OBJECTIVE: To study the in vitro and in vivo antitumor effects of the polysaccharide of Alocasia cucullata (PAC) and the underlying mechanism. METHODS: B16F10 and 4T1 cells were cultured with PAC of 40 µg/mL, and PAC was withdrawn after 40 days of administration. The cell viability was detected by cell counting kit-8. The expression of Bcl-2 and Caspase-3 proteins were detected by Western blot and the expressions of ERK1/2 mRNA were detected by quantitative real-time polymerase chain reaction (qRT-PCR). A mouse melanoma model was established to study the effect of PAC during long-time administration. Mice were divided into 3 treatment groups: control group treated with saline water, positive control group (LNT group) treated with lentinan at 100 mg/(kg·d), and PAC group treated with PAC at 120 mg/(kg·d). The pathological changes of tumor tissues were observed by hematoxylin-eosin staining. The apoptosis of tumor tissues was detected by TUNEL staining. Bcl-2 and Caspase-3 protein expressions were detected by immunohistochemistry, and the expressions of ERK1/2, JNK1 and p38 mRNA were detected by qRT-PCR. RESULTS: In vitro, no strong inhibitory effects of PAC were found in various tumor cells after 48 or 72 h of administration. Interestingly however, after 40 days of cultivation under PAC, an inhibitory effect on B16F10 cells was found. Correspondingly, the long-time administration of PAC led to downregulation of Bcl-2 protein (P<0.05), up-regulation of Caspase-3 protein (P<0.05) and ERK1 mRNA (P<0.05) in B16F10 cells. The above results were verified by in vivo experiments. In addition, viability of B16F10 cells under long-time administration culture in vitro decreased after drug withdrawal, and similar results were also observed in 4T1 cells. CONCLUSIONS Long-time administration of PAC can significantly inhibit viability and promote apoptosis of tumor cells, and had obvious antitumor effect in tumor-bearing mice.
Mice ; Animals ; Alocasia/metabolism* ; MAP Kinase Signaling System ; Caspase 3/metabolism* ; Apoptosis ; RNA, Messenger/metabolism*

Objective: To investigate the therapeutic effect and mechanism of Liangge Powder against sepsis-induced acute lung injury (ALI) . Methods: From April to December 2021, the key components of Liangge Powder and its targets against sepsis-induced ALI were analyzed by network pharmacology, and to enrich for relevant signaling pathways. A total of 90 male Sprague-Dawley rats were randomly assigned to sham-operated group, sepsis-induced ALI model group (model group), Liangge Powder low, medium and high dose group, ten rats in the sham-operated group and 20 rats in each of the remaining four groups. Sepsis-induced ALI model was established by cecal ligation and puncture. Sham-operated group: gavage with 2 ml saline and no surgical treatment. Model group: surgery was performed and 2 ml saline was gavaged. Liangge Powder low, medium and high dose groups: surgery and gavage of Liangge Powder 3.9, 7.8 and 15.6 g/kg, respectively. To measure the wet/dry mass ratio of rats lung tissue and evaluate the permeability of alveolar capillary barrier. Lung tissue were stained with hematoxylin and eosin for histomorphological analysis. The levels of tumor necrosis factor-alpha (TNF-α), interleukin (IL) -6 and IL-1β in bronchoalveolar lavage fluid (BALF) were measured by enzyme-linked immunosorbent assay. The relative protein expression levels of p-phosphatidylinositol 3-kinase (PI3K), p-protein kinase B (AKT), and p-ertracellular regulated protein kinases (ERK) were detected via Western blot analysis. Results: Network pharmacology analysis indicated that 177 active compounds of Liangge Powder were selected. A total of 88 potential targets of Liangge Powder on sepsis-induced ALI were identified. 