20(S)-Ginsenoside Rh2 inhibits hepatocellular carcinoma by suppressing angiogenesis and the GPC3-mediated Wnt/ß­catenin signaling pathway.

20(S)-Ginsenoside Rh2 has significant anti-tumor effects in various types of cancers, including human hepatocellular carcinoma (HCC). However, its molecular targets and mechanisms of action remain largely unknown. Here, we aim to elucidate the potential mechanisms by which Rh2 suppresses HCC growth. We first demonstrate the role of Rh2 in inhibiting angiogenesis. We observe that Rh2 effectively suppresses cell proliferation and induces apoptosis in HUVECs. Furthermore, Rh2 significantly inhibits HepG2-stimulated HUVEC proliferation, migration and tube formation, accompanied by the downregulation of VEGF and MMP-2 expressions. We also reveal that Rh2 inhibits HCC growth through the downregulation of glypican-3-mediated activation of the Wnt/ß-catenin pathway. We observe a dose-dependent inhibition of proliferation and induction of apoptosis in HepG2 cells upon Rh2 treatment, which is mediated by the inhibition of glypican-3/Wnt/ß-catenin signaling. Moreover, downregulation of glypican-3 expression enhances the effects of Rh2 on the glypican-3/Wnt/ß-catenin signaling pathway, resulting in greater suppression of tumor growth in HepG2 cells. Collectively, our findings shed light on the molecular mechanisms through which Rh2 modulates HCC growth, which involve the regulation of angiogenesis and the glypican-3/Wnt/ß-catenin pathway. These insights may pave the way for the development of novel therapeutic strategies targeting these pathways for the treatment of HCC.

Integrated Bioinformatics Analysis for the Identification of Key lncRNAs, mRNAs, and Potential Drugs in Clear Cell Renal Cell Carcinomas.

Purpose: The overall survival of clear cell renal cell carcinoma (ccRCC) is poor. Markers for early detection and progression could improve disease outcomes. This study aims to reveal the potential pathogenesis of ccRCC by integrative bioinformatics analysis and to further develop new therapeutic strategies. Patients and Methods: RNA-seq data of 530 ccRCC cases in TCGA were downloaded, and a comprehensive analysis was carried out using bioinformatics tools. Another 14 tissue samples were included to verify the expression of selected lncRNAs by qRT-PCR. DGIdb database was used to screen out potential drugs, and molecular docking was used to explore the interaction and mechanism between candidate drugs and targets. Results: A total of 58 differentially expressed lncRNAs (DElncRNAs) and 660 differentially expressed mRNAs (DEmRNAs) were identified in ccRCC. LINC02038, FAM242C, LINC01762, and PVT1 were identified as the optimal diagnostic lncRNAs, of which PVT1 was significantly correlated with the survival rate of ccRCC. GO analysis of cell components showed that DEmRNAs co-expressed with 4 DElncRNAs were mainly distributed in the extracellular area and the plasma membrane, involved in the transport of metal ions, the transport of proteins across membranes, and the binding of immunoglobulins. Immune infiltration analysis showed that MDSC was the most correlated immune cells with PVT1 and key mRNA SIGLEC8. Validation analysis showed that GABRD, SIGLEC8 and CDKN2A were significantly overexpressed, while ESRRB, ELF5 and UMOD were significantly down-regulated, which was consistent with the expression in our analysis. Furthermore, 84 potential drugs were screened by 6 key mRNAs, of which ABEMACICLIB and RIBOCICLIB were selected for molecular docking with CDKN2A, with stable binding affinity. Conclusion: In summary, 4 key lncRNAs and key mRNAs of ccRCC were identified by integrative bioinformatics analysis. Potential drugs were screened for the treatment of ccRCC, providing a new perspective for disease diagnosis and treatment.

Advances in understanding the role and mechanisms of tumor stem cells in HER2-positive breast cancer treatment resistance (Review).

Approximately 15-20% of breast carcinomas exhibit human epidermal growth factor receptor (HER2) protein overexpression. HER2-positive breast cancer (BC) is a heterogeneous and aggressive subtype with poor prognosis and high relapse risk. Although several anti-HER2 drugs have achieved substantial efficacy, certain patients with HER2-positive BC relapse due to drug resistance after a treatment period. There is increasing evidence that BC stem cells (BCSCs) drive therapeutic resistance and a high rate of BC recurrence. BCSCs may regulate cellular self-renewal and differentiation, as well as invasive metastasis and treatment resistance. Efforts to target BCSCs may yield new methods to improve patient outcomes. In the present review, the roles of BCSCs in the occurrence, development and management of BC treatment resistance were summarized; BCSC-targeted strategies for the treatment of HER2-positive BC were also discussed.

