Down-regulation of CYTL1 attenuates bleomycin-induced pulmonary fibrosis in mice by inhibiting M2 macrophage polarization via the TGF-ß/CCN2 axis.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by chronic inflammation, lung tissue fibrotic changes and impaired lung function. Pulmonary fibrosis 's pathological process is thought to be influenced by macrophage-associated phenotypes. IPF treatment requires specific targets that target macrophage polarization. Cytokine-like 1(CYTL1) is a secreted protein with multiple biological functions first discovered in CD34+ haematopoietic cells. However, its possible effects on IPF progression remain unclear. This study investigated the role of CYTL1 in IPF progression in a bleomycin-induced lung injury and fibrosis model. In bleomycin-induced mice, CYTL1 is highly expressed. Moreover, CYTL1 ablation alleviates lung injury and fibrosis in vivo. Further, downregulating CYTL1 reduces macrophage M2 polarization. Mechanically, CYTL1 regulates transforming growth factor ß (TGF-ß)/connective tissue growth factor (CCN2) axis and inhibition of TGF-ß pathway alleviates bleomycin-induced lung injury and fibrosis. In conclusion, highly expressed CYTL1 inhibits macrophage M2 polarization by regulating TGF-ß/CCN2 expression, alleviating bleomycin-induced lung injury and fibrosis. CYTL1 could, therefore, serve as a promising IPF target.

Prenatal methamphetamine hydrochloride exposure downregulates miRNA-151-3p and CACNA1C in testis rats' offspring.

Due to the widespread use of methamphetamine (METH) among reproductive-aged women, the effects of intrauterine exposure to METH need to be investigated, as previous studies on this topic have been limited. The goal of this study is to examine the influence of two regulatory genes (miRNA-151-3p and CACNA1C) on the intrauterine life of mice exposed to METH. Pregnant mice received doses of 2 and 5 mg/kg of METH and saline from day 10 of pregnancy until the end. Their offspring were then evaluated for miRNA-151-3p and CACNA1C gene expression levels using real-time PCR. The findings indicated that exposure to METH reduced the expression levels of both miRNA-151-3p and CACNA1C genes in offspring compared to the control group (p≤0.001). In conclusion, intrauterine exposure to METH leads to a decrease in expression levels of both miRNA-151-3p and CACNA1C genes, potentially disrupting regulatory pathways involving these genes and having an impact on male reproductive health.

p27 specifically decreases in squamous carcinoma, and mediates NNK-induced transformation of human bronchial epithelial cells.

Lung cancer remains the leading cause of cancer-related deaths, with cigarette smoking being the most critical factor, linked to nearly 90% of lung cancer cases. NNK, a highly carcinogenic nitrosamine found in tobacco, is implicated in the lung cancer-causing effects of cigarette smoke. Although NNK is known to mutate or activate certain oncogenes, its potential interaction with p27 in modulating these carcinogenic effects is currently unexplored. Recent studies have identified specific downregulation of p27 in human squamous cell carcinoma, in contrast to adenocarcinoma. Additionally, exposure to NNK significantly suppresses p27 expression in human bronchial epithelial cells. Subsequent studies indicates that the downregulation of p27 is pivotal in NNK-induced cell transformation. Mechanistic investigations have shown that reduced p27 expression leads to increased level of ITCH, which facilitates the degradation of Jun B protein. This degradation in turn, augments miR-494 expression and its direct regulation of JAK1 mRNA stability and protein expression, ultimately activating STAT3 and driving cell transformation. In summary, our findings reveal that: (1) the downregulation of p27 increases Jun B expression by upregulating Jun B E3 ligase ITCH, which then boosts miR-494 transcription; (2) Elevated miR-494 directly binds to 3'-UTR of JAK1 mRNA, enhancing its stability and protein expression; and (3) The JAK1/STAT3 pathway is a downstream effector of p27, mediating the oncogenic effect of NNK in lung cancer. These findings provide significant insight into understanding the participation of mechanisms underlying p27 inhibition of NNK induced lung squamous cell carcinogenic effect.