354 GO terms of Liangge Powder on sepsis-induced ALI and 108 pathways were identified using GO and KEGG analysis. PI3K/AKT signaling pathway was recognized to play an important role for Liangge Powder against sepsis-induced ALI. Compared with the sham-operated group, the lung tissue wet/dry weight ratio of rats in the model group (6.35±0.95) was increased (P<0.001). HE staining showed the destruction of normal structure of lung tissue. The levels of IL-6 [ (392.36±66.83) pg/ml], IL-1β [ (137.11±26.83) pg/ml] and TNF-α [ (238.34±59.36) pg/ml] were increased in the BALF (P<0.001, =0.001, <0.001), and the expression levels of p-PI3K, p-AKT and p-ERK1/2 proteins (1.04±0.15, 0.51±0.04, 2.31±0.41) were increased in lung tissue (P=0.002, 0.003, 0.005). The lung histopathological changes were reduced in each dose group of Liangge Powder compared with the model group. Compared with the model group, the wet/dry weight ratio of lung tissue (4.29±1.26) was reduced in the Liangge Powder medium dose group (P=0.019). TNF-α level [ (147.85±39.05) pg/ml] was reduced (P=0.022), and the relative protein expression levels of p-PI3K (0.37±0.18) and p-ERK1/2 (1.36±0.07) were reduced (P=0.008, 0.017). The wet/dry weight ratio of lung tissue (4.16±0.66) was reduced in the high-dose group (P=0.003). Levels of IL-6, IL-1β and TNF-α[ (187.98±53.28) pg/ml, (92.45±25.39) pg/ml, (129.77±55.94) pg/ml] were reduced (P=0.001, 0.027, 0.018), and relative protein expression levels of p-PI3K, p-AKT and p-ERK1/2 (0.65±0.05, 0.31±0.08, 1.30±0.12) were reduced (P=0.013, 0.018, 0.015) . Conclusion: Liangge Powder has therapeutic effects in rats with sepsis-induced ALI, and the mechanism may be related to the inhibition of ERK1/2 and PI3K/AKT pathway activation in lung tissue.
Male ; Animals ; Rats ; Rats, Sprague-Dawley ; Proto-Oncogene Proteins c-akt ; Phosphatidylinositol 3-Kinase ; Phosphatidylinositol 3-Kinases ; Powders ; Animal Experimentation ; Interleukin-6 ; MAP Kinase Signaling System ; Network Pharmacology ; Tumor Necrosis Factor-alpha ; Acute Lung Injury/drug therapy* ; Sepsis/drug therapy*

OBJECTIVE: To investigate the effect of midazolam on pain in lumbar disc herniation model rats based on p38 MAPK signaling pathway. METHODS: Fifty SPF-grade Sprague-Dawley healthy rats, half male and half female, were selected and randomly divided into normal group, model group, and low-dose, medium-dose, high-dose groups. Model group and low-dose, medium-dose, high-dose groups were initially modeled for lumbar disc herniation. Intraperitoneal injection of saline was performed in rats of normal and model groups; and in the low-dose, medium-dose, and high-dose groups, intraperitoneal injection of midazolam was performed with doses of 30, 60, and 90 mg/kg, respectively. Interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), 5-hydroxytryptamine (5-HT), β-endorphin (β-EP), substance P (SP), neuropeptide Y (NPY) were detected in the serum of rats by enzyme-linked immunoassay. The expression of p38 MAPK and matrix metalloproteinase-3(MMP-3) protein were detected by Western blot in the tissues of rats of each group. RESULTS: The levels of TNF-α, IL-1β and β-EP were higher and the level of 5-HT was lower in the model group than in the normal group(P<0.05);the levels of TNF-α, IL-1β and β-EP were lower and the level of 5-HT was higher in the low-dose, medium-dose and high-dose groups than in the model group(P<0.05). The levels of SP and NPY increased in the model group compared with the normal group (P<0.05) and the levels of SP and NPY decreased in the low-dose, medium-dose and high-dose groups compared with the model group (P<0.05). The expression of p38 MAPK and MMP-3 increased in the model group compared with the normal group (P<0.05); the expression of p38 MAPK and MMP-3 decreased in the low-dose, medium-dose and high-dose compared with the model group(P<0.05). CONCLUSION Midazolam may ameliorate the immune inflammatory response in rats with a model of lumbar disc herniation, possibly regulated through the p38MAPK signaling pathway.
