The present study was designed to evaluate the anti-fatigue activity and the behavioral and biochemical effects of Kai-Xin-San (KXS) extracts on fatigued rats. The rats were randomly divided into six groups: untreated control (UC), running control (RC), RC treated with 13 mg/kg/day modafinil and RC treated with KXS at dosages of 125, 250 and 500 mg/kg/day, respectively. The treatments were administered orally. Anti-fatigue activity was assessed using the treadmill running test and serum biochemical parameters were determined using an autoanalyzer and commercially available kits. Furthermore, the standardization of the KXS extracts was ensured using a high-performance liquid chromatography (HPLC)-fingerprint. The extracts were shown to increase exhaustive running time in the treadmill running test and reverse the fatigue-induced reduction in hepatic/muscle glycogen and testosterone, in addition to reducing the lactate dehydrogenase (LDH), serum urea nitrogen (SUN), blood lactic acid (BLA) and β-endorphin levels in the serum of the fatigued rats. Moreover, the extracts enhanced superoxide dismutase (SOD) activity and decreased the malondialdehyde (MDA) levels in the serum of the fatigued rats. The results of this preliminary study indicated that KXS exhibits anti-fatigue activity. This was reflected in the effects on the biochemical markers for fatigue.

Previous studies have reported that excess activation of autophagy in cardiomyocytes is associated with an increase in myocardial oxygen-glucose deprivation/reperfusion (OGD/R) injury. Ozone therapy affords significant cardio-protection against myocardial OGD/R injury. The present study was designed to determine whether ozone-induced tolerance to myocardial OGD/R injury was mediated by inhibiting autophagy. Subsequently, the rat cardio myoblast H9C2 cell line was used in the present study. A model of H9C2 cells under OGD/R was established. The cells were incubated with different concentrations of ozone (10-60 µg/ml) during reperfusion. Furthermore, to investigate the role of autophagy in OGD/R-induced injury, the autophagy inducer and inhibitor were applied. Cell viability was detected by Cell Counting kit-8 assay. Cell apoptosis was evaluated by flow cytometry. Oxidative stress was examined by superoxide dismutase, lactate dehydrogenase and malondialdehyde levels. The expressions of apoptosis regulator B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (BAX), cleaved caspase-3, markers of autophagy microtuble-associated protein 1 light chain 3 (LC3), autophagy-related protein 5 (Atg5) and Beclin-1 were measured by western blot analysis. As a result, OGD/R notably decreased cell viability and induced apoptosis in H9C2 cells, while ozone (10-40 µg/ml) reversed the noxious effects of OGD/R on H9C2 cells, and 20 µg/ml ozone was the most effective. Ozone inhibited the decrease in the ratio of Bcl-2/BAX and the expression of cleaved caspase-3, and inhibited the increase in the ratio of LC3-II/LC3-I and the expression of Atg5 and Beclin-1 elicited by OGD/R, as well as dose-dependently preventing OGD/R-induced oxidative stress. Furthermore, rapamycin markedly reversed the effects of ozone (20 µg/ml) on OGD/R-induced expression of autophagy marker proteins and 3-methyladenine further improved the effect of ozone. Taken together, the results of the present study provided a credible mechanism by which ozone treatment at low concentrations could protect the myocardium from OGD/R-induced injury by inhibiting autophagy.

The aim of the present study was to investigate the effects of gallic acid (GA) on the proliferation and apoptosis of colon cancer cells and to further clarify the mechanism of GA function associated with SRC and EGFR phosphorylation. HCT116 and HT29 cells were treated with different concentrations of GA for 24 h. Cell proliferation and apoptosis were analyzed using plate clone formation and flow cytometry assays, respectively. In addition, the expression of apoptosis-related proteins was examined by western blotting. Furthermore, the level of STAT3, AKT, SRC and EGFR phosphorylation was analyzed by western blotting and immunofluorescence. Subsequently, the SRC inhibitor PP2 and the EGFR inhibitor gefitinib were used to analyze the GA-associated mechanisms. In addition, a xenograft tumor model was established to confirm the effects of GA in vivo. The results indicated that GA inhibited cell proliferation and promoted cell apoptosis by upregulating the ratio of cleaved caspase-3/pro-caspase-3 and cleaved caspase-9/pro-caspase-9. Concurrently, GA decreased the level of phosphorylated (p)-SRC, p-EGFR, p-AKT and p-STAT3. Following treatment with PP2 and gefitinib in both cancer cell lines and animal model, GA was demonstrated to inhibit EGFR and SRC phosphorylation to downregulate STAT3 and AKT phosphorylation. In vivo, GA prevented tumor growth, promoted tumor apoptosis and decreased the level of p-SRC, p-EGFR, p-STAT3 and p-AKT. In conclusion, GA was indicated to suppress colon cancer proliferation by inhibiting SRC and EGFR phosphorylation.