Human esophageal cancer stem-like cells escape the cytotoxicity of natural killer cells via down-regulation of ULBP-1.

BACKGROUND: Cancer stem-like cells (CSCs) play an important role in initiation and progression of aggressive cancers, including esophageal cancer. Natural killer (NK) cells are key effector lymphocytes of innate immunity that directly attack a wide variety of cancer cells. NK cell-based therapy may provide a new treatment option for targeting CSCs. In this study, we aimed to investigate the sensitivity of human esophageal CSCs to NK cell-mediated cytotoxicity. METHODS: CSCs were enriched from human esophageal squamous cell carcinoma cell lines via sphere formation culture. Human NK cells were selectively expanded from the peripheral blood of healthy donors. qRT-PCR, flow cytometry and ELISA assays were performed to examine RNA expression and protein levels, respectively. CFSE-labeled target cells were co-cultured with human activated NK cells to detect the cytotoxicity of NK cells by flow cytometry. RESULTS: We observed that esophageal CSCs were more resistant to NK cell-mediated cytotoxicity compared with adherent counterparts. Consistently, esophageal CSCs showed down-regulated expression of ULBP-1, a ligand for NK cells stimulatory receptor NKG2D. Knockdown of ULBP-1 resulted in significant inhibition of NK cell cytotoxicity against esophageal CSCs, whereas ULBP-1 overexpression led to the opposite effect. Finally, the pro-differentiation agent all-trans retinoic acid was found to enhance the sensitivity of esophageal CSCs to NK cell cytotoxicity. CONCLUSIONS: This study reveals that esophageal CSCs are more resistant to NK cells through down-regulation of ULBP-1 and provides a promising approach to promote the activity of NK cells targeting esophageal CSCs.

Hyperglycemia-induced Sirt3 downregulation increases microglial aerobic glycolysis and inflammation in diabetic neuropathic pain pathogenesis.

BACKGROUND: Hyperglycemia-induced neuroinflammation significantly contributes to diabetic neuropathic pain (DNP), but the underlying mechanisms remain unclear. OBJECTIVE: To investigate the role of Sirt3, a mitochondrial deacetylase, in hyperglycemia-induced neuroinflammation and DNP and to explore potential therapeutic interventions. METHOD AND RESULTS: Here, we found that Sirt3 was downregulated in spinal dorsal horn (SDH) of diabetic mice by RNA-sequencing, which was further confirmed at the mRNA and protein level. Sirt3 deficiency exacerbated hyperglycemia-induced neuroinflammation and DNP by enhancing microglial aerobic glycolysis in vivo and in vitro. Overexpression of Sirt3 in microglia alleviated inflammation by reducing aerobic glycolysis. Mechanistically, high-glucose stimulation activated Akt, which phosphorylates and inactivates FoxO1. The inactivation of FoxO1 diminished the transcription of Sirt3. Besides that, we also found that hyperglycemia induced Sirt3 degradation via the mitophagy-lysosomal pathway. Blocking Akt activation by GSK69093 or metformin rescued the degradation of Sirt3 protein and transcription inhibition of Sirt3 mRNA, which substantially diminished hyperglycemia-induced inflammation. Metformin in vivo treatment alleviated neuroinflammation and diabetic neuropathic pain by rescuing hyperglycemia-induced Sirt3 downregulation. CONCLUSION: Hyperglycemia induces metabolic reprogramming and inflammatory activation in microglia through the regulation of Sirt3 transcription and degradation. This novel mechanism identifies Sirt3 as a potential drug target for treating DNP.

2-methoxyestradiol sensitizes tamoxifen-resistant MCF-7 breast cancer cells via downregulating HIF-1α.