Rats ; Male ; Female ; Animals ; Intervertebral Disc Displacement/pathology* ; Rats, Sprague-Dawley ; Matrix Metalloproteinase 3/metabolism* ; Midazolam ; Tumor Necrosis Factor-alpha/metabolism* ; Serotonin/metabolism* ; MAP Kinase Signaling System/physiology* ; Pain ; p38 Mitogen-Activated Protein Kinases/metabolism*

Cardiac fibrosis is a cause of morbidity and mortality in people with heart disease. Anti-fibrosis treatment is a significant therapy for heart disease, but there is still no thorough understanding of fibrotic mechanisms. This study was carried out to ascertain the functions of cytokine receptor-like factor 1 (CRLF1) in cardiac fibrosis and clarify its regulatory mechanisms. We found that CRLF1 was expressed predominantly in cardiac fibroblasts. Its expression was up-regulated not only in a mouse heart fibrotic model induced by myocardial infarction, but also in mouse and human cardiac fibroblasts provoked by transforming growth factor-‍β1 (TGF‍-‍β1). Gain- and loss-of-function experiments of CRLF1 were carried out in neonatal mice cardiac fibroblasts (NMCFs) with or without TGF-‍β1 stimulation. CRLF1 overexpression increased cell viability, collagen production, cell proliferation capacity, and myofibroblast transformation of NMCFs with or without TGF‍-‍β1 stimulation, while silencing of CRLF1 had the opposite effects. An inhibitor of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway and different inhibitors of TGF-‍β1 signaling cascades, comprising mothers against decapentaplegic homolog (SMAD)‍-dependent and SMAD-independent pathways, were applied to investigate the mechanisms involved. CRLF1 exerted its functions by activating the ERK1/2 signaling pathway. Furthermore, the SMAD-dependent pathway, not the SMAD-independent pathway, was responsible for CRLF1 up-regulation in NMCFs treated with TGF-‍β1. In summary, activation of the TGF-‍β1/SMAD signaling pathway in cardiac fibrosis increased CRLF1 expression. CRLF1 then aggravated cardiac fibrosis by activating the ERK1/2 signaling pathway. CRLF1 could become a novel potential target for intervention and remedy of cardiac fibrosis.
Animals ; Humans ; Mice ; Disease Models, Animal ; Fibroblasts/metabolism* ; Fibrosis ; MAP Kinase Signaling System ; Mitogen-Activated Protein Kinase 3/metabolism* ; Myocardial Infarction/metabolism* ; Receptors, Cytokine/metabolism* ; Signal Transduction ; Transforming Growth Factor beta1/pharmacology*

Objective To investigate the neuroprotective effect of methylene blue on diabetic retinopathy in rats. Methods Thirty SD rats were randomly divided into blank, control and experimental groups. The control and experimental groups were induced with diabetes by streptozotocin (STZ) intraperitoneal injection. After 6 weeks of successful modeling, the experimental group received intravitreal injection of methylene blue at a dose of [0.2 mg/(kg.d)], while the control group received an equal amount of dimethyl sulfoxide (DMSO) intravitreal injection, both continuously injected for 7 days. ELISA was used to detect the levels of retinal superoxide dismutase (SOD), 8-iso-prostaglandin F2alpha (iPF2α) and interleukin-1β (IL-1β) in rats. Western blot analysis was used to detect the expression of retinal extracellular signal-regulated kinase 1/2 phosphorylation (p-ERK1/2) and phosphorylated protein kinase B (p-AKT), and PAS staining was used to detect retinal morphological changes. Results Compared with the blank group rats, the retinal SOD activity in the control and experimental group rats was significantly reduced. iPF2α, IL-1β and p-ERK1/2 level increased, while p-AKT level decreased. Compared with the control group, the SOD activity of the experimental group rats increased. iPF2α and IL-1β level went down, while p-ERK1/2 and p-AKT level went up significantly. The overall thickness of the retinal layer and the number of retinal ganglion cells were significantly reduced. Conclusion Methylene blue improves diabetic retinopathy in rats by reducing retinal oxidative stress and enhancing ERK1/2 and AKT phosphorylation.
