Bacteria/Nanozyme Composites: New Therapeutics for Disease Treatment.

Bacterial therapy is recognized as a cost-effective treatment for several diseases. However, its development is hindered by limited functionality, weak inherent therapeutic effects, and vulnerability to harsh microenvironmental conditions, leading to suboptimal treatment activity. Enhancing bacterial activity and therapeutic outcomes emerges as a pivotal challenge. Nanozymes have garnered significant attention due to their enzyme-mimic activities and high stability. They enable bacteria to mimic the functions of gene-edited bacteria expressing the same functional enzymes, thereby improving bacterial activity and therapeutic efficacy. This review delineates the therapeutic mechanisms of bacteria and nanozymes, followed by a summary of strategies for preparing bacteria/nanozyme composites. Additionally, the synergistic effects of such composites in biomedical applications such as gastrointestinal diseases and tumors are highlighted. Finally, the challenges of bacteria/nanozyme composites are discussed and propose potential solutions. This study aims to provide valuable insights to offer theoretical guidance for the advancement of nanomaterial-assisted bacterial therapy.

Traditional Chinese Medicine Shi-Bi-Man ameliorates psoriasis via inhibiting IL-23/Th17 axis and CXCL16-mediated endothelial activation.

BACKGROUND: Psoriasis is a chronic inflammatory genetic disease, mainly manifesting in the skin. Conventional therapies, such as glucocorticosteroids and corticosteroids, have adverse effects that limit drug use. Hence, it is imperative to identify a new therapeutic strategy that exhibits a favorable safety profile. Shi-Bi-Man (SBM) is a safe herbal supplement sourced from various natural plants, including ginseng, angelica sinensis, polygonum multiflorum, and aloe vera. PURPOSE: We aimed to find a potential treatment for psoriasis and investigate the underlying mechanism through which SBM alleviates psoriatic-like skin inflammation in mice. METHODS: We investigated the effects of supplementing with SBM through intragastric administration or smear administration in a murine model of imiquimod-induced psoriasis. The changes in body weight and Psoriasis Area and Severity Index (PASI) score were recorded throughout the entire process. Additionally, we used hematoxylin-eosin staining to observe the skin structure and performed single-cell RNA sequencing to explore the underlying mechanism of SBM in influencing the psoriasis-like phenotype. Immunofluorescence was conducted to verify our findings. Furthermore, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was employed to investigate the impact of Tetrahydroxy stilbene glycoside (TSG) on the expression levels of IL23 in HaCaT cells. RESULTS: SBM remarkably alleviated the psoriasis-like phenotype by inhibiting IL-23/Th17 cell axis. Single-cell RNA sequencing analysis revealed a decrease in the expression of Il17 and Il23 in keratinocytes and T cells, concomitant with a reduction in the proportion of Th17 cells. Meanwhile, the activation of endothelial cells was inhibited, accompanied by a decrease in the expression of Cxcl16. In vitro, the addition of TSG to HaCaT cells resulted in significant suppression of IL23 expression stimulated by tumor necrosis factor-alpha (TNF-α).

Gallic acid ameliorates endometrial hyperplasia through the inhibition of the PI3K/AKT pathway and the down-regulation of cyclin D1 expression.