The clinical studies for breast cancer (BC) are now assessing the efficacy of 2-Methoxyestradiol (2-ME), a naturally occurring derivative of estradiol. Our study aimed to explore the potential of combining the 2-ME and tamoxifen (TAM) on sensitization of TAM-resistant cells using LCC2 the TAM-resistant cells as a model and comparing the results to the sensitive cells MCF-7. Sulphorhodamine-B (SRB) assay is used to examine the 2-ME chemo-sensitizing impact on the cytotoxicity of TAM on LCC2 cells. Colorimetric assay kits were used to assess the level of the apoptosis-related markers caspases 3, Bcl2, and Bax in cell lysate. Hypoxia-inducible factor 1 alpha (HIF-1α) expression was measured using western blotting. Total cholesterol and triglyceride (TG) levels were examined colorimetrically, using the BIOLABO kit. The use of 2-ME enhanced the cytotoxic effects of TAM and effectively reversed TAM resistance. This was achieved by inhibiting the expression of HIF-1α, while concurrently increasing the levels of apoptotic marker caspase-3, as well as the pro-apoptotic protein Bax. Additionally, there was a reduction in the levels of Bcl2, an anti-apoptotic protein. Furthermore, a reduction in TG and cholesterol levels was noted. Our findings show that HIF-1α plays an important role in TAM resistance and that suppression of HIF-1α by 2-ME-mediated sensitization of BC-resistant cells to TAM. Therefore, the concurrent administration of TAM/2-ME might potentially serve as a viable therapeutic approach to address TAM resistance and enhance the overall therapy efficacy for patients with BC.

AD16 attenuates neuroinflammation induced by cerebral ischemia through down-regulating astrocytes A1 polarization.

A1 polarization of astrocytes mediated prolonged inflammation contributing to brain injury in ischemic stroke. We have previously shown that AD16 protects against neonatal hypoxic-ischemic brain damage in vivo and oxygen-glucose deprivation in vitro. More recently, AD16 has demonstrated safety, tolerability, and favorable pharmacokinetics in a randomized controlled phase I trial. In this study, we utilized a rat model of transient middle cerebral artery occlusion (tMCAO) to explore whether the anti-inflammatory compound AD16 protects against ischemic brain injury by regulating A1 polarization and its underlying mechanisms. Our results showed that AD16 treatment significantly reduced the brain infarcted volume and improved neurological function in tMCAO rats. GO analysis results show that differential genes among the Sham, tMCAO and AD16 treatment groups are involved in the regulation of cytokine and inflammatory response. KEGG enrichment pathways analysis mainly enriched in cytokine-cytokine receptor interaction, viral protein interaction with cytokine-cytokine receptor, TNF, chemokine, NF-κB and IL-17 signaling pathway. Furthermore, AD16 treatment decreased the permeability of the blood-brain barrier and suppressed neuroinflammation. AD16 treatment also significantly reduced the polarization of A1 and inhibited NF-κB and JAK2/STAT3 signaling pathways. This study demonstrates that AD16 protects against brain injury in ischemic stroke by reducing A1 polarization to suppress neuroinflammation through downregulating NF-κB and JAK2/STAT3 signaling. Our findings uncover a potential molecular mechanism for AD16 and suggest that AD16 holds promising therapeutic potential against cerebral ischemia.

Arsenic-induced downregulation of BRWD3 suppresses proliferation and induces apoptosis in lung adenocarcinoma cells through the p53 and p65 pathways.

Bromodomain and WD-repeat domain-containing protein 3 (BRWD3) exhibits high expression in lung adenocarcinoma (LUAD) tissues and cells; however, its function in arsenic-induced toxicological responses remains unclear. This study aimed to investigate BRWD3 expression in response to arsenic-induced conditions and its impact on the proliferation and apoptosis of LUAD cell line SPC-A1 upon BRWD3 knockdown. The results revealed a decrease in BRWD3 expression in SPC-A1 cells treated with sodium arsenite (NaAsO2), but not sodium arsenite's metabolites. BRWD3 knockdown suppressed cell proliferation and induced apoptosis in SPC-A1 cells. Western blot analysis revealed that BRWD3 knockdown resulted in the upregulation of p53, phospho-p53-Ser392, and its downstream factors including MDM2, Bak, and Bax. Additionally, we observed the downregulation of p65, phospho-p65-Ser276, phospho-p65-Ser536, and its downstream factors, including IκBα, BIRC3, XIAP and CIAP1. Moreover, polymerase chain reaction analysis showed that BRWD3 knockdown also resulted in the downregulation of proliferation-related genes and upregulation of apoptosis-related genes. In conclusion, BRWD3 mediated proliferation and apoptosis via the p53 and p65 pathways in response to arsenic exposure, suggesting potential implications for LUAD treatment through BRWD3 downregulation by arsenic.