Rats ; Animals ; Diabetic Retinopathy/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Mitogen-Activated Protein Kinase 3/metabolism* ; Interleukin-1beta/metabolism* ; Methylene Blue/pharmacology* ; Phosphorylation ; Rats, Sprague-Dawley ; MAP Kinase Signaling System ; Diabetes Mellitus, Experimental/drug therapy* ; Superoxide Dismutase/metabolism*

Activated fibroblasts and M2-polarized macrophages may contribute to the progression of pulmonary fibrosis by forming a positive feedback loop. This study was aimed to investigate whether fibroblasts and macrophages form this loop by secreting SDF-1 and TGF-β and the impacts of neotuberostemonine (NTS) and tuberostemonine (TS). Mice were intratracheally injected with 3 U·kg^-1 bleomycin and orally administered with 30 mg·kg^-1 NTS or TS. Primary pulmonary fibroblasts (PFBs) and MH-S cells (alveolar macrophages) were used in vitro. The animal experiments showed that NTS and TS improved fibrosis related indicators, inhibited fibroblast activation and macrophage M2 polarization, and reduced the levels of TGF-β and SDF-1 in alveolar lavage fluid. Cell experiments showed that TGF-β1 may activated fibroblasts into myofibroblasts secreting SDF-1 by activating the PI3K/AKT/HIF-1α and PI3K/PAK/RAF/ERK/HIF-1α pathways. It was also found for the first time that SDF-1 was able to directly polarize macrophages into M2 phenotype secreting TGF-β through the same pathways as mentioned above. Moreover, the results of the cell coculture confirmed that fibroblasts and macrophages actually developed a feedback loop to promote fibrosis, and the secretion of TGF-β and SDF-1 was crucial for maintaining this loop. NTS and TS may disturb this loop through inhibiting both the PI3K/AKT/HIF-1α and PI3K/PAK/RAF/ERK/HIF-1α pathways to improve pulmonary fibrosis. NTS and TS are stereoisomeric alkaloids with pyrrole[1,2-a]azapine skeleton, and their effect on improving pulmonary fibrosis may be largely attributed to their parent nucleus. Moreover, this study found that inhibition of both the AKT and ERK pathways is essential for maximizing the improvement of pulmonary fibrosis.