Advances in Understanding the Roles of Mesenchymal Stem Cells in Lung Cancer.

Lung cancer is the most common and deadliest type of cancer worldwide. Research concerning lung cancer has made considerable progress in recent decades, but lung cancer remains the leading cause of malignancy-related mortality rate. Mesenchymal stem cells (MSCs) mainly exist in fat, umbilical cord blood, bone marrow, bone, and muscle. MSCs are a primary component of the tumor microenvironment (TME). Recent studies have shown that MSCs have roles in lung cancer-related proliferation, invasion, migration, and angiogenesis, but the underlying mechanisms are poorly understood. Because MSCs can migrate to the TME, there is increasing attention toward the use of MSCs in drugs or gene vectors for cancer treatment. This review summarizes the roles and effects of MSCs in lung cancer, while addressing clinical applications of MSCs in lung cancer treatment.

The promotion of active aging through older adult education in the context of population aging.

We have entered an era of population aging, and many public health problems associated with aging are becoming more serious. Older adults have earlier onset of chronic diseases and suffer more disability. Therefore, it is extremely important to promote active aging and enhance health literacy. These involves full consideration of the need for education and the provision of solutions to problems associated with aging. The development of OAE is an important measure for implementing the strategy of active aging, and curriculum construction is a fundamental component of achieving OAE. Various subjective and objective factors have limited the development of OAE. To overcome these difficulties and ensure both active and healthy aging, the requirements for active aging should be implemented, the limitations of current OAE should be addressed, system integration should be increased, and the curriculum system should be improved. These approaches will help to achieve the goal of active aging. This paper discusses OAE from the perspective of active aging, based on the promotion of health literacy and provides suggestions to protect physical and mental health among older adults, while promoting their social participation. The provision of various social guarantees for normal life in older adults is a new educational concept.

Overexpression of IRF3 Predicts Poor Prognosis in Clear Cell Renal Cell Carcinoma.

BACKGROUND: Growing findings have demonstrated that interferon regulatory transcription factor (IRF) family members are linked to the progression of various cancers. However, the roles of IRFs in clear cell renal cell carcinoma (ccRCC) remain undefined. Herein, we conducted a comprehensive analysis using the bioinformatics method to evaluate the expression patterns, clinical significance, and regulation of IRFs-related mechanisms in patients with ccRCC. METHODS: Data from the Cancer Genome Atlas (TCGA), International Cancer Genome Consortium (ICGA), and Gene Expression Omnibus (GEO) databases were used for investigation comprehensively. Specifically, we carried out a series of analyses to identify the candidate IRF and to explore its potential action mechanisms using the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. What is more, we emphatically investigate the association of candidate IRF with tumor immunity in ccRCC through the CIBERSORT algorithm, TIMER and GEPIA databases. RESULTS: Herein, IRF3 was identified as candidate IRF, which was highly expressed in ccRCC, and its overexpression was significantly associated with worse clinical outcomes and adverse overall survival. Uni- and multi-variate Cox regression analysis demonstrated that IRF3 overexpression was an independent predictor of worse prognosis. Functional enrichment analysis showed that IRF3 might participate in several cancer-related biological processes and signaling pathways, thereby promoting the progression of ccRCC. Additionally, we found that IRF3 was remarkably associated with tumor-infiltrating immune cells (TIICs) and various immune-related genes. CONCLUSION: Herein, we identified IRF3 from the IRF gene family members, which could serve as promising prognostic marker and therapeutic target in ccRCC.

[Influence of endoplasmic reticulum stress on smoking-induced nucleus pulposus cells apoptosis and inflammatory response].

