β) was used to construct the intervertebral CEP cell degeneration model caused by inflammation and the 7-8-week-old SD rat intervertebral disc degeneration model, and pcDNA3.1-HMGB1 (pcDNA3.1) was used as the control of RES effect. Flow cytometry and TUNEL staining were used to detect the apoptotic rate of intervertebral CEP cells and rat intervertebral disc tissue cells, respectively. ELISA kit was used to detect the content of interleukin 10 (IL-10) and tumor necrosis factor α (TNF-α) in the cell supernatant and rat serum. Western blot was used to detect the expressions of HMGB1, extracellular signal-regulated protein kinase (ERK), phosphorylated ERK (p-ERK), B cell lymphoma/leukemia 2 gene (Bcl-2), and Bcl-2-associated X protein (Bax). ResultsThe extracted cells were identified as rat intervertebral CEP cells. CCK-8 method screened out the highest activity of intervertebral CEP cells treated with 30 μmol/L RES. The gene chip analysis confirmed that the HMGB1-ERK signal was the target of RES. Both cell experiments and animal experiments showed that RES treatment can significantly down-regulate the apoptosis rate of intervertebral CEP cells, inhibit the release of TNF-α, and increase the content of IL-10; and down-regulate the expressions of HMGB1, p-ERK, and Bax, and increase Bcl-2; and pcDNA3.1 could partially reverse these effects of RES, and the differences were all significant (P<0.05). ConclusionRES can significantly inhibit the apoptosis of intervertebral CEP cells induced by inflammation, which is related to inhibiting the expression of HMGB1.
ObjectiveTo understand the research status of phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) signaling pathway in the thyroid cancer (TC), as well as its role in the occurrence, cell differentiation, invasion, and metastasis of the TC, so as to find potential targets for treatment of TC. MethodThe literature about the research of PI3K/AKT signaling pathway in the TC was searched and summarized. ResultsThe PI3K/AKT signaling pathway was abnormally activated directly or indirectly in the TC, resulting in inhibition of cell apoptosis, malignant proliferation, accelerated cycle progression, invasion, and metastasis, etc., which promoted the occurrence and development of the TC. There were also some tumor suppressor genes, microRNAs, long chain non-coding RNAs, etc., which indirectly inhibited the activation of PI3K/AKT signaling pathway, or directly acted on it inhibiting its activity to inhibit the occurrence and development of the TC. ConclusionsFor the TC, some proteins, genes, microRNAs, and long chain non-coding RNAs directly or indirectly activate the PI3K/AKT signaling pathway through different targets to promote the occurrence and development of TC. At the same time, many targets inhibit the activation of the PI3K/AKT signaling pathway, which inhibits the malignant proliferation, invasion, and metastasis of TC. At present, there have been studies trying to use PI3K/AKT signaling pathway as a breakthrough for the treatment of TC. In-depth exploration of the role of PI3K/AKT signaling pathway in different TC is of great significance to find new targets for the treatment of TC.
To investigate the mechanism of cAMP/Ca2+ signaling pathway inducing bone marrow mesenchymal stem cells to differentiate into neuronal cells, we cultured the bone marrow mesenchymal stem cells D1 cells in the present study. D1 cells were divided into two groups: control group and salidroside inducing groups. Control group was cultured with complete culture solution D/F12, while salidroside inducing groups were induced with 100 mg·L–1 salidroside for different time periods (24, 48 and 72 hours). PCR-array assay was used to detect expression of 84 calcium related mRNA, and significantly different genes were chosen to analyse. As a result, there were 4 significantly upregulated mRNAs inclu-ding DNA damage-inducible transcript 3 (Ddit3), heat shock protein 5 (Hspa5), protein phosphatase 1 regulatory subunit (Ppp1r15a) and prostaglandin-endoperoxide synthase 2 (Ptgs-2), and 4 significantly downregulated mRNAs including glucagon (Gcg), interleukin 2 (Il2), tumor necrosis factor (Tnf) and somatostatin (Sst) in the cAMP/Ca2+ signaling pathway. They probably had an effect on the process of salidroside induced D1 cells differentiating into neuronal cells.