Animals ; Mice ; Pulmonary Fibrosis/metabolism* ; Transforming Growth Factor beta/pharmacology* ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; MAP Kinase Signaling System ; Alkaloids/pharmacology* ; Fibroblasts ; Macrophages/metabolism*

OBJECTIVE: To investigate whether the antihypertensive mechanism of electroacupuncture (EA) is associated with attenuating phenotype transformation of vascular smooth muscle cells (VSMCs) via phosphoinositide3-kinase (PI3K)/protein kinase B (Akt) and mitogen-activated protein kinase (MAPK) signaling pathways. METHODS: Eight Wistar-ktoyo (WKY) rats were set as normal blood pressure group (normal group). A total of 32 spontaneous hypertensive rats (SHRs) were randomly divided into 4 groups using random number tables: a model group, an EA group, an EA+PI3K antagonist group (EA+P group), and an EA+p38 MAPK agonist+extracellular signal-regulated kinase (ERK) agonist group (EA+M group) (n=8/group). SHRs in EA group, EA+P group and EA+M group received EA treatment 5 sessions per week for continuous 4 weeks, while rats in the normal and model groups were bundled in same condition. The systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP) of each rat was measured at 0 week and the 4th week. After 4-week intervention, thoracic aorta was collected for hematoxylin-eosin (HE) staining, immunohistochemistry [the contractile markers α-smooth muscle actin (α-SMA) and calponin and the synthetic marker osteopontin (OPN)] and Western blot [α-SMA, calponin, OPN, PI3K, phosphorylated-Akt (p-Akt), Akt, p-p42/44 ERK, total p42/44 ERK, p-p38 MAPK and total p38 MAPK]. RESULTS: EA significantly reduced SBP, DBP and MAP (P<0.01). HE staining showed that the wall thickness of thoracic aorta in EA group was significantly decreased (P<0.01). From results of immunohistochemistry and Western blot, EA increased the expression of α-SMA and calponin, and decreased the expression of OPN (P<0.01). In addition, the expression of PI3K and p-Akt increased (P<0.01), while the expression of p-p42/44 ERK and p-p38 MAPK decreased in EA group (P<0.01). However, these effects were reversed by PI3K antagonist, p38 MAPK agonist and ERK agonist. CONCLUSIONS EA was an effective treatment for BP management. The antihypertensive effect of EA may be related with inhibition of phenotypic transformation of VSMCs, in which the activation of PI3K/Akt and the repression of MAPK pathway were involved.
Animals ; Electroacupuncture ; Extracellular Signal-Regulated MAP Kinases/metabolism* ; MAP Kinase Signaling System ; Muscle, Smooth, Vascular ; Phenotype ; Phosphatidylinositol 3-Kinases/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Rats ; Rats, Inbred SHR

To investigate the protective effect of Tongqiao Huoxue Decoction containing cerebrospinal fluid(TQHXD-CSF) on HT22 cells damaged by oxygen-glucose deprivation/reoxygenation(OGD/R) and whether the mechanism is related to the regulation of ASK1/MKK4/JNK signaling pathway. HT22 cells were subjected to OGD/R to simulate cerebral ischemia-reperfusion injury(CIRI). Then the cells were randomly divided into five groups: blank cerebrospinal fluid(control group), OGD/R group, TQHXD-CSF group, Z-VAD-FMK group(20 μmol·L~(-1)) and TQHXD-CSF+Z-VAD-FMK group. Except the control group, cells in the other groups were reoxygenated for 12 h after 6 h of oxygen and glucose deprivation for modeling OGD/R, and group administration was performed. Cell viability and cytotoxicity were detected by CCK8 and LDH assay kit, respectively and the morphology of HT22 cells was observed by inverted microscope. Western blot and qRT-PCR were employed to detect the protein and mRNA expression levels of Bax, Bcl-2 and caspase-3, respectively. Then HT22 cells were assigned into the control group, OGD/R group, si-NC group, si-ASK1 group, TQHXD-CSF group and TQHXD-CSF+si-ASK1 group. Cell viability, proliferation and apoptosis were determined by CCK8, electric cell-substrate impedance sensing(ECIS), and Hoechst staining and flow cytometry, respectively. The protein expression of MKK4, p-MKK4, JNK, p-JNK, c-Jun, p-c-Jun, Cyt C, Bax, Bcl-2 and caspase-3 was tested by Western blot. The results showed that compared with OGD/R group, TQHXD-CSF significantly enhanced cell viability, improved cell morphology and reduced the protein and mRNA expression levels of Bax, Bcl-2 and caspase-3. In addition, when ASK1 was silenced, compared with OGD/R group, TQHXD-CSF remarkably improved cell viability, and decreased apoptosis rate and the protein expression levels of p-MKK4, p-JNK, p-c-Jun, Cyt C, Bax/Bcl-2 and caspase-3, but the effect was not as good as that of TQHXD-CSF+si-ASK1 group. In conclusion, TQHXD-CSF can inhibit apoptosis mediated by ASK1/MKK4/JNK signaling pathway in OGD/R-damaged HT22 cells, and has protective effect on ischemia-reperfusion injury.