OBJECTIVE: To investigate the influence of endoplasmic reticulum stress (ERS) on smoking-induced nucleus pulposus cells apoptosis and inflammatory response. METHODS: Between October 2016 and October 2018, 25 patients with cervical disc herniation receiving discectomy were collected and divided into smoking group (14 cases) and non-smoking group (11 cases). The baseline data of age, gender, herniated segment, and Pfirrmann grading showed no significant difference between the two groups ( P>0.05). The obtained nucelus pulposus tissues were harvested to observe the cell apoptosis via detecting the apoptosis-related proteins (Caspase-3 and PRAP) by TUNEL staining and Western blot test. The nucleus pulposus cells were isolated and cultured with enzyme digestion, of which the third generation cells were used in follow-up experiments. Then, the expressions of inflammatory factors [interleukin 1ß (IL-1ß) and tumor necrosis factor α (TNF-α)] were detected by ELISA; the nuclear translocation of P65 was monitored by cell immunofluorescence staining. Furthermore, ERS-related proteins (GRP78 and CHOP) were detected by Western blot; and endoplasmic reticulum ultrastructure was observed under transmission electron microscope. To verify the regulatory effect of ERS, cells were pretreated by ERS specific inhibitor (4-PBA), then cell apoptosis and inflammatory response were tested. RESULTS: The nucleus pulposus tissue observation showed that the cell apoptotic rate and the expressions of apoptosis-related proteins (Caspase-3 and PARP) were obviously higher in smoking group than in non-smoking group ( P<0.05). The nucleus pulposus cells observation indicated that the expressions of the inflammatory factors (IL-1ß and TNF-α) and the ERS-related proteins (GRP78 and CHOP) were also higher in smoking group than in non-smoking group ( P<0.05). The results of cell immunofluorescence staining further confirmed that smoking stimulated nuclear translocation of P65 in nucleus pulposus cells. The ERS injury was much more serious in smoking group than in non-smoking group. Furthermore, after 4-PBA inhibiting ERS, the expressions of GRP78, CHOP, IL-1ß, TNF-α, and P65 were significantly decreased ( P<0.05), and flow cytometry results showed that cell apoptotic rate in smoking group was decreased, showing significant difference compared with the non-smoking group ( P<0.05). CONCLUSION: Somking can stimulate cell apoptosis and inflammatory response in nucleus pulposus cells via ESR pathway. Suppressing ESR may be a novel target to suspend smoking-induced intervertebral disc degeneration.

[Effect of heme oxygenase 1 on the apoptosis of human degenerated nucleus pulposus cells induced by tumor necrosis factor α].

Objective: To investigate the effect of heme oxygenase 1 (HO-1) on the apoptosis of human degenerated nucleus pulposus (NP) cells induced by tumor necrosis factor α (TNF-α), and explore its possible molecular mechanism. Methods: The intervertebral disc tissues were derived from patients with lumbar intervertebral disc herniation. Then, the NP cells were cultured in vitro and the third generation of NP cells were used for subsequent experiments. Cell counting kit 8 (CCK-8) method was used to observe the proliferative effect of TNF-α on the NP cells in vitro at the concentration of 10, 20, 50, 100, and 200 ng/mL. The most apropriate concentration was selected according to the result of CCK-8. The NP cells were cultured with basal medium (control group), TNF-α (TNF-α group), TNF-α and CoPP 10 µmol/L (CoPP group), and TNF-α and ZnPP 15 µmol/L (ZnPP group), respectively. After cultured, the cell poptosis was detected by Hoechst staining and flow cytometry; the expression of cleaved Caspase-3, epithelial membrane protein 1 (EMP-1), HO-1, and p-P65 proteins were detected by Western blot. In order to further explore the potential molecular mechanisms of HO-1 for cell apoptosis, the NP cells were cultured with TNF-α (TNF-α stimulated group), TNF-α and pyrrolidine dithiocarbamate (PDTC) 5 µmol/L (TNF-α+PDTC stimulated group), respectively. Then the cell apoptosis rate was measured by flow cytometry at 24 hours after cultured. Results: The optimal concentration of TNF-α was 100 ng/mL. Hoechst staining showed that a few apoptotic cells could be observed in control group and CoPP group; the apoptosis-like nucleis were observed in TNF-α group and ZnPP group, which was the most significant in ZnPP group. Flow cytometry showed that the cell apoptosis rates of TNF-α group, CoPP group, and ZnPP group were significantly increased when compared with the control group ( P<0.05). Compared with TNF-α group, the cell apoptosis rate in CoPP group decreased ( P<0.05), while in ZnPP group it increased ( P<0.05). Western blot showed that the expression of HO-1 protein in TNF-α group was decreased, and the expressions of cleaved Caspase-3, EMP-1, and p-P65 proteins were increased when compared with the control group ( P<0.05). Compared with TNF-α group, the expression of HO-1 protein in CoPP group increased, and the expressions of cleaved Caspase-3, EMP-1, and p-P65 proteins were reduced ( P<0.05); the expression of HO-1 protein in ZnPP group decreased ( P<0.05), the expressions of cleaved Caspase-3 and EMP-1 proteins increased ( P<0.05), and the expression of p-P65 protein was not significantly changed ( P>0.05). Compared with TNF-α stimulated group, the cell apoptosis rate in TNF-α+PDTC stimulated group was significantly reduced ( t=3.076, P=0.031). Conclusion: HO-1 can inhibit the apoptosis of degerated NP cells induced by TNF-α, and its mechanism effect is by inhibiting the nuclear factor кB signaling pathway.