ObjectiveTo summarize the active changes of Wnt signaling pathway in osteoarthritis (OA) as well as the influence and mechanism of dual-targeted regulation on cartilage and subchondral bone and the role of crosstalk between them on OA process.MethodsThe relevant literature concerning the articular cartilage, subchondral bone, and crosstalk between them in OA and non-OA states by Wnt signaling pathway in vivo and vitro experimental studies and clinical studies in recent years was reviewed, and the mechanism was analyzed and summarized.ResultsWnt signaling can regulate the differentiation and function of chondrocytes and osteoblasts through the classic β-catenin-dependent or non-classical β-catenin-independent Wnt signaling pathway and its cross-linking with other signaling pathways, thereby affecting the cartilage and bone metabolism. Moreover, Wnt signaling pathway can activate the downstream protein Wnt1-inducible-signaling pathway protein 1 to regulate the progress of OA and it also can be established gap junctions between different cells in cartilage and subchondral bone to communicate molecules directly to regulate OA occurrence and development. Intra-articular injection of Wnt signaling inhibitor SM04690 can inhibit the progress of OA, and overexpression of Wnt signaling pathway inhibitor Dickkopf in osteoblasts can antagonize the role of vascular endothelial growth factor work on chondrocytes and inhibit the catabolism of its matrix.ConclusionThe regulation of metabolism and function of cartilage and subchondral bone and crosstalk between them is through interactions among Wnt signaling pathway and molecules of other signaling. Therefore, it plays an vital role in the occurrence and development of OA and is expected to become a new target of OA treatment through intervention and regulation of Wnt signaling pathway.
Objective To investigate the effects of growth differentiation factor 15 (GDF15) on lung adenocarcinoma bone metastasis in nude mice by regulating phosphatase and tensin homology/phosphatidylinositol 3 kinase/protein kinase B (PTEN/PI3K/AKT) signaling pathway. Methods The bone metastasis model of lung adenocarcinoma in nude mice was established and mice were randomly divided into Control group, sh-NC group, sh-GDF15 group, sh-GDF15+sh-NC group and sh-GDF15+sh-PTEN group. The changes of 50% pain withdrawl threshold (PWT) and thermal withdrawal latency (TWL) were observed. bone destruction was analyzed by Micro-CT. Bone morphology was observed by HE staining. The expression of osteoclast-related factors was detected by immunohistochemistry. The expression of PTEN/PI3K/AKT signaling pathway was detected by Western blot. Results The tibia tissue of nude mice in Control group and sh-NC group was destroyed, the continuity of bone cortex was interrupted, obvious bone tumor lesions were observed, the tumor cells were irregular in shape, densely distributed and disordered, and the nuclei were obviously deformed. Compared with sh-NC group, sh-GDF15 group had less tibial tissue destruction, the bone tumor cell density was significantly reduced, the pathological morphology was significantly improved, and 50% PWT, TWL, bone mineral density (BMD), trabecular bone volume/total volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and PTEN expression were increased, the expressions of trabecular separation (Tb.Sp), cathepsin K (CTSK), matrix metalloproteinase 9 (MMP-9), p-PI3K PI3K and p-Akt/Akt were decreased (P<0.05). Compared with sh-GDF15+sh-NC group, sh-GDF15+sh-PTEN group had severe tibial tissue damage, the bone tissue tumor cells were dense and the pathological injury was aggravated, and 50%PWT, TWL, BMD, BV/TV, Tb.N, Tb.Th and PTEN expression were decreased, the expressions of Tb.Sp, CTSK, MMP-9, p-PI3K /PI3K and p-Akt /Akt were increased (P<0.05). Conclusion GDF15 can promote bone metastasis of lung adenocarcinoma in nude mice, and its mechanism is related to inhibition of PTEN/PI3K/AKT signaling pathway.