Clustering of the Membrane Protein by Molecular Self-Assembly Downregulates the Signaling Pathway for Cancer Cell Inhibition.

This work reports a cyclic peptide appended self-assembled scaffold that recognizes the membrane protein EGFR and arrests the EGFR signaling through multivalent interactions by assembly-induced aggregation. When incubated with cells, the oligomers of PAD-1 first recognize the overexpressed EGFR on cancer cell membranes for arresting EGFR, which then initiates cellular uptake through endocytosis. The accumulation of PAD-1 and EGFR in the lysosome results in the formation of nanofibers, leading to the lysosomal membrane permeabilization (LMP). These processes disrupt the homeostasis of EGFR and inhibit the downstream signaling transduction of EGFR for cancer cell survival. Moreover, LMP induced the release of protein aggregates that could generate endoplasmic reticulum (ER) stress, resulting in cancer cell death selectively. In vivo studies indicate the efficient antitumor efficiency of PAD-1 in tumor-bearing mice. As a first example, this work provides an alternative strategy for controlling protein behavior for tuning cellular events in living cells.

Lonicerin alleviates intestinal myenteric neuron injury induced by hypoxia/reoxygenation treated macrophages by downregulating EZH2.

Intestinal ischemia-reperfusion (IR) injury is a common gastrointestinal disease that induces severe intestinal dysfunction. Intestinal myenteric neurons participate in maintaining the intestinal function, which will be severely injured by IR. Macrophages are widely reported to be involved in the pathogenesis of organ IR injury, including intestine, which is activated by NLRP3 signaling. Lonicerin (LCR) is a natural extracted monomer with inhibitory efficacy against the NLRP3 pathway in macrophages. The present study aims to explore the potential protective function of LCR in intestinal IR injury. Myenteric neurons were extracted from mice. RAW 264.7 cells were stimulated by H/R with or without 10 µM and 30 µM LCR. Remarkable increased release of IL-6, MCP-1, and TNF-α were observed in H/R treated RAW 264.7 cells, along with an upregulation of NLRP3, cleaved-caspase-1, IL-1ß, and EZH2, which were sharply repressed by LCR. Myenteric neurons were cultured with the supernatant collected from each group. Markedly decreased neuron number and shortened length of neuron axon were observed in the H/R group, which were signally reversed by LCR. RAW 264.7 cells were stimulated by H/R, followed by incubated with 30 µM LCR with or without pcDNA3.1-EZH2. The inhibition of LCR on NLRP3 signaling in H/R treated RAW 264.7 cells was abolished by EZH2 overexpression. Furthermore, the impact of LCR on neuron number and neuron axon length in myenteric neurons in the H/R group was abated by EZH2 overexpression. Collectively, LCR alleviated intestinal myenteric neuron injury induced by H/R treated macrophages via downregulating EZH2.

Sitagliptin synergizes 5-fluorouracil efficacy in colon cancer cells through MDR1-mediated flux impairment and down regulation of NFκB2 and p-AKT survival proteins.

