BACKGROUND:β-Caryophyllene has a variety of pharmacological activities such as antioxidant,anti-inflammatory and anti-apoptotic,which may have a better therapeutic effect on osteoarthritis.OBJECTIVE:To investigate the effect and mechanism of β-caryophyllene on mouse osteoarthritis.METHODS:Forty C57BL/6J mice were randomly divided into sham group,model group,low-dose β-caryophyllene group and high-dose β-caryophyllene group,with 10 mice in each group.Hulth method was used to construct an osteoarthritis model in the latter three groups.Four weeks after modeling,70 and 140 mg/kg/d β-caryophyllene was intragastrically given in the low-and high-dose β-caryophyllene groups,respectively,and normal saline was given by gavage in the sham group and the model group,once a day,for 4 weeks.After administration,knee joint morphological changes were observed by hematoxylin-eosin staining,serum levels of inflammatory factors(tumor necrosis factor-α,interleukin-1β,interleukin-6,and interleukin-10)were detected by ELISA,and oxidative stress indexes(glutathione peroxidase,superoxide dismutase,and malondialdehyde)were detected by chemiluminescence.The expression levels of key proteins in the Sonic hedgehog(Shh)/glioma associated oncogene homolog 1(Gli1)signaling pathway were detected by immunohistochemistry and western blot.RESULTS AND CONCLUSION:(1)Compared with the sham group,a large number of inflammatory cells infiltrated in the knee joint of mice in the model group,cartilage tissue was seriously damaged,serum levels of tumor necrosis factor-α,interleukin-1β,interleukin-6,interleukin-10 and malondialdehyde were significantly increased(P＜0.01),the activities of glutathione peroxidase and superoxide dismutase were significantly decreased(P＜0.01),and the relative expression levels of Shh and Gli1 in the knee joint were significantly increased(P＜0.01).(2)Compared with the model group,in the low-and high-dose β-caryophyllene groups,inflammatory cell infiltration in the mouse knee joint was decreased,cartilage tissue injury was alleviated,serum levels of tumor necrosis factor-α,interleukin-1 β,interleukin-6 and malondialdehyde were significantly decreased(P＜0.05),the activities of glutathione peroxidase and superoxide dismutase were significantly increased(P＜0.01),and the expression levels of Shh and Gli1 in the knee joint were significantly decreased(P＜0.01).The above-mentioned improvements were more significant in the high-dose β-caryophyllene group than the low-dose β-caryophyllene group.To conclude,β-caryophyllene can improve osteoarthritis,and its mechanism may be related to reducing inflammation and oxidative stress damage by regulating the Shh/Gli1 signaling pathway.

BACKGROUND:β-Caryophyllene has a variety of pharmacological activities such as antioxidant,anti-inflammatory and anti-apoptotic,which may have a better therapeutic effect on osteoarthritis.OBJECTIVE:To investigate the effect and mechanism of β-caryophyllene on mouse osteoarthritis.METHODS:Forty C57BL/6J mice were randomly divided into sham group,model group,low-dose β-caryophyllene group and high-dose β-caryophyllene group,with 10 mice in each group.Hulth method was used to construct an osteoarthritis model in the latter three groups.Four weeks after modeling,70 and 140 mg/kg/d β-caryophyllene was intragastrically given in the low-and high-dose β-caryophyllene groups,respectively,and normal saline was given by gavage in the sham group and the model group,once a day,for 4 weeks.After administration,knee joint morphological changes were observed by hematoxylin-eosin staining,serum levels of inflammatory factors(tumor necrosis factor-α,interleukin-1β,interleukin-6,and interleukin-10)were detected by ELISA,and oxidative stress indexes(glutathione peroxidase,superoxide dismutase,and malondialdehyde)were detected by chemiluminescence.The expression levels of key proteins in the Sonic hedgehog(Shh)/glioma associated oncogene homolog 1(Gli1)signaling pathway were detected by immunohistochemistry and western blot.RESULTS AND CONCLUSION:(1)Compared with the sham group,a large number of inflammatory cells infiltrated in the knee joint of mice in the model group,cartilage tissue was seriously damaged,serum levels of tumor necrosis factor-α,interleukin-1β,interleukin-6,interleukin-10 and malondialdehyde were significantly increased(P＜0.01),the activities of glutathione peroxidase and superoxide dismutase were significantly decreased(P＜0.01),and the relative expression levels of Shh and Gli1 in the knee joint were significantly increased(P＜0.01).