Objective To explore the effect of natural hirudin on proliferation of human microvascular endothelial cells (HMVECs) and its preliminary mechanism of promoting angiogenesis. Methods Three-dimensional culture models of HMVECs were established in vitro and observed by inverted phase contrast microscopy after 24 hours of culturing. Then, the three-dimensional culture models of HMVECs were treated with different concentrations (1, 4, and 7 ATU/mL) of the natural hirudin, respectively, and Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum as control. The cell proliferations of 4 groups were detected by cell counting kit 8 (CCK-8) method at 24, 48, and 72 hours; the angiogenesis of 4 groups were observed by tube formation assay at 24 hours; the expressions of vascular endothelial growth factor (VEGF) and Notch1 of HMVECs in 4 groups were observed by immunofluorescence staining at 24 hours. Results The observation of cells in three-dimensional culture models showed that HMVECs attached to Matrigel well, and the cells formed tube structure completely after 24 hours. The results of CCK-8 test showed that the absorbance (A) value of 1 and 4 ATU/mL groups were higher than that of control group at each time point (P<0.05), andA value of 4 ATU/mL group was the highest. The A value of 7 ATU/mL group was significantly lower than those of 1 and 4 ATU/mL groups and control group (P<0.05). The tube formation assay showed that the tube structure was more in 1 and 4 ATU/mL groups than in 7 ATU/mL group and control group, and in 4 ATU/mL group than in 1 ATU/mL group, showing significant differences (P<0.05). There was no significant difference between 7 ATU/mL group and control group (P>0.05). The results of immunofluorescence staining showed that compared with control group, the Notch1 expression was higher in 1 and 4 ATU/mL groups and lower in 7 ATU/mL group; and there was significant difference between 4 and 7 ATU/mL groups and control group (P<0.05). The VEGF expression was higher in 1, 4, and 7 ATU/mL groups than in control group, in 4 ATU/mL group than in 1 and 7 ATU/mL groups, showing significant differences (P<0.05). Conclusion Natural hirudin can promote angiogenesis at low and medium concentrations, but suppress angiogenesis at high concentrations. Its mechanism may be related to the VEGF-Notch signal pathway.
ObjectiveTo investigate the expression pattern and significance of Sonic Hedgehog (Shh) signaling pathway by observing whether the Shh signaling pathway components express in the adult rat after spinal cord injury (SCI). MethodsSixty-four healthy male Sprague-Dawley rats were randomly divided into normal group (group A, 8 rats), sham group (group B, 8 rats), and SCI group (group C, 48 rats). In group A, the rats served as controls without any treatment; a decompressive laminectomy was performed on T7-9 levels without SCI in group B; and modified Allen's method was used to make SCI model in group C. Basso Beattie Bresnahan (BBB) scale was used to assess the hind limb motor function at 12 hours, 1 day, 3 days, 7 days, 14 days, and 21 days after SCI; the immunofluorescence staining, real-time PCR, and Western blot were performed to detect the mRNA and protein expression levels of Shh and Glioma-associated oncogene homolog-1 (Gli-1) in SCI zone. ResultsThe BBB score slowly increased with time in group C, but the scores at each time point in group C were significantly lower than those in group A and group B (P<0.05). The results of immunofluorescence staining showed that Shh and Gli-1 rapidly increased after SCI in astrocytes. Real-time PCR and Western blot showed that the relative expression levels of Shh and Gli-1 mRNA and protein were gradually increased in group C and reached a maximum at 7 days. In addition, the relative expression levels of Shh and Gli-1 mRNA and protein in group C were significantly higher than those in group A and group B (P<0.05). On the other hand, compared with group A, the expression of Gli-1 protein was reduced in the cytoplasm but increased in nucleus in group C. ConclusionAstrocytes synthesize and secrete Shh and Gli-1 signaling molecules after SCI, both Shh and Gli-1 significantly up-regulate and exhibit dynamic changes, which suggests Shh signaling pathway may be involved in nerve cell regeneration after SCI.