BACKGROUND: Gallic acid (GA) is an organic compound with phenolic properties that occurs naturally and can be found in Guizhi Fuling capsules, showcasing a wide range of biological functionalities. PURPOSE: The objective of this study was to examine the influence of GA on endometrial hyperplasia (EH) and elucidate its underlying mechanism. METHODS: Initially, the induction of EH was achieved by administering estradiol to mice via continuous subcutaneous injection for a duration of 21 days. Concurrently, GA treatment was administered, and subsequently, the uterine tissue structure was assessed using hematoxylin and eosin (H&E) staining. Following this, the proliferation of human endometrial cells treated by GA was determined utilizing the CCK-8 method. Furthermore, network pharmacology and single-cell-RNA-seq data were employed to identify the target of GA action. In addition, we will employ immunofluorescence (IF), immunohistochemistry (IHC), flow cytometry, western blot and RT-qPCR methodologies to investigate the impact of GA on the expression level of cyclin D1, PI3K, p-PI3K, AKT, p-AKT. RESULTS: GA treatment ameliorated histopathological alterations in the uterus and suppress proliferation. Estradiol stimulation can activate the PI3K/AKT pathway, leading to up-regulation of cyclin D1 expression, whereas GA treatment results in down-regulation of its expression. CONCLUSIONS: The expression of cyclin D1 is down-regulated by GA through the inhibition of the PI3K/AKT pathway, effectively mitigating estradiol-induced EH in mice.

Recent developments of hydroxamic acid hybrids as potential anti-breast cancer agents.

Histone deacetylase inhibitors not only possess favorable effects on modulating tumor microenvironment and host immune cells but also can reactivate the genes silenced due to deacetylation and chromatin condensation. Hydroxamic acid hybrids as promising histone deacetylase inhibitors have the potential to address drug resistance and reduce severe side effects associated with a single drug molecule due to their capacity to simultaneously modulate multiple targets in cancer cells. Accordingly, rational design of hydroxamic acid hybrids may provide valuable therapeutic interventions for the treatment of breast cancer. This review aimed to provide insights into the in vitro and in vivo anti-breast cancer therapeutic potential of hydroxamic acid hybrids, together with their mechanisms of action and structure-activity relationships, covering articles published from 2020 to the present.

Study on Targeted Drugs for Non-Small Cell NSCLC with PD-1PD-L1 Signaling Pathway.

OBJECTIVE: To study the biological function and clinical significance of PD-1/PD-L1 representation in non-small cell lung cancer (NSCLC), and the representation and correlation of PD-1/PD-L1 and epidermal growth factor receptor (EGFR) in NSCLC. METHODS: The pathological specimens and clinical data of 108 patients with NSCLC were collected. The representation of PD-1/PD-L1 was detected by immunohistochemistry, and the representation of EGFR was detected by PCR. Logistic regression analysis was used to assess the correlation between PD-1/PD-L1 and EGFR. RESULTS: The representations of PD-L1 and PD-1 in NSCLC tissues were higher than those in normal lung tissues (P<0.05), and the positive rates were 53.70% and 55.56% respectively, which were related to lymph node metastasis and TNM staging (P<0.05). CONCLUSION: The risk of EGFR mutation in NSCLC patients is high, which is closely related to the positive of PD1/PD-L1. PD1/PD-L1 can be used as a predictor of EGFR mutation.

Oral Immunotherapy Reshapes Intestinal Immunosuppression via Metabolic Reprogramming to Enhance Systemic Anti-Tumor Immunity.

Tumor immunotherapy offers a new paradigm to treat cancer; however, the existing regimens are accompanied by the dilemma of insufficient therapeutic outcomes and off-target adverse effects. The intestinal immune system contains a bulk of immune cells, which can be important contributors to the maintenance of systemic immune homeostasis. However, manipulating intestinal immunity to achieve systemic anti-tumor immunity is extremely challenging. Here, an oral immunotherapy strategy is reported using immune-enhancing fullerenes (IEF) that can reinvigorate anti-tumor immunity via immune cell-metabolic reprogramming of intestinal immune cells. Findings show that IEF can remodel anti-inflammatory macrophages into tumor-killing macrophages by regulating the energy metabolism pathway from oxidative phosphorylation (OXPHOS) to glycolysis. Consequently, IEF can reprogram the immunosuppressive intestinal immunity and enhance sys temic immunity in vivo, thereby boosting anti-tumor immunity and converting "cold" tumors into "hot" tumors. Oral immunotherapy strategy, modulating autoimmune cells in the intestine and achieving systemic anti-tumor immunity, can ensure safe and efficient tumor immunotherapy.