(2)Compared with the model group,in the low-and high-dose β-caryophyllene groups,inflammatory cell infiltration in the mouse knee joint was decreased,cartilage tissue injury was alleviated,serum levels of tumor necrosis factor-α,interleukin-1 β,interleukin-6 and malondialdehyde were significantly decreased(P＜0.05),the activities of glutathione peroxidase and superoxide dismutase were significantly increased(P＜0.01),and the expression levels of Shh and Gli1 in the knee joint were significantly decreased(P＜0.01).The above-mentioned improvements were more significant in the high-dose β-caryophyllene group than the low-dose β-caryophyllene group.To conclude,β-caryophyllene can improve osteoarthritis,and its mechanism may be related to reducing inflammation and oxidative stress damage by regulating the Shh/Gli1 signaling pathway.

In this study, serum metabolomics techniques and molecular biology methods were used to investigate the intervention effect of kaji-ichigoside F1 on chronic unpredictable mild stress(CUMS) depression mouse model and its mechanism. The CUMS depression mouse model was constructed, and the mice were divided into blank group, model group, escitalopram(ESC, 10 mg·kg~(-1)) group, and low-dose, medium-dose, and high-dose kaji-ichigoside F1 groups(1, 2, and 4 mg·kg~(-1)). CUMS modeling was performed on all mice except the blank group, and the cycle was four weeks. At the end of modelling, ESC and kaji-ichigoside F1 were administered by gavage once a day for 28 days. After the end of the administration, behavioral testing(sucrose preference test, open field test, forced swimming test, and tail suspension test) was conducted to evaluate the improvement of depression symptoms of different doses of kaji-ichigoside F1 on CUMS depression mouse model. The morphology of neurons and the number of Nissl bodies in the hippocampus were observed by Nissl staining. Metabolomics technique was used to analyze the changes in serum differential metabolites in mice. Protein expression levels of P2X7 purinergic receptor(P2X7R), adenosine A1 receptor(A1R), and adenosine receptor A2A(A2AR) in mouse hippocampus were detected by Western blot. The results showed that compared with that in the blank group, the body weight of mice in the model group was significantly decreased, and the sucrose preference rate was significantly decreased. The immobility time was significantly increased in the forced swimming and tail suspension tests, and the total moving distance was significantly decreased in the open field test. The number of Nissl bodies was significantly decreased, and the depression-like behavior and the number of Nissl bodies in the hippocampus of mice were significantly improved after administration of kaji-ichigoside F1. In the metabonomics analysis, the purine metabolism of serum after kaji-ichigoside F1 administration was involved in the metabolic passage of depression, and Western blot analysis verified the expression of P2X7R, A1R, and A2AR proteins in purine metabolic pathways. The results show that kaji-ichigoside F1 significantly decreases the expression of P2X7R and A2AR proteins in the hippocampus of CUMS model mice and increases the expression level of A1R proteins. It is suggested that kaji-ichigoside F1 may play an antidepressant role by regulating the expression of P2X7R, A1R, and A2AR proteins in the purine metabolism pathway.
Animals ; Mice ; Antidepressive Agents/administration & dosage* ; Metabolomics ; Depression/genetics* ; Male ; Drugs, Chinese Herbal/administration & dosage* ; Disease Models, Animal ; Hippocampus/metabolism* ; Behavior, Animal/drug effects* ; Humans

Prostate cancer is the second most common malignancy and the sixth leading cause of cancer-related death in men worldwide. Radical prostatectomy (RP) is the standard treatment for localized prostate cancer, but the procedure often results in postoperative erectile dysfunction (ED). The poor efficacy of phosphodiesterase 5 inhibitors after surgery highlights the need to develop new therapies to enhance cavernous nerve regeneration and improve the erectile function of these patients. In the present study, we aimed to examine the potential of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in preserving erectile function in cavernous nerve injury (CNI) mice. We found that HB-EGF expression was reduced significantly on the 1 st day after CNI in penile tissue. Ex vivo and in vitro studies showed that HB-EGF promotes major pelvic ganglion neurite sprouting and neuro-2a (N2a) cell migration. In vivo studies showed that exogenous HB-EGF treatment significantly restored the erectile function of CNI mice to 86.9% of sham levels. Immunofluorescence staining showed that mural and neuronal cells were preserved by inducing cell proliferation and reducing apoptosis and reactive oxygen species production. Western blot analysis showed that HB-EGF upregulated protein kinase B and extracellular signal-regulated kinase activation and neurotrophic factor expression. Overall, HB-EGF is a major promising therapeutic agent for treating ED in postoperative RP.
Animals ; Male ; Heparin-binding EGF-like Growth Factor/therapeutic use* ; Erectile Dysfunction/etiology* ; Mice ; Penis/drug effects* ; Nerve Regeneration/drug effects* ; Penile Erection/drug effects* ; Peripheral Nerve Injuries/drug therapy* ; Cell Proliferation/drug effects* ; Apoptosis/drug effects* ; Cell Movement/drug effects* ; Prostatectomy/adverse effects* ; Mice, Inbred C57BL ; Reactive Oxygen Species/metabolism*

OBJECTIVES: To characterize the biological properties of double-negative T (DNT) cells isolated from leukoreduction filter residues. METHODS: Leukoreduction filters containing residues from 400 mL whole blood units (n=6) were collected from a blood center. Filters were back-flushed with normal saline, and the eluate was concentrated to obtain leukoreduction filter residues. Leukocytes in the residues were counted by dual-fluorescence staining. DNT cells were then isolated from the residues using antibody-mediated adsorption and density gradient centrifugation. Both cryopreserved and fresh unstimulated DNT cells derived from the residues were subjected to in vitro culture. Following culture, cells were assessed for expansion fold, viability, immunophenotype, differentiation status, and cytotoxicity against target cells using dual-fluorescence staining and flow cytometry, with comparisons made to DNT cells derived from whole blood. RESULTS: The leukocyte recovery rate achieved through reverse flushing of the leukocyte reduction filter was (41.9±14.7)%. Compared to whole blood, the DNT cell starting material obtained from filter residues showed no significant difference in total T-cell content (P>0.05). However, the viability and purity of the resulting DNT cell starting materials were significantly lower (both P<0.05). After 17 days of culture, DNT cells from filter residues and whole blood showed no significant differences in expansion fold, immunophenotype, differentiation status, or cytotoxicity toward target cells (all P>0.05). However, the viability of DNT cells from residues was significantly lower than that of whole blood-derived DNT cells [(86.0±4.2)% vs. (92.2±1.2)%, P<0.05]. After thawing (post 3 or 15 days of cryopreservation) and 17 days of culture, DNT cell starting materials from residues showed comparable immunophenotype, expansion fold, and differentiation status to their non-cryopreserved counterparts from the same source (all P>0.05). However, the viability of DNT cells cryopreserved for 3 days [92.4% (91.8%, 92.8%)] and the cytotoxicity against target cells of those cryopreserved for 15 days [91.3% (89.4%, 95.1%)] were significantly higher than those of non-cryopreserved DNT cells [87.8% (82.0%, 89.0%) and 70.9% (67.3%, 80.2%), respectively] (P<0.05). CONCLUSIONS DNT cells derived from leukoreduction filter residues exhibited highly comparable characteristics to those from whole blood in terms of expansion, purity, differentiation, and biological potency. Furthermore, their biological activity post-cryopreservation and revival remained largely similar to non-cryopreserved cells. These findings suggest that leukoreduction filter residues represent a promising alternative source of starting material for manufacturing off-the-shelf, allogeneic DNT cell therapeutics.

OBJECTIVE: To explore the effect and mechanism of DNA methyltransferase 1 (DNMT1) knockout on the inhibition of acute myeloid leukemia (AML) by natural killer (NK) cells. METHODS: The peripheral blood NK cells of AML patients and controls were collected, and the mRNA and protein level of DNMT1 were measured by PCR and Western blot, respectively. The DNMT1 knockout mice were constructed to obtain NK^DNMT1-/- cells. The NK cells were stimulated with interleukin (IL)-12, IL-15, and IL-18 to construct memory NK cells, and then the interferon-γ (IFN-γ) levels were measured by ELISA. After co-culturing with memory NK cells and HL60 cells, the killing effect of NK^DNMT1-/- cells on HL60 cells was detected by LDH assay. Then, the HL60 cell apoptosis and NK cell NKG2D level were measured by flow cytometry. The perforin and granzyme B protein levels of NK cells were measured by Western blot. The AML model mice were constructed by injecting HL60 cells into the tail vein, meanwhile, memory NK cells were also injected, and then the mouse weights, CD33 positive rates, and survival time were detected. RESULTS: The mRNA and protein levels of DNMT1 in NK cells of AML patients were significantly higher than those in the control group (both P < 0.01), while the IFN-γ level induced by interleukin was significantly lower than that in the control group (P < 0.05). Compared with NK^DNMT1+/+ cells, the ability of NK^DNMT1-/- cells to secrete IFN-γ after interleukin stimulation was significantly increased (P < 0.05). The killing and apoptosis-inducing effects of NK^DNMT1-/- cells on HL60 cells were significantly stronger than those of NK^DNMT1+/+ cells (both P < 0.05). The NKG2D level and expression of perforin and granzyme B of NK^DNMT1-/- cells were significantly increased compared with NK^DNMT1+/+ cells (all P < 0.05). Compared with AML mice injected with NK^DNMT1+/+ cells, AML mice injected with NK^DNMT1-/- cells showed significantly increased body weight, decreased CD33 positive rate, and prolonged survival time (all P < 0.05). CONCLUSION Knocking out DNMT1 can enhance the inhibitory effect of NK cells on AML, which may be related to enhancing NK cell memory function.
Killer Cells, Natural/metabolism* ; Animals ; Leukemia, Myeloid, Acute ; Humans ; DNA (Cytosine-5-)-Methyltransferase 1 ; Mice ; Mice, Knockout ; HL-60 Cells ; Apoptosis ; Interferon-gamma/metabolism* ; Granzymes/metabolism* ; Perforin/metabolism* ; NK Cell Lectin-Like Receptor Subfamily K/metabolism*

OBJECTIVE: To assess the efficacy of Qingda Granule (QDG) in ameliorating hypertension-induced cardiac damage and investigate the underlying mechanisms involved. METHODS: Twenty spontaneously hypertensive rats (SHRs) were used to develope a hypertension-induced cardiac damage model. Another 10 Wistar Kyoto (WKY) rats were used as normotension group. Rats were administrated intragastrically QDG [0.9 g/(kg•d)] or an equivalent volume of pure water for 8 weeks. Blood pressure, histopathological changes, cardiac function, levels of oxidative stress and inflammatory response markers were measured. Furthermore, to gain insights into the potential mechanisms underlying the protective effects of QDG against hypertension-induced cardiac injury, a network pharmacology study was conducted. Predicted results were validated by Western blot, radioimmunoassay immunohistochemistry and quantitative polymerase chain reaction, respectively. RESULTS: The administration of QDG resulted in a significant decrease in blood pressure levels in SHRs (P<0.01). Histological examinations, including hematoxylin-eosin staining and Masson trichrome staining revealed that QDG effectively attenuated hypertension-induced cardiac damage. Furthermore, echocardiography demonstrated that QDG improved hypertension-associated cardiac dysfunction. Enzyme-linked immunosorbent assay and colorimetric method indicated that QDG significantly reduced oxidative stress and inflammatory response levels in both myocardial tissue and serum (P<0.01). CONCLUSIONS Both network pharmacology and experimental investigations confirmed that QDG exerted its beneficial effects in decreasing hypertension-induced cardiac damage by regulating the angiotensin converting enzyme (ACE)/angiotensin II (Ang II)/Ang II receptor type 1 axis and ACE/Ang II/Ang II receptor type 2 axis.
Animals ; Drugs, Chinese Herbal/therapeutic use* ; Hypertension/pathology* ; Renin-Angiotensin System/drug effects* ; Rats, Inbred SHR ; Oxidative Stress/drug effects* ; Male ; Rats, Inbred WKY ; Blood Pressure/drug effects* ; Myocardium/pathology* ; Rats ; Inflammation/pathology*

The polymerase 1 and transcript release factor (PTRF)-cytoplasmic phospholipase A2 (cPLA2) phospholipid remodeling pathway facilitates tumor proliferation in glioma. Nevertheless, blockade of this pathway leads to the excessive activation of oncogenic receptors on the plasma membrane and subsequent drug resistance. Here, CD26/dipeptidyl peptidase 4 (DPP4) was identified through screening of CRISPR/Cas9 libraries. Suppressing PTRF-cPLA2 signaling resulted in the activation of the epidermal growth factor receptor (EGFR) pathway through phosphatidylcholine and lysophosphatidylcholine remodeling, which ultimately increased DPP4 transcription. In turn, DPP4 interacted with EGFR and prevented its ubiquitination. Linagliptin, a DPP4 inhibitor, facilitated the degradation of EGFR by blocking its interaction with DPP4. When combined with the cPLA2 inhibitor AACOCF3, it exhibited synergistic effects and led to a decrease in energy metabolism in glioblastoma cells. Subsequent in vivo investigations provided further evidence of a synergistic impact of linagliptin by augmenting the sensitivity of AACOCF3 and strengthening the efficacy of temozolomide. DPP4 serves as a novel target and establishes a constructive feedback loop with EGFR. Linagliptin is a potent inhibitor that promotes EGFR degradation by blocking the DPP4-EGFR interaction. This study presents innovative approaches for treating glioma by combining linagliptin with AACOCF3 and temozolomide.

OBJECTIVES: To investigate the therapeutic mechanism of nodakenin for Crohn's disease (CD)-like colitis in mice. METHODS: Using a colonic organoid model with lipopolysaccharide (LPS)- and ATP-induced pyroptosis, we investigated the effects of nodakenin on pyroptosis, intestinal barrier function and inflammatory response by detecting key pyroptosis-regulating factors and assessing changes in permeability and pro-inflammatory factors. In a mouse model of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced CD-like colitis, the therapeutic effect of nodakenin was evaluated by measuring changes in body weight, DAI score, colonic histopathologies, inflammation score, intestinal barrier function and intestinal epithelial cell pyroptosis. The mechanism of nodakenin protection against pyroptosis of intestinal epithelial cells was explored using network pharmacology analysis and in vivo and in vitro experiments. RESULTS: In LPS- and ATP-induced colonic organoids, treatment with nodakenin significantly inhibited the expressions of NLRP3, GSDMD-N, cleaved caspase-1 and caspase-11, improved intestinal FITC-dextran (FD4, 4000) permeability, and decreased the levels of IL-1β and IL-18. In the mouse model of TNBS-induced colitis, nodakenin treatment significantly alleviated weight loss, reduced DAI score, inflammatory cell infiltration and inflammation score, and decreased serum FD4 and I-FABP levels and bacteria translocation to the mesenteric lymph nodes, spleen and liver. The mice with nodakenin treatment had also lowered expressions of NLRP3, GSDMD-N, cleaved caspase-1 and caspase-11 in the intestinal mucosa. Network pharmacology analysis suggested that the inhibitory effect of nodakenin on colitis was associated with the PI3K/Akt pathway. In both the colonic organoid model and mouse models of colitis, nodakenin effectively inhibited the activation of the PI3K/Akt pathway, and the application of IGF-1, a PI3K/Akt pathway activator, strongly attenuated the protective effect of nodakenin against intestinal epithelial cell pyroptosis and intestinal barrier dysfunction. CONCLUSIONS Nodakenin protects intestinal barrier function and alleviates CD-like colitis in mice at least partly by inhibiting PI3K/Akt signaling to reduce intestinal epithelial cell pyroptosis.
Animals ; Pyroptosis/drug effects* ; Mice ; Trinitrobenzenesulfonic Acid ; Colitis/drug therapy* ; Epithelial Cells/drug effects* ; Intestinal Mucosa/cytology* ; Disease Models, Animal ; Coumarins/pharmacology* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Crohn Disease/drug therapy*

OBJECTIVES: To investigate the mechanism through which pinostrobin (PSB) alleviates dextran sulfate sodium (DSS)-induced colitis in mice. METHODS: C57BL/6 mice were randomized into control group, DSS model group, and PSB intervention (30, 60, and 120 mg/kg) groups. Colitis severity of the mice was assessed by examining body weight changes, disease activity index (DAI), colon length, and histopathology. The expressions of tight junction proteins ZO-1 and claudin-1 in the colon tissues were examined using immunofluorescence staining, and macrophage infiltration and polarization were analyzed with flow cytometry. ELISA and RT-qPCR were used for detecting the expressions of inflammatory factors (TNF‑α and IL-6) and chemokines (CCL₂, CXCL₁₀, and CX₃CL₁) in the colon tissues, and PI3K/AKT phosphorylation levels were analyzed with Western blotting. In cultured Caco-2 and RAW264.7 cells, the effect of PSB on CCL₂-mediated macrophage migration was assessed using Transwell assay. Network pharmacology analysis was performed to predict the key pathways that mediate the therapeutic effect of PSB. RESULTS: In DSS-induced mouse models, PSB at 60 mg/kg optimally alleviated colitis, shown by reduced weight loss and DAI scores and increased colon length. PSB treatment significantly upregulated ZO-1 and claudin-1 expressions in the colon tissues, inhibited colonic macrophage infiltration, and promoted the shift of macrophage polarization from M1 to M2 type. In cultured intestinal epithelial cells, PSB significantly inhibited PI3K/AKT phosphorylation and suppressed chemokine CCL₂ expression. PSB treatment obviously blocked CCL₂-mediated macrophage migration of RAW264.7 cells, which could be reversed by exogenous CCL₂. Network pharmacology analysis and rescue experiments confirmed PI3K/AKT and CCL₂ signaling as the core targets of PSB. CONCLUSIONS PSB alleviates DSS-induced colitis in mice by targeting intestinal epithelial PI3K/AKT signaling, reducing CCL₂ secretion, and blocking macrophage chemotaxis and migration, highlighting the potential of PSB as a novel natural compound for treatment of inflammatory bowel disease.
Animals ; Mice ; Mice, Inbred C57BL ; Phosphatidylinositol 3-Kinases/metabolism* ; Colitis/drug therapy* ; Dextran Sulfate ; Proto-Oncogene Proteins c-akt/metabolism* ; Macrophages ; Chemokine CCL2/metabolism* ; Humans ; Signal Transduction/drug effects* ; Caco-2 Cells ; RAW 264.7 Cells ; Epithelial Cells/drug effects* ; Intestinal Mucosa/metabolism*