Humans ; Apoptosis ; bcl-2-Associated X Protein/metabolism* ; Caspase 3/metabolism* ; Glucose ; MAP Kinase Signaling System ; Oxygen/metabolism* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Reperfusion Injury/metabolism* ; RNA, Messenger/metabolism*

Deep venous thrombosis (DVT) poses a major challenge to public health worldwide. Endothelial cell injury evokes inflammatory and oxidative responses that contribute to thrombus formation. Tea polyphenol (TP) in the form of epigallocatechin-3-gallate (EGCG) has anti-inflammatory and oxidative effect that may ameliorate DVT. However, the precise mechanism remains incompletely understood. The current study was designed to investigate the anti-DVT mechanism of EGCG in combination with warfarin (an oral anticoagulant). Rabbits were randomly divided into five groups. A DVT model of rats was established through ligation of the inferior vena cava (IVC) and left common iliac vein, and the animals were orally administered with EGCG, warfarin, or vehicle for seven days. In vitro studies included pretreatment of human umbilical vein endothelial cells (HUVECs) with different concentrations of EGCG for 2 h before exposure to hydrogen peroxide. Thrombus weight and length were examined. Histopathological changes were observed by hematoxylin-eosin staining. Blood samples were collected for detecting coagulation function, including thrombin and prothrombin times, activated partial thromboplastin time, and fibrinogen levels. Protein expression in thrombosed IVCs and HUVECs was evaluated by Western blot, immunohistochemical analysis, and/or immunofluorescence staining. RT-qPCR was used to determine the levels of AGTR-1 and VEGF mRNA in IVCs and HUVECs. The viability of HUVECs was examined by CCK-8 assay. Flow cytometry was performed to detect cell apoptosis and ROS generation was assessed by 2',7'-dichlorofluorescein diacetate reagent. In vitro and invivo studies showed that EGCG combined with warfarin significantly reduced thrombus weight and length, and apoptosis in HUVECs. Our findings indicated that the combination of EGCG and warfarin protects HUVECs from oxidative stress and prevents apoptosis. However, HIF-1α silencing weakened these effects, which indicated that HIF-1α may participate in DVT. Furthermore, HIF-1α silencing significantly up-regulated cell apoptosis and ROS generation, and enhanced VEGF expression and the activation of the PI3K/AKT and ERK1/2 signaling pathways. In conclusion, our results indicate that EGCG combined with warfarin modifies HIF-1α and VEGF to prevent DVT in rabbits through anti-inflammation via the PI3K/AKT and ERK1/2 signaling pathways.
Animals ; Anticoagulants/pharmacology* ; Catechin/analogs & derivatives* ; Eosine Yellowish-(YS)/pharmacology* ; Fibrinogen/pharmacology* ; Hematoxylin/pharmacology* ; Human Umbilical Vein Endothelial Cells ; Humans ; Hydrogen Peroxide/pharmacology* ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism* ; MAP Kinase Signaling System ; Phosphatidylinositol 3-Kinases/metabolism* ; Polyphenols/pharmacology* ; Proto-Oncogene Proteins c-akt/metabolism* ; RNA, Messenger ; Rabbits ; Rats ; Reactive Oxygen Species/metabolism* ; Signal Transduction ; Sincalide/pharmacology* ; Tea ; Thrombin/pharmacology* ; Vascular Endothelial Growth Factor A/metabolism* ; Venous Thrombosis/pathology* ; Warfarin/pharmacology*

Amino Acid Substitution ; Antineoplastic Agents/pharmacology* ; Cell Line, Tumor ; Drug Resistance, Neoplasm/genetics* ; Gastrointestinal Neoplasms/pathology* ; Humans ; MAP Kinase Signaling System/genetics* ; Mutation, Missense ; Receptor, ErbB-3/metabolism* ; Receptor, Fibroblast Growth Factor, Type 1/metabolism*