Single-cell analyses reveal cannabidiol rewires tumor microenvironment via inhibiting alternative activation of macrophage and synergizes with anti-PD-1 in colon cancer.

Colorectal tumors often create an immunosuppressive microenvironment that prevents them from responding to immunotherapy. Cannabidiol (CBD) is a non-psychoactive natural active ingredient from the cannabis plant that has various pharmacological effects, including neuroprotective, antiemetic, anti-inflammatory, and antineoplastic activities. This study aimed to elucidate the specific anticancer mechanism of CBD by single-cell RNA sequencing (scRNA-seq) and single-cell ATAC sequencing (scATAC-seq) technologies. Here, we report that CBD inhibits colorectal cancer progression by modulating the suppressive tumor microenvironment (TME). Our single-cell transcriptome and ATAC sequencing results showed that CBD suppressed M2-like macrophages and promoted M1-like macrophages in tumors both in strength and quantity. Furthermore, CBD significantly enhanced the interaction between M1-like macrophages and tumor cells and restored the intrinsic anti-tumor properties of macrophages, thereby preventing tumor progression. Mechanistically, CBD altered the metabolic pattern of macrophages and related anti-tumor signaling pathways. We found that CBD inhibited the alternative activation of macrophages and shifted the metabolic process from oxidative phosphorylation and fatty acid oxidation to glycolysis by inhibiting the phosphatidylinositol 3-kinase-protein kinase B signaling pathway and related downstream target genes. Furthermore, CBD-mediated macrophage plasticity enhanced the response to anti-programmed cell death protein-1 (PD-1) immunotherapy in xenografted mice. Taken together, we provide new insights into the anti-tumor effects of CBD.

The double-edged role of neutrophil heterogeneity in inflammatory diseases and cancers.

Neutrophils are important immune cells act as the body's first line of defense against infection and respond to diverse inflammatory cues. Many studies have demonstrated that neutrophils display plasticity in inflammatory diseases and cancers. Clarifying the role of neutrophil heterogeneity in inflammatory diseases and cancers will contribute to the development of novel treatment strategies. In this review, we have presented a review on the development of the understanding on neutrophil heterogeneity from the traditional perspective and a high-resolution viewpoint. A growing body of evidence has confirmed the double-edged role of neutrophils in inflammatory diseases and tumors. This may be due to a lack of precise understanding of the role of specific neutrophil subsets in the disease. Thus, elucidating specific neutrophil subsets involved in diseases would benefit the development of precision medicine. Thusly, we have summarized the relevance and actions of neutrophil heterogeneity in inflammatory diseases and cancers comprehensively. Meanwhile, we also discussed the potential intervention strategy for neutrophils. This review is intended to deepen our understanding of neutrophil heterogeneity in inflammatory diseases and cancers, while hold promise for precise treatment of neutrophil-related diseases.

Inhibiting redox-mediated endothelial migration by gadofullerenes for inducing tumor vascular normalization and improving chemotherapy.

Tumor vascular normalization (TVN) reverses abnormal tumor vasculatures, which could boost anti-cancer efficiency and especially increase drug intratumoral delivery. Endothelial cells play a vital role in angiogenesis, yet continuous modulating endothelial cell migration to improve TVN is ingenious but challenging. Here we propose a potential strategy for TVN based on inhibiting endothelial migration using antioxidative fullerene nanoparticles (FNPs). We demonstrate that FNPs inhibit cell migration upon their anti-oxidation effects in vitro. The optimized alanine-modified gadofullerene (GFA) exhibits superior TVN ability and inhibits tumor growth in vivo. Mechanically, facilitated with the protein microarray, we confirm that GFA could suppress the focal adhesion pathway to restrain endothelial migration. Subsequently, remarkable anti-tumor efficacy of chemotherapy synergy was obtained, which benefited from a more normalized vascular network by GFA. Together, our study introduces the potential of FNPs as promising TVN boosters to consider in cancer nanomedicine design.