Long non-coding RNAs (lncRNAs) participate in the development of ovarian cancer (OC). The present study aimed to explore the roles of long intergenic non-protein coding RNA 1094 (LINC01094) in OC. LINC01094 and microRNA (miR)-532-3p expression in OC tissues and cells were measured using reverse transcription-quantitative PCR. Cell migration and invasion were detected using wound healing assays and Transwell assays, respectively. The binding of LINC01094 or β-catenin to miR-126-5p was detected using a Dual-luciferase reporter assay, and protein expression was confirmed using western blot analysis. The expression level of LINC01094 in patients with OC was higher in OC tissues compared with in adjacent tissues, and LINC01094 was upregulated in OC cell lines. In addition, LINC01094 overexpression promoted the viability, migration, invasion and cell cycle progression of OC cells, and inhibited OC cell apoptosis. Moreover, LINC01094 negatively regulated miR-532-3p in OC cells and tissues. miR-532-3p overexpression decreased the viability, migration, invasion and cell cycle progression of OC cells alongside downregulation of Wnt/β-catenin signaling pathway protein expression, as well as increasing OC cell apoptosis. Inhibition of LINC01094 with small interfering (si)-LINC01094 and overexpression of LINC01094 respectively reversed the effect of miR-532-3p inhibitor and mimics on OC cells. miR-532-3p could directly target β-catenin, and miR-532-3p inhibitor increased β-catenin expression, while si-LINC01094 attenuated this effect. In addition, LINC01094 overexpression promoted tumor growth in vivo by regulating miR-532-3p. Taken together, LINC01094 promoted the growth, migration, invasion and Wnt/β-catenin signaling pathway expression of OC cells by modulating miR-532-3p.

Protective role of lncRNA HOXB-AS3 in doxorubicin (DOX)-induced cardiotoxicity and its mechanism were studied. Viability of PC and H9c2 cells treated with different doses of DOX was determined through CCK-8 assay. Relative level of HOXB-AS3 in DOX-treated cardiomyocytes was detected. Regulatory effect of HOXB-AS3 on the proliferative ability of DOX-treated cardiomyocytes was assessed. Through dual-luciferase reporter gene assay, the binding relationship between HOXB-AS3 and miRNA-875-3p was verified. Rescue experiments were conducted to explore the role of HOXB-AS3/miRNA-875-3p in influencing the proliferation of DOX-treated cardiomyocytes. The proliferative ability of cardiomyocytes was dose-dependently downregulated after DOX treatment. Relative level of HOXB-AS3 was upregulated in DOX-treated cardiomyocytes. Silence of HOXB-AS3 in cardiomyocytes undergoing DOX treatment markedly elevated their proliferative ability. miRNA-875-3p was the direct target of HOXB-AS3. Knockdown of miRNA-875-3p reversed the role of HOXB-AS3 in regulating the proliferative ability of cardiomyocytes. HOXB-AS3 protects DOX-induced suppression in the proliferation of cardiomyocytes through targeting and downregulating miRNA-875-3p.

Kai-Xin-San (KXS) is a traditional Chinese medicine that has been widely used for the treatment of emotion-related disease. However, the underlying mechanism remains largely unknown. The present study aimed to examine whether phospho-cAMP response element-binding protein (pCREB) and upstream components, such as extracellular signal-regulated kinase (ERK), phospho-ERK (pERK), phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt), glycogen synthase kinase 3β (GSK3β) and pGSK3β are associated with the antidepressive effect of KXS. In total, 24 male Wistar rats were randomly divided into three groups, including control (n=8, no treatment), induced with chronic unpredictable mild stress (CMS) (n=8), and CMS rats treated with KXS at dosage of 370 mg/kg/day orally. Primary hippocampal neuronal cultures were prepared from Wistar rats for cell survival and proliferation assays. In KXS rats, increased protein expression levels of pCREB, BDNF and tyrosine receptor kinase B (TrkB) were observed in the hippocampus and prefrontal cortex, compared with the CMS model group. Furthermore, increased expression levels of ERK, pERK, PI3K, Akt, and GSK3β were also detected in the hippocampus and prefrontal cortex of KXS-treated rats compared with CMS model rats and in primary hippocampal neuronal cells treated with KXS. These results suggest that pCREB and upstream components, including TrkB/ERK/CREB and TrkB/PI3 K/CREB, may contribute to the antidepressive effect induced by KXS. Further studies are required to confirm these findings.