Resveratrol protects against mitochondrial dysfunction through autophagy activation in human nucleus pulposus cells.

Intervertebral disc degeneration (IVDD) is closely related with aging, whereas mitochondrial damage is a common feature of aging that results in cell apoptosis. Resveratrol (RES) is a natural antioxidant that protects against mitochondrial dysfunction in various cells. This study aimed to investigate the protective role of RES against mitochondrial dysfunction and human nucleus pulposus cell (NPC) apoptosis. We found that mitochondrial dysfunction and NPC apoptosis could be induced under oxidative stress by 100 µmol/l of H2O2. However, RES tended to attenuate the H2O2-mediated cytotoxicity. Therefore, autophagic state was evaluated in NPCs to further reveal the underlying mechanism. Results showed that RES reversed the impaired autophagy induced by H2O2, and this increased autophagic flux was confirmed by the addition of bafilomycin A1. Moreover, pretreatment with 3-methyladenine showed that the potential mechanism of RES to prevent deteriorating mitochondrial function and cell apoptosis was related to autophagy activation. Furthermore, MRI and histological detection were employed to provide more solid evidence that RES injection in an IVDD rabbit model effectively retards the degenerative process of the intervertebral discs in vivo. In summary, these results suggested that RES could alleviate mitochondrial dysfunction and cell apoptosis under oxidative stress and may delay the progression of disc degeneration, whose mechanism is associated with an advantageous role of autophagy induced by RES.

Risk Factors for the Failure of Spinal Burst Fractures Treated Conservatively According to the Thoracolumbar Injury Classification and Severity Score (TLICS): A Retrospective Cohort Trial.

BACKGROUND: The management of thoracolumbar (TL) burst fractures is still controversial. The thoracolumbar injury classification and severity score (TLICS) algorithm is now widely used to guide clinical decision making, however, in clinical practice, we come to realize that TLICS also has its limitations for treating patients with total scores less than 4, for which conservative treatment may not be optimal in all cases. PURPOSE: The aim of this study is to identify several risk factors for the failure of conservative treatment of TL burst fractures according to TLICS algorithm. METHODS: From June 2008 to December 2013, a cohort of 129 patients with T10-l2 TL burst fractures with a TLISC score ≤3 treated non-operatively were identified and included into this retrospective study. Age, sex, pain intensity, interpedicular distance (IPD), canal compromise, loss of vertebral body height and kyphotic angle (KA) were selected as potential risk factors and compared between the non-operative success group and the non-operative failure group. RESULTS: One hundred and four patients successfully completed non-operative treatment, the other 25 patients were converted to surgical treatment because of persistent local back pain or progressive neurological deficits during follow-up. Our results showed that age, visual analogue scale (VAS) score and IPD, KA were significantly different between the two groups. Furthermore, regression analysis indicated that VAS score and IPD could be considered as significant predictors for the failure of conservative treatment. CONCLUSION: The recommendation of non-operative treatment for TLICS score ≤3 has limitations in some patients, and VAS score and IPD could be considered as risk factors for the failure of conservative treatment. Thus, conservative treatment should be decided with caution in patients with greater VAS scores or IPD. If non-operative management is decided, a close follow-up is necessary.

The effect of L-carnosine on the circadian resetting of clock genes in the heart of rats.