Identification of a novel dicistro-like virus associated with the roots of tomato plants.

Viruses belonging to the family Dicistroviridae have a monopartite positive-sense single-stranded RNA genome and infect a variety of arthropods. Using high-throughput sequencing, we detected a novel dicistro-like virus, tentatively named "tomato root-associated dicistro-like virus" (TRaDLV), in the roots of tomato plants showing yellow mosaic symptoms on the leaves. The diseased tomato plants were coinfected with multiple plant viruses, and TRaDLV was present in the roots but not in the leaves. The genome of TRaDLV is 8726 nucleotides in length, excluding the poly(A) tail, and contains two open reading frames (ORFs) separated by an intergenic region (IGR). The TRaDLV genome showed characteristics similar to those of dicistroviruses, including the presence of a 3C-like protease domain, repeated amino acid sequences representing multiple copies of viral genome-linked protein (VPg)-like sequences in the ORF1 polyprotein, and a series of stem-loop structures resembling an internal ribosome entry site in the IGR. Phylogenetic analysis revealed that TRaDLV clustered with unclassified dicistro-like viruses from invertebrates or identified in samples of plant-derived material. These findings indicate the existence of a novel dicistro-like virus that may associate with plant roots or a root-inhabiting organism.

Paeonol ameliorates endometrial hyperplasia in mice via inhibiting PI3K/AKT pathway-related ferroptosis.

BACKGROUND: Paeonol (Pae) is one of the active ingredients from components of Guizhi Fuling Capsule, a traditional Chinese medicine widely used for the treatment of women's diseases, which exhibits various biological and pharmacological activities. PURPOSE: The objective of this study was to investigate the molecular mechanism underlying the role of Pae in protecting against endometrial hyperplasia (EH). METHODS: CCK-8 assay was performed to detect the effect of Pae on cell proliferation. Hematoxylin and eosin (H&E) staining was performed to evaluate uterine tissue structure. A network pharmacology study was performed to search the disease targets. Single-cell transcriptome analysis was performed with uterine tissues from 3 healthy donors and 3 EH patients on 10X Genomics platform. Changes in lipid peroxidation were detected by the MDA reaction. IHC assay, Western blot, immunofluorescence and RT-qPCR were used to study the effects of estradiol and Pae on the expression levels of GPX4, PI3K, AKT, p-PI3K, p-AKT in mice. RESULTS: Pae treatment resulted in a decrease in cell viability of endometrial epithelial cells. Loss of uterus weight and morphology changes were observed in mice. In addition, Fe iron concentration and MDA levels increased, while the expression of GPX4, p-PI3K and p-AKT diminished. CONCLUSIONS: Pae exhibited obvious alleviative activity in estradiol-induced mice via PI3K/AKT signaling pathway-regulated ferroptosis.

Oral fullerene tablets for colorectal cancer therapy based on modulation of tumor inflammatory microenvironments.

The development and progression of colorectal cancer (CRC) are highly dependent on the long-term inflammatory microenvironment with immune dysregulation in the colorectum. However, effective therapeutics are limited to targeting CRC. Here, we developed oral fullerene tablets (OFTs) that can act directly on the colorectal site by oral administration and reduce the inflammatory state at the tumor site for effective CRC therapy. In detail, OFTs scavenged reactive oxygen species (ROS), restrained the mutation of the wild-type P53, inhibited the activation of the inflammatory pathway nuclear factor-κB (NF-κB) and the signal transducer and activator of transcription 3 (STAT3) in the colorectum of CRC mice. Subsequently, OFTs could greatly reduce the infiltration of pro-inflammatory M1 macrophages and neutrophils at the tumor site, restoring the inflammatory microenvironment and immune homeostasis in the colorectal region, and ultimately achieving the inhibition of CRC. In addition, there were no significant toxic side effects of the long-term administration of OFTs. Our work provides an effective oral therapeutic strategy for CRC therapy by modulating the colorectal tumor inflammatory microenvironment and sheds light on the route for oral nano-materials in the clinical treatment of CRC.

Amphiphilic Aminated Derivatives of [60]Fullerene as Potent Inhibitors of Tumor Growth and Metastasis.

Malignant proliferation and metastasis are the hallmarks of cancer cells. Aminated [70]fullerene exhibits notable antineoplastic effects, promoting it a candidate for multi-targeted cancer drugs. It is an urgent need to reveal the structure-activity relationship for antineoplastic aminated fullerenes. Herein, three amphiphilic derivatives of [60]fullerene with clarified molecular structures are synthesized: TAPC-4, TAPC-3, and TCPC-4. TAPC-4 inhibits the proliferation of diverse tumor cells via G0/G1 cell cycle arrest, reverses the epithelial-mesenchymal transition, and abrogates the high mobility of tumor cells. TAPC-4 can be excreted from the organism and achieves an in vivo inhibition index of 75.5% in tumor proliferation and 87.5% in metastatic melanoma with a wide safety margin. Molecular dynamics simulations reveal that the amphiphilic molecular structure and the ending amino groups promote the targeting of TAPC-4 to heat shock protein Hsp90-beta, vimentin, and myosin heavy chain 9 (MYH9), probably resulting in the alteration of cyclin D1 translation, vimentin expression, and MYH9 location, respectively. This work initially emphasizes the dominant role of the amphiphilic structure and the terminal amino moieties in the antineoplastic effects of aminated fullerenes, providing fundamental support for their anti-tumor drug development.

Dapagliflozin Alleviates Coxsackievirus B3-induced Acute Viral Myocarditis by Regulating the Macrophage Polarization Through Stat3-related Pathways.

Viral myocarditis (VMC), which is most prevalently caused by Coxsackievirus B3 (CVB3) infection, is a serious clinical condition characterized by cardiac inflammation. Dapagliflozin, a kind of sodium glucose co-transporters 2(SGLT-2) inhibitor, exhibited protective effects on plenty of inflammatory diseases, while its effect on viral myocarditis has not been studied. Recently, we found the protective effect of dapagliflozin on VMC. After CVB3 infection, dapagliflozin and STATTIC (a kind of stat3 inhibitor) were given to Balb/c male mice for 8 days, and then the severity of myocarditis was assessed. Our results indicated that dapagliflozin significantly alleviated the severity of viral myocarditis, elevated the survival rate, and ameliorated cardiac function. Besides, dapagliflozin can decrease the level of pro-inflammatory cytokines including IL-1ß, IL-6, and TNF-α. Furthermore, dapagliflozin can inhibit macrophages differentiate to classically activated macrophages (M1) in cardiac tissue and activate the Stat3 signal pathway which is reported to promote polarization of the alternatively activated macrophage (M2). And STATTIC can reverse these changes caused by dapagliflozin. In conclusion, we found that dapagliflozin treatment increased anti-inflammatory macrophage polarization and reduced cardiac injury following VMC via activating Stat3 signal pathway.

Fullerene nanoparticles for the treatment of ulcerative colitis.

Ulcerative colitis (UC) is a long-term, recurrent inflammatory bowel disease for which no effective cure is yet available in the clinical setting. Repairing the barrier dysfunction of the colon and reducing intestinal inflammation are considered key objectives to cure UC. Here we demonstrate a novel therapeutic strategy based on a C60 fullerene suspension (C60FS) to treat dinitrobenzene sulfonic acid-induced UC in an animal model. C60FS can repair the barrier dysfunction of UC and effectively promote the healing of ulcers; it also manifests better treatment effects compared with mesalazine enema. C60FS can reduce the numbers of basophils in the blood of UC rats and mast cells in the colorectal tissue, thereby effectively alleviating inflammation. The expression of H1R, H4R, and VEGFR2 receptors in colorectal tissues is inhibited by C60FS, and the levels of histamine and prostaglandin in the rat blood are reduced. This work presents a reliable strategy based on fullerene to cure UC and provides a novel guide for UC treatment.

AVE0991, a nonpeptide angiotensin-(1-7) mimic, inhibits angiotensin II-induced abdominal aortic aneurysm formation in apolipoprotein E knockout mice.

AVE0991, a nonpeptide angiotensin-(1-7) mimic, has similar protective effects for cardiovascular system to Ang-(1-7). In this article, we aimed to explore the effects of AVE0991 and Ang-(1-7) on abdominal aortic aneurysm (AAA) induced by Ang II in apolipoprotein E knockout mice. The mice AAA model was established by Ang II infusion, and then mice received different treatment with saline, Ang II (1.44 mg/kg/day), different dose AVE0991 (0.58 or 1.16 µmol/kg/day), or Ang-(1-7) (400 ng/kg/min). The incidence of AAA was 76%, 48%, 28%, and 24% in the vehicle, the low-dose AVE0991, high-dose AVE0991, and the Ang-(1-7) group, respectively. In comparison with control group, AVE0991 and Ang-(1-7) treatment significantly increased smooth muscle cells and decreased macrophage accumulation, the expression levels of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor α (TNF-α), and the expression and activity of metalloproteinases 2 and 9 in mice AAA model or in human smooth muscle cells (hVSMCs). The therapeutic effects may be contributed to reduction of oxidative stress and downregulation of P38 and ERK1/2 signal pathways via Mas receptor activation, whereas the positive impacts were reversed by co-administration with the Mas antagonist A779 (400 ng/kg/min) and AVE0991 in Ang II-infused mice or in hVSMCs. Therefore, AVE0991 and Ang-(1-7) might be novel and promising interventions in the prevention and treatment of AAA. KEY MESSAGES: • AVE0991 dose-dependently inhibited Ang II-induced AAA formation in Apoe-/- mice. • Ang-(1-7) played the same protective role as high-dose AVE0991. • Inhibition of Mas receptor with A779 could reverse the protective effect of AVE0991. • The therapeutic effects may be contributed to reduction of oxidative stress and downregulation of P38 and ERK1/2 signal pathways via Mas receptor activation.

Pro-Renin Receptor Overexpression Promotes Angiotensin II-Induced Abdominal Aortic Aneurysm Formation in Apolipoprotein E-Knockout Mice.

The pro-renin receptor (PRR) is an important novel component of the renin-angiotensin (Ang) system that has multiple functions, which are not yet completely understood. In this study, we aimed to explore the effect of PRR on the formation of Ang II-induced abdominal aortic aneurysm (AAA) in apolipoprotein E-knockout mice. We used Ang II (1.44 mg/kg/day) infusion to induce AAA followed by a treatment of saline, telmisartan, no treatment, Ad-EGFP, Ad-PRR, or Ad-PRR plus telmisartan. The incidence of AAA was 35%, 60%, 65%, 90%, and 55% in the Telmisartan, Vehicle, Ad-EGFP, Ad-PRR, and Ad-PRR+Telmisartan groups, respectively. Compared with the Vehicle and Ad-EGFP groups, PRR overexpression markedly increased macrophage infiltration; levels of proinflammatory cytokines, including monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α); the expression and activity of MMP2 and MMP9; NOX2 and NOX4 protein and mRNA expression; nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity; extracellular-signal-regulated kinase (ERK) and P38MAPK expression; but decreased smooth muscle cells content in AAA. However, telmisartan reversed the adverse effects of PRR. In addition, ERK inhibitor PD98059 eliminated the acceleration of Ang II-induced AAA formation by PRR, and coadministration of telmisartan and PD98059 further abolished the adverse effects of PRR on Ang II-induced AAA formation. Thus, PRR plays an important role in the pathological development of AAA via both Ang II-dependent and Ang II-independent activation of ERK pathways. These results suggest that inhibition of PRR activation may be a promising approach to the treatment of AAA.

(Pro)renin Receptor is Involved in Myocardial Damage in Alcoholic Cardiomyopathy.

BACKGROUND: (Pro)renin receptor (PRR), a novel member of the renin-angiotensin system, participates in various cardiovascular diseases. However, the role of PRR in alcoholic cardiomyopathy (ACM), which is caused by alcohol intake and manifests as myocardial damage and cardiac dysfunction, remains unclear. METHODS: PRR gene silencing was achieved by transfecting recombinant adenovirus expressing anti-PRR short hairpin RNA (PRR-shRNA). In vitro, primary rat cardiac fibroblasts (CFs) were cultured with the stimulation of alcohol (200 mM), with or without PRR-shRNA and PD98059. Immunofluorescence, RT-PCR, and Western blot were used to measure the protein and messenger (mRNA) expression of PRR, fibrotic factors, and members of related signaling pathways. In vivo, Wistar rats were fed a diet containing 9% (v/v) alcohol or a normal diet for 3 months, with or without PRR-shRNA. Sirius Red staining, immunohistochemical staining, and toluidine blue staining were used to evaluate myocardial fibrosis, oxidative stress, and inflammation response. RESULTS: Alcohol markedly increased PRR mRNA and protein expression in a time- and concentration-dependent manner in CFs. The increased expression of fibrotic factors induced by alcohol was prevented by PRR-shRNA and PD98059. Moreover, PRR-shRNA decreased the phosphorylation of extracellular regulated protein kinases (ERK) 1/2 in CFs. Furthermore, PRR-shRNA decreased cardiac fibrosis, reduced oxidative stress, and alleviated inflammation response in the myocardial tissue. CONCLUSIONS: Our results show that PRR-ERK1/2 signaling was involved in the development of ACM and that PRR could be a new target for the treatment of ACM.

Effects of the (Pro)renin Receptor on Cardiac Remodeling and Function in a Rat Alcoholic Cardiomyopathy Model via the PRR-ERK1/2-NOX4 Pathway.

Alcoholic cardiomyopathy (ACM) caused by alcohol consumption manifests mainly as by maladaptive myocardial function, which eventually leads to heart failure and causes serious public health problems. The (pro)renin receptor (PRR) is an important member of the local tissue renin-angiotensin system and plays a vital role in many cardiovascular diseases. However, the mechanism responsible for the effects of PRR on ACM remains unclear. The purpose of this study was to determine the role of PRR in myocardial fibrosis and the deterioration of cardiac function in alcoholic cardiomyopathy. Wistar rats were fed a liquid diet containing 9% v/v alcohol to establish an alcoholic cardiomyopathy model. Eight weeks later, rats were injected with 1 × 109v.g./100 µl of recombinant adenovirus containing EGFP (scramble-shRNA), PRR, and PRR-shRNA via the tail vein. Cardiac function was assessed by echocardiography. Cardiac histopathology was measured by Masson's trichrome staining, immunohistochemical staining, and dihydroethidium staining. In addition, cardiac fibroblasts (CFs) were cultured to evaluate the effects of alcohol stimulation on the production of the extracellular matrix and their underlying mechanisms. Our results indicated that overexpression of PRR in rats with alcoholic cardiomyopathy exacerbates myocardial oxidative stress and myocardial fibrosis. Silencing of PRR expression with short hairpin RNA (shRNA) technology reversed the myocardial damage mediated by PRR. Additionally, PRR activated phosphorylation of ERK1/2 and increased NOX4-derived reactive oxygen species and collagen expression in CFs with alcohol stimulation. Administration of the ERK kinase inhibitor (PD98059) significantly reduced NOX4 protein expression and collagen production, which indicated that PRR increases collagen production primarily through the PRR-ERK1/2-NOX4 pathway in CFs. In conclusion, our study demonstrated that PRR induces myocardial fibrosis and deteriorates cardiac function through ROS from the PRR-ERK1/2-NOX4 pathway during ACM development.