Brain-derived neurotrophic factor (BDNF), tropomyosin receptor kinase B (TrkB), vascular endothelial growth factor (VEGF) and CD105 are highly expressed in several types of cancer. The present study aimed to determine whether BDNF, TrkB, VEGF and CD105 are associated with the prognosis and metastasis of patients with cervical squamous cell carcinoma (SCC) at the IB2 stage. A total of 79 patients with IB2-stage SCC were enrolled in the present study. The expression levels of BDNF, TrkB, VEGF and CD105 in IB2-stage cervical cancer tissue were detected by immunohistochemistry and their association with clinicopathological indexes or prognostic factors was statistically analyzed. Reverse transcription quantitative PCR was used to detect whether the expression of VEGF was affected in SiHa cells co-cultured with BDNF. In addition, BDNF-induced SiHa cell migration and invasion were examined. BDNF expression in the cervical cancer samples was significantly associated with positive lymphovascular space invasion (P<0.001) and pelvic lymph node metastasis (P<0.05). In addition, microvessel density was verified as an independent prognostic factor for overall survival (P<0.05). In vitro analysis indicated that BDNF significantly induced cellular migration and invasion of SiHa cells in a dose-dependent manner (P<0.001). BDNF induced the expression of VEGF in SiHa cells, which was inhibited by BDNF antibodies or an inhibitor of TrkB receptor (P<0.05). BDNF may be considered a useful indicator of pelvic metastasis, which is involved in the aggressive spread of IB2-stage SCC. BDNF-induced upregulation of VEGF was revealed to act as a pro-angiogenic factor in SCC (Trial registration no. http://apps.who.int/trialsearch/; ChiCTR1800017778).

The aim of the present study was to investigate the effects and the underlying mechanisms of Yinchenhao Decoction (YCHD), a traditional Chinese medicine formulation, on C57BL/6 mice with lithogenic diet (LD)-induced cholelithiasis. The condition of cholelithiasis was evaluated using a six-level criteria. Levels of alanine aminotransferase (ALT), alkaline phosphatase (ALP), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) in the serum and liver tissue were measured using enzyme colorimetry. Concentrations of TC, phospholipids (PL) and total bile acids (TBA) in the bile were measured to calculate the cholesterol saturation index. Liver histopathology was microscopically observed and mRNA expression levels of ABCG5, ABCG8, SRBI, ABCB4, ABCB11 and NPC1L1 involved in cholesterol metabolism were measured using reverse transcription-quantitative PCR. The results showed that feeding mice the LD induced cholelithiasis, along with abnormal serum biochemical indices and imbalances in biliary cholesterol homeostasis. Increased ALT and ALP levels in the serum and ALT, ALP, TC and LDL-C levels in the serum and liver indicated the existence of hepatocyte injury, which were consistent with the pathological changes. YCHD treatment ameliorated the serum and hepatic biochemical abnormalities and adjusted the biliary imbalance. In addition, elevated expression of ATP-binding cassette subfamily G member 5/8, scavenger receptor class B type I and Niemann-Pick C1 Like 1 in the liver and small intestine were observed at the onset of cholelithiasis but were reversed by YCHD. Taken together, results from the present study suggest that YCHD ameliorated LD-induced cholelithiasis mice, which may be caused by improvements in biliary cholesterol supersaturation and regulation of cholesterol metabolism.

The aim of the present study was to investigate the roles of matrix metalloproteinase 14 (MMP-14) in corneal inflammatory lymphangiogenesis. The expression of MMP-14 in vivo was detected by immunohistochemistry, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot assays, under various corneal conditions. pCMV-MMP-14 or empty pCMV vectors were injected into mouse corneal stroma, 3 days after suture placement in a standard suture-induced inflammatory corneal neovascularization assay. The outgrowth of blood and lymphatic vessels and macrophage recruitment were analyzed using immunofluorescence. The expression levels of vascular endothelial growth factor (VEGF) subtypes were tested by RT-qPCR. MMP-14 expression was upregulated significantly following various corneal injuries. The results demonstrated, for the first time, that MMP-14 strongly promotes corneal lymphangiogenesis and macrophage infiltration during inflammation. Furthermore, expression levels of VEGF-C and VEGF receptor-3, but not other VEGF components, were significantly upregulated by the intrastromal delivery of MMP-14 during corneal lymphangiogenesis. In conclusion, this study indicates that MMP-14 is critically involved in the processes of lymphangiogenesis. Inhibition of MMP-14 may provide a viable treatment for transplant rejection and other lymphatic disorders.