It is reported that the circadian timing system may be included in the mechanism by which L-carnosine (Car) affects multiple physiological alterations including blood glucose, cardiovascular functions etc. However, it is not clear whether Car would affect the circadian rhythm of clock genes in the heart and what is the possible mechanism underlying. To clarify these issues, we compared the effects of Car on the expression of circadian genes in the heart of normal and vagotomized rats under control and jet lag conditions. The normal and vagotomized (va) male Wistar rats were divided into three groups respectively. The control and va-Control groups (fed with regular chow) were sampled before the reversal of LD cycle and feeding schedule (day 0). The normal and va-Normal resetting groups (fed with regular chow) as well as the Car and va-Car resetting groups (fed with Car-containing diet) were sampled on day 3 and day 5 after the experimental jet lag. Car-feeding obviously enhanced the resetting rates of clock genes (Bmal1, Dec1, Cry1) in the heart of normal rats after the experimental jet lag. The unilateral surgical vagotomy didn't alter the diurnal expression patterns and resetting rates of the examined clock genes in normal diet feeding rats. In contrast, it abolished the Car-induced rapid resetting of the clock genes in the heart. Therefore, Car feeding plays a positive role in the circadian resynchronization of the heart clock, which is underlied by the autonomic nervous system.

[Restoring phenotype of dedifferentiated normal nucleus pulposus cells by resveratrol].

OBJECTIVE: To investigate the effects of in-vitro monolayer culture and three-dimensional (3-D) alginate microsphere culture on the differentiation of normal human nucleus pulposus cells (NPCs), and to discuss the regulatory mechanism of restoring the phenotype of dedifferentiated NPCs by culturing resveratrol (RES) in 3-D alginate microsphere. METHODS: Normal human nucleus pulposus tissues were harvested for culture and identification of NPCs from 6 patients with burst lumbar vertebra fracture. NPCs at passages 1, 3, 5, and 7 in the in-vitro monolayer culture were harvested to observe the morphology, cell aging, and proteoglycan expression. The cell proliferation rates of NPCs at passage 1 in-vitro in monolayer culture and in 3-D alginate microsphere culture were detected. NPCs at passage 7 were randomly divided into 3-D alginate microsphere control group (group A), RES group (group B), silent mating type information regulation 2 homolog 1 (SIRT1)- small interfering RNA (siRNA) + RES group (group C), and negative control-siRNA + RES group (group D); and NPCs in the in-vitro monolayer culture was monolayer control group (group E). After corresponding treatment, Western blot was used for determining the protein expressions of SIRT1, Aggrecan, and collagen type II; real-time fluorescence quantitative PCR was used for detecting SIRT1 mRNA expression. RESULTS: The cultured cells were identified to be NPCs. Morphological observation, senescence-associated P-galactosidase (SA-P-gal) staining, and toluidine blue staining showed that dedifferentiation of normal NPCs tended to occur under continuous in-vitro monolayer culture, which was more obvious with increase of passage number. NPCs in 3-D alginate microsphere culture showed significantly lower proliferation rate than NPCs in the in-vitro monolayer culture (P < 0.05), but it could significantly improve the protein expressions of collagen type II and Aggrecan in dedifferentiated NPCs, showing significantly difference between groups E and A (P < 0.05). The protein expressions of SIRT1, collagen type II, and Aggrecan in group B were significantly improved when compared with that in group A (P < 0.05). Real-time fluorescence quantitative PCR and Western blot showed that the expressions of SIRT1 mRNA and proteins in group C were significantly inhibited after transfected with SIRT1-siRNA when compared with those in groups B and D (P < 0.05), and the protein expressions of collagen type II and Aggrecan in group C were significantly lower than those in groups B and D (P < 0.05). CONCLUSION: Continuous in-vitro monolayer culture could efficiently cultivate numerous seeding NPCs, but it is liable to dedifferentiate. In 3-D alginate microsphere culture, RES could restore the phenotype of dedifferentiated NPCs and synthesize more extracellular matrix, which is related to the regulation of SIRT1.

Adenovirus-Mediated th Gene Therapy of Parkinson's Disease in a Experimental Rat Model.

A recombinant adenovirus (AdCMV th) encoding tyrosine hydroxylase (TH) gene with CMV promoter was constructed and propagated. Southern blot analyses was used to identify positive plaques. Virus titer was about 1.4x10(14) pfu/L as determined by plaque forming assay. In glial cells infected with AdCMV th, the TH expression was demonstrated by immunohistochemical staining and HPLC-ECD. 678.8 ng DA was detected in the extract of 1x10(6) AdCMV th infected glial cells, but no detectable DA was found in AdCMVLacZ-infected glial cells. Injection of AdCMV th (1x10(7) pfu/rat) into the striatum of PD rats significantly improved the apomorphine-induced rotation movement(approximately 60%). The improvement in rotation movement remained up to 5 months after injection, and TH expression positive cells were found in the vicinity of injection. These results indicate that adenovirus may be a useful carrier for in vivo gene therapy in the PD patients.