5-fluorouracil (5-FU) is an inexpensive treatment for colon cancer; however, its efficacy is limited by chemoresistance. This study investigates the combination therapy approach of 5-FU with Sitagliptin (Sita), a diabetic drug with potential cancer-modulating effects. The combination was evaluated in vitro and in silico, focusing on the effects of Sita and 5-FU on colon cancer cells. The results showed that the addition of Sita significantly decreased the IC50 of 5-FU compared to 5-Fu monotherapy. The study also found that Sita and 5-FU interact synergistically, with a combination index below 1. Sita successfully lowered the 5-FU dosage reduction index, decreasing the expression of MDR1 mRNA and p-AKT and NFκB2 subunits p100/p52 protein. Molecular docking analyses confirmed Sita's antagonistic action on MDR1 and thymidylate synthase proteins. The study concludes that sitagliptin can target MDR1, increase apoptosis, and significantly reduce the expression of p-AKT and NFκB2 cell-survival proteins. These effects sensitize colon cancer cells to 5-FU. Repurposing sitagliptin may enhance the anticancer effects of 5-FU at lower dosages.

Doxorubicin downregulates cell cycle regulatory hub genes in breast cancer cells.

Breast cancer (BC) is the leading commonly diagnosed cancer in the world, with complex mechanisms underlying its development. There is an urgent need to enlighten key genes as potential therapeutic targets crucial to advancing BC treatment. This study sought to investigate the influence of doxorubicin (DOX) on identified key genes consistent across numerous BC datasets obtained through bioinformatic analysis. To date, a meta-analysis of publicly available coding datasets for expression profiling by array from the Gene Expression Omnibus (GEO) has been carried out. Differentially Expressed Genes (DEGs) identified using GEO2R revealed a total of 23 common DEGs, including nine upregulated genes and 14 downregulated genes among the datasets of three platforms (GPL570, GPL6244, and GPL17586), and the commonly upregulated DEGs, showed significant enrichment in the cell cycle in KEGG analysis. The top nine genes, NUSAP1, CENPF, TPX2, PRC1, ANLN, BUB1B, AURKA, CCNB2, and CDK-1, with higher degree values and MCODE scores in the cytoscape program, were regarded as hub genes. The hub genes were activated in disease states commonly across all the subclasses of BC and correlated with the unfavorable overall survival of BC patients, as verified by the GEPIA and UALCAN databases. qRT-PCR confirmed that DOX treatment resulted in reduced expression of these genes in BC cell lines, which reinforces the evidence that DOX remains an effective drug for BC and suggests that developing modified formulations of doxorubicin to reduce toxicity and resistance, could enhance its efficacy as an effective therapeutic option for BC.

PM2.5 Exposure Inhibits Transepithelial Anion Short-circuit Current by Downregulating P2Y2 Receptor/CFTR Pathway.

Fine particulate matter (PM2.5) can damage airway epithelial barriers. The anion transport system plays a crucial role in airway epithelial barriers. However, the detrimental effect and mechanism of PM2.5 on the anion transport system are still unclear. In this study, airway epithelial cells and ovalbumin (OVA)-induced asthmatic mice were used. In transwell model, the adenosine triphosphate (ATP)-induced transepithelial anion short-circuit current (Isc) and airway surface liquid (ASL) significantly decreased after PM2.5 exposure. In addition, PM2.5 exposure decreased the expression levels of P2Y2R, CFTR and cytoplasmic free-calcium, but ATP can increase the expressions of these proteins. PM2.5 exposure increased the levels of Th2-related cytokines of bronchoalveolar lavage fluid, lung inflammation, collagen deposition and hyperplasisa of goblet cells. Interestingly, the administration of ATP showed an inhibitory effect on lung inflammation induced by PM2.5. Together, our study reveals that PM2.5 impairs the ATP-induced transepithelial anion Isc through downregulating P2Y2R/CFTR pathway, and this process may participate in aggravating airway hyperresponsiveness and airway inflammation. These findings may provide important insights on PM2.5-mediated airway epithelial injury.

Therapeutic effects of tilorone on mammary carcinogenesis through downregulation of pro-inflammatory cytokines and oxidative stress.

