SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2.

We show that SARS-CoV-2 spike protein interacts with both cellular heparan sulfate and angiotensin-converting enzyme 2 (ACE2) through its receptor-binding domain (RBD). Docking studies suggest a heparin/heparan sulfate-binding site adjacent to the ACE2-binding site. Both ACE2 and heparin can bind independently to spike protein in vitro, and a ternary complex can be generated using heparin as a scaffold. Electron micrographs of spike protein suggests that heparin enhances the open conformation of the RBD that binds ACE2. On cells, spike protein binding depends on both heparan sulfate and ACE2. Unfractionated heparin, non-anticoagulant heparin, heparin lyases, and lung heparan sulfate potently block spike protein binding and/or infection by pseudotyped virus and authentic SARS-CoV-2 virus. We suggest a model in which viral attachment and infection involves heparan sulfate-dependent enhancement of binding to ACE2. Manipulation of heparan sulfate or inhibition of viral adhesion by exogenous heparin presents new therapeutic opportunities.

Ribosome-rescuer PELO catalyzes the oligomeric assembly of NOD-like receptor family proteins via activating their ATPase enzymatic activity.

NOD-like receptors (NLRs) are pattern recognition receptors for diverse innate immune responses. Self-oligomerization after engagement with a ligand is a generally accepted model for the activation of each NLR. We report here that a catalyzer was required for NLR self-oligomerization. PELO, a well-known surveillance factor in translational quality control and/or ribosome rescue, interacted with all cytosolic NLRs and activated their ATPase activity. In the case of flagellin-initiated NLRC4 inflammasome activation, flagellin-bound NAIP5 recruited the first NLRC4 and then PELO was required for correctly assembling the rest of NLRC4s into the NLRC4 complex, one by one, by activating the NLRC4 ATPase activity. Stoichiometric and functional data revealed that PELO was not a structural constituent of the NLRC4 inflammasome but a powerful catalyzer for its assembly. The catalytic role of PELO in the activation of cytosolic NLRs provides insight into NLR activation and provides a direction for future studies of NLR family members.

A Non-canonical PDK1-RSK Signal Diminishes Pro-caspase-8-Mediated Necroptosis Blockade.

Necroptosis induction in vitro often requires caspase-8 (Casp8) inhibition by zVAD because pro-Casp8 cleaves RIP1 to disintegrate the necrosome. It has been unclear how the Casp8 blockade of necroptosis is eliminated naturally. Here, we show that pro-Casp8 within the necrosome can be inactivated by phosphorylation at Thr265 (pC8T265). pC8T265 occurs in vitro in various necroptotic cells and in the cecum of TNF-treated mice. p90 RSK is the kinase of pro-Casp8. It is activated by a mechanism that does not need ERK but PDK1, which is recruited to the RIP1-RIP3-MLKL-containing necrosome. Phosphorylation of pro-Casp8 at Thr265 can substitute for zVAD to permit necroptosis in vitro. pC8T265 mimic T265E knockin mice are embryonic lethal due to unconstrained necroptosis, and the pharmaceutical inhibition of RSK-mediated pC8T265 diminishes TNF-induced cecum damage and lethality in mice by halting necroptosis. Thus, phosphorylation of pro-Casp8 at Thr265 by RSK is an intrinsic mechanism for passing the Casp8 checkpoint of necroptosis.

An Atlas of Human Glycosylation Pathways Enables Display of the Human Glycome by Gene Engineered Cells.

The structural diversity of glycans on cells-the glycome-is vast and complex to decipher. Glycan arrays display oligosaccharides and are used to report glycan hapten binding epitopes. Glycan arrays are limited resources and present saccharides without the context of other glycans and glycoconjugates. We used maps of glycosylation pathways to generate a library of isogenic HEK293 cells with combinatorially engineered glycosylation capacities designed to display and dissect the genetic, biosynthetic, and structural basis for glycan binding in a natural context. The cell-based glycan array is self-renewable and reports glycosyltransferase genes required (or blocking) for interactions through logical sequential biosynthetic steps, which is predictive of structural glycan features involved and provides instructions for synthesis, recombinant production, and genetic dissection strategies. Broad utility of the cell-based glycan array is demonstrated, and we uncover higher order binding of microbial adhesins to clustered patches of O-glycans organized by their presentation on proteins.

Gut stem cell necroptosis by genome instability triggers bowel inflammation.

The aetiology of inflammatory bowel disease (IBD) is a multifactorial interplay between heredity and environment1,2. Here we report that deficiency in SETDB1, a histone methyltransferase that mediates the trimethylation of histone H3 at lysine 9, participates in the pathogenesis of IBD. We found that levels of SETDB1 are decreased in patients with IBD, and that mice with reduced SETDB1 in intestinal stem cells developed spontaneous terminal ileitis and colitis. SETDB1 safeguards genome stability3, and the loss of SETDB1 in intestinal stem cells released repression of endogenous retroviruses (retrovirus-like elements with long repeats that, in humans, comprise approximately 8% of the genome). Excessive viral mimicry generated by motivated endogenous retroviruses triggered Z-DNA-binding protein 1 (ZBP1)-dependent necroptosis, which irreversibly disrupted homeostasis of the epithelial barrier and promoted bowel inflammation. Genome instability, reactive endogenous retroviruses, upregulation of ZBP1 and necroptosis were all seen in patients with IBD. Pharmaceutical inhibition of RIP3 showed a curative effect in SETDB1-deficient mice, which suggests that targeting necroptosis of intestinal stem cells may represent an approach for the treatment of severe IBD.

Lactate contributes to remote ischemic preconditioning-mediated protection against myocardial ischemia reperfusion injury by facilitating autophagy via AMPK-mTOR-TFEB-CX43 axis.

Remote ischemic preconditioning (RIPC) exerts a protective role on myocardial ischemia reperfusion (I/R) injury by the release of various humoral factors. Lactate is a common metabolite in ischemic tissues. Nevertheless, little is known about the role lactate plays in myocardial I/R injury and its underlying mechanism. This investigation revealed that RIPC elevated the level of lactate in blood and myocardium. Furthermore, AZD3965, a selective monocarboxylate transporter 1 (MCT1) inhibitor and 2-Deoxy-D-glucose (2-DG), a glycolysis inhibitor, mitigated the effects of RIPC-induced elevated lactate in the myocardium and prevented RIPC against myocardial I/R injury. In an in vitro hypoxia reoxygenation (H/R) model, lactate markedly mitigated H/R-induced cell damage in H9c2 cells. Meanwhile, further studies suggested that lactate contributed to RIPC rescuing I/R-induced autophagy deficiency by promoting TFEB translocation to the nucleus through activating the AMPK-mTOR pathway without influencing the PI3K-Akt pathway, thus reducing cardiomyocytes damage. Interestingly, we also found that lactate upregulated the mRNA and protein expression of CX43 by facilitating the binding of TFEB to CX43 promoter in the myocardium. Functionally, silencing of TFEB attenuated the protective effect of lactate on cell damage, which was reversed by overexpression of CX43. Further mechanistic studies suggested lactate facilitated CX43-regulated autophagy via AMPK-mTOR-TFEB signaling pathway. Collectively, our research demonstrates that RIPC protects against myocardial I/R injury through lactate-mediated myocardial autophagy via AMPK-mTOR-TFEB-CX43 axis.

"Glyco-sulfo barcodes" regulate chemokine receptor function.

Chemokine ligands and receptors regulate the directional migration of leukocytes. Post-translational modifications of chemokine receptors including O-glycosylation and tyrosine sulfation have been reported to regulate ligand binding and resulting signaling. Through in silico analyses, we determined potential conserved O-glycosylation and sulfation sites on human and murine CC chemokine receptors. Glyco-engineered CHO cell lines were used to measure the impact of O-glycosylation on CC chemokine receptor CCR5, while mutation of tyrosine residues and treatment with sodium chlorate were performed to determine the effect of tyrosine sulfation. Changing the glycosylation or tyrosine sulfation on CCR5 reduced the receptor signaling by the more positively charged CCL5 and CCL8 more profoundly compared to the less charged CCL3. The loss of negatively charged sialic acids resulted only in a minor effect on CCL3-induced signal transduction. The enzymes GalNAc-T1 and GalNAc-T11 were shown to be involved in the process of chemokine receptor O-glycosylation. These results indicate that O-glycosylation and tyrosine sulfation are involved in the fine-tuning and recognition of chemokine interactions with CCR5 and the resulting signaling.

Probing the binding specificities of human Siglecs by cell-based glycan arrays.

Siglecs are a family of sialic acid-binding receptors expressed by cells of the immune system and a few other cell types capable of modulating immune cell functions upon recognition of sialoglycan ligands. While human Siglecs primarily bind to sialic acid residues on diverse types of glycoproteins and glycolipids that constitute the sialome, their fine binding specificities for elaborated complex glycan structures and the contribution of the glycoconjugate and protein context for recognition of sialoglycans at the cell surface are not fully elucidated. Here, we generated a library of isogenic human HEK293 cells with combinatorial loss/gain of individual sialyltransferase genes and the introduction of sulfotransferases for display of the human sialome and to dissect Siglec interactions in the natural context of glycoconjugates at the cell surface. We found that Siglec-4/7/15 all have distinct binding preferences for sialylated GalNAc-type O-glycans but exhibit selectivity for patterns of O-glycans as presented on distinct protein sequences. We discovered that the sulfotransferase CHST1 drives sialoglycan binding of Siglec-3/8/7/15 and that sulfation can impact the preferences for binding to O-glycan patterns. In particular, the branched Neu5Acα2-3(6-O-sulfo)Galß1-4GlcNAc (6'-Su-SLacNAc) epitope was discovered as the binding epitope for Siglec-3 (CD33) implicated in late-onset Alzheimer's disease. The cell-based display of the human sialome provides a versatile discovery platform that enables dissection of the genetic and biosynthetic basis for the Siglec glycan interactome and other sialic acid-binding proteins.

Installation of O-glycan sulfation capacities in human HEK293 cells for display of sulfated mucins.

The human genome contains at least 35 genes that encode Golgi sulfotransferases that function in the secretory pathway, where they are involved in decorating glycosaminoglycans, glycolipids, and glycoproteins with sulfate groups. Although a number of important interactions by proteins such as selectins, galectins, and sialic acid-binding immunoglobulin-like lectins are thought to mainly rely on sulfated O-glycans, our insight into the sulfotransferases that modify these glycoproteins, and in particular GalNAc-type O-glycoproteins, is limited. Moreover, sulfated mucins appear to accumulate in respiratory diseases, arthritis, and cancer. To explore further the genetic and biosynthetic regulation of sulfated O-glycans, here we expanded a cell-based glycan array in the human embryonic kidney 293 (HEK293) cell line with sulfation capacities. We stably engineered O-glycan sulfation capacities in HEK