Tilorone dihydrochloride (tilorone) is an orally active interferon inducer with anticancer effects. The present study aimed to evaluate the anticancer effects of tilorone in breast cancer. MTT assay was done to measure the proliferation of MCF-7 and MDA-MB-231 breast cancer cells after treatment with tilorone. Mammary carcinogenesis was induced by subcutaneous injection (35 mg/kg, 0.5 mL) of dimethylbenz[a]anthracene (DMBA) in mammary pads of Sprague Dawley (SD) rats. Tumors were allowed to grow for 16 weeks till their sizes reached to 550-700 mm3, and then treated with 10 and 20 mg/kg of tilorone and standard drug doxorubicin (4 mg/kg) twice a week for 3 weeks. Normal and disease-control animals received normal saline. Tumor volumes and body weights were measured. Tumors were isolated to measure the levels of interferon-ß (IFN-ß), vascular endothelial growth factor-A (VEGF-A), P53 and inflammatory markers by enzyme-linked immunosorbent assay (ELISA). Serum biochemistry, lipid peroxidation (LPO) and antioxidant enzymes were measured by standard methods. Histopathology and immunohistochemistry (IHC) of P53 was done in tumor sections. Tilorone reduced the proliferation of MCF-7 and MDA-MB-231 cells with IC50 concentrations at 34.08 µM and 14.27 µM, respectively. Tilorone treatment showed reduced tumor volume, and increased survival with no significant changes in the body weights. Tilorone treatment also decreased levels of inflammatory markers and VEGF-A and increased IFN-ß and P53 levels. Further, treatment with tilorone also decreased LPO and increased antioxidants levels. Histopathology of tumor sections showed normalizing morphology of treated animals. IHC of tumor sections showed increased levels of P53. In conclusion, tilorone has potential anticancer effects against breast cancer.

Osteoblast-specific down-regulation of NLRP3 inflammasome by aptamer-functionalized liposome nanoparticles improves bone quality in postmenopausal osteoporosis rats.

Rationale: NLRP3 inflammasome is critical in the development and progression of many metabolic diseases driven by chronic inflammation, but its effect on the pathology of postmenopausal osteoporosis (PMOP) remains poorly understood. Methods: We here firstly examined the levels of NLRP3 inflammasome in PMOP patients by ELISA. Then we investigated the possible mechanisms underlying the effect of NLRP3 inflammasome on PMOP by RNA sequencing of osteoblasts treated with NLRP3 siRNA and qPCR. Lastly, we accessed the effect of decreased NLRP3 levels on ovariectomized (OVX) rats. To specifically deliver NLRP3 siRNA to osteoblasts, we constructed NLRP3 siRNA wrapping osteoblast-specific aptamer (CH6)-functionalized lipid nanoparticles (termed as CH6-LNPs-siNLRP3). Results: We found that the levels of NLRP3 inflammasome were significantly increased in patients with PMOP, and were negatively correlated with estradiol levels. NLRP3 knock-down influenced signal pathways including immune system process, interferon signal pathway. Notably, of the top ten up-regulated genes in NLRP3-reduced osteoblasts, nine genes (except Mx2) were enriched in immune system process, and five genes were related to interferon signal pathway. The in vitro results showed that CH6-LNPs-siNLRP3 was relatively uniform with a dimeter of 96.64 ± 16.83 nm and zeta potential of 38.37 ± 1.86 mV. CH6-LNPs-siNLRP3 did not show obvious cytotoxicity and selectively delivered siRNA to bone tissue. Moreover, CH6-LNPs-siNLRP3 stimulated osteoblast differentiation by activating ALP and enhancing osteoblast matrix mineralization. When administrated to OVX rats, CH6-LNPs-siNLRP3 promoted bone formation and bone mass, improved bone microarchitecture and mechanical properties by decreasing the levels of NLRP3, IL-1ß and IL-18 and increasing the levels of OCN and Runx2. Conclusion: NLRP3 inflammasome may be a new biomarker for PMOP diagnosis and plays a key role in the pathology of PMOP. CH6-LNPs-siNLRP3 has potential application for the treatment of PMOP.

Catalpol alleviates heat stroke-induced liver injury in mice by downregulating the JAK/STAT signaling pathway.

BACKGROUND: Heat stroke (HS) generated liver injury is a lethal emergency that occurs when the body is exposed to temperatures up to 40 °C for a few hours. PURPOSE: This study aimed to evaluate the therapeutic prospects of Catalpol (CA) from the blood-cooling herb Rehamanniae Radix on liver injury by HS. STUDY DESIGN AND METHODS: A murine HS model (41 ± 0.5 °C, 60 ± 5 % relative humidity) and two cell lines (lipopolysaccharide + 42 °C) were used to assess the protective effects of CA on physiological, pathological, and biochemical features in silico, in vivo, and in vitro. RESULTS: CA treatment significantly improved survival rates in vivo and cell viability in vitro over those of the untreated group. Additionally, CA treatment reduced core body temperature, enhanced survival time, and mitigated liver tissue damage. Furthermore, CA treatment also reduced the activities of AST and ALT enzymes in the serum samples of HS mice. Molecular docking analysis of the 28 overlapping targets between HS and CA revealed that CA has strong binding affinities for the top 15 targets. These targets are primarily involved in nine major signaling pathways, with the JAK-STAT pathway being highly associated with the other eight pathways. Our findings also indicate that CA treatment significantly downregulated the expression of proinflammatory cytokines both in vivo and in vitro while upregulating the expression of anti-inflammatory cytokines. Moreover, CA treatment reduced the levels of JAK2, phospho-STAT5, and phospho-STAT3 both in vivo and in vitro, which is consistent with its inhibition of the apoptotic markers p53, Bcl2, and Bax. CONCLUSIONS: Heat stroke-induced liver injury was inhibited by CA through the downregulation of JAK/STAT signaling.

Discovery of Novel HDAC3 Inhibitors with PD-L1 Downregulating/Degrading and Antitumor Immune Effects.

Targeting the programmed cell death-1/ligand 1 (PD-1/PD-L1) pathway is one of the most promising cancer treatment strategies. Studies have shown that HDAC inhibitors can enhance the antitumor immune response by modulating the expression of PD-L1. Herein, we designed and synthesized a series of novel hydrazide-based small molecule HDAC inhibitors; among them, compound HQ-30 showed selective HDAC3 inhibition (IC50 = 89 nM) and remarkable PD-L1-degrading activity (DC50 = 5.7 µM, Dmax = 80% at 10 µM). Further studies revealed that HQ-30 induced the degradation of PD-L1 by regulating cathepsin B (CTSB) in the lysosomes. Further, HQ-30 could enhance the infiltration of CD3+ CD4+ helper T and CD3+ CD8+ cytotoxic T cells in tumors, thus activating the tumor immune microenvironment. Moreover, HQ-30 possessed a benign toxicity profile (LD50 > 1000 mg/kg) and favorable pharmacokinetic properties (F = 57%). Taken together, HQ-30 is worthy of further investigation as a small molecule-based epigenetic modulator of tumor immunotherapy.

Supplementation of Vitamin D3 and Fructooligosaccharides Downregulates Intestinal Defensins and Reduces the Species Abundance of Romboutsia ilealis in C57BL/6J Mice.

The activation of the vitamin D receptor (VDR) in the ileum has been shown to regulate Paneth cell-specific defensins, a large family of antimicrobial peptides; hence, this may serve as a potential mechanism to maintain intestinal homeostasis. Previously, we have demonstrated that a combination of vitamin D3 (VD) and fructooligosaccharides (FOSs) upregulates colonic Vdr in mice. Here, we aim to examine the effect of VD, alone or in combination with FOSs, on intestinal barrier integrity and the secretion of antimicrobial peptides, as well as the gut microbial community. Male and female C57BL/6J mice at 6 weeks old were randomized into three groups to receive the following dietary regimens (n = 10/sex/group) for 8 weeks: (1) standard AIN-93G control diet (CTR), (2) CTR + 5000 IU vitamin D3 (VD), and (3) VD + 5% fructooligosaccharides (VF). VD and VF differentially regulated the mRNA expressions of tight junction proteins in the colon and ileum. VF suppressed the upregulation of colonic ZO-1 and occludin, which was induced by VD supplementation alone. In the ileum, occludin but not ZO-1 was upregulated 20-fold in the VF-treated mice. While VD did not alter the mRNA expressions of Vdr and defensins in the ileum, these targets were downregulated by VF. Microbial analysis further reveals a shift of microbial beta diversity and a reduction in Romboutsia ilealis, a pathobiont, in VF-treated mice. Though the implications of these phenotypical and microbial changes remain to be determined, the administration of FOSs in the presence of VD may serve as an effective dietary intervention for maintaining intestinal homeostasis.

Liquidambaric acid inhibits the proliferation of hepatocellular carcinoma cells by targeting PPARα-RXRα to down-regulate fatty acid metabolism.

Hepatocellular carcinoma (HCC) is a primary malignant tumor of the liver. As the global obesity rate rises, non-alcoholic fatty liver disease (NAFLD) has emerged as the most rapidly increasing cause of HCC. Consequently, the regulation of lipid metabolism has become a crucial target for the prevention and treatment of HCC. Liquidambaric acid (LDA), a pentacyclic triterpenoid compound derived from various plants, exhibits diverse biological activities. We found that LDA could inhibit HCC cell proliferation by arresting cell cycle and prompting apoptosis. Additionally, LDA can augment the therapeutic efficacy of Regorafenib in HCC in vitro and vivo. Our study utilized transcriptome analysis, luciferase reporter assays, and co-immunocoprecipitation experiments to elucidate the anti-HCC mechanism of LDA. We discovered that LDA disrupts the formation of the PPARα-RXRα heterodimer, leading to the down-regulation of the ACSL4 gene and subsequently impacting the fatty acid metabolism of HCC cells, ultimately inhibiting HCC proliferation. Our research contributes to the identification of novel therapeutic agents and targets for the treatment of HCC.

BI-7273, a BRD9 inhibitor, reduces lipid accumulation by downregulating the AKT/mTOR/SREBP1 signaling pathway.

Increases in de novo lipogenesis that disturbed lipid homeostasis and caused lipid accumulation are a major cause of NAFLD and obesity. SREBP1 is a crucial regulatory factor controlling the expression of rate-limiting enzymes of lipid synthesis. A reduction in SREBP1expression can reduce lipid accumulation. Thus, we utilized an SREBP1-luciferase-KI HEK293 cell line constructed by our lab to screen 200 kinds of epigenetic drugs for their ability to downregulate SREBP1expression. BI-7273, an inhibitor of bromodomain-containing protein 9 (BRD9), was screened and found to decrease SREBP1 expression. What is more, BI-7273 has been confirmed that it could reduce lipid accumulation in HepG2 cells by BODIPY staining, and significantly decrease the protein expression of SREBP1 and FASN. To explore the potential mechanism BI-7273 reducing lipid accumulation, RNA sequencing (RNA-seq) was performed and demonstrated that BI-7273 reduced lipid accumulation by downregulating the AKT/mTOR/SREBP1 pathway in vitro. Finally, these results were verified in NAFLD and obesity mouse model induced by high fat diet (HFD). The results indicated that BI-7273 could decrease mouse body weight and improve insulin sensitivity, but also exhibited a strong negative correlation with serum lipid levels, and also demonstrated that BI-7273 reduced lipid accumulation via AKT/mTOR/SREBP1 pathway in vivo. In conclusion, our results revealed that BI-7273 decreases lipid accumulation by downregulating the AKT/mTOR/SREBP1 pathway in vivo and in vitro. This is the first report demonstrating the protective effect of this BRD9 inhibitor against NAFLD and obesity. BRD9 may be a novel target for the discovery of effective drugs to treat lipid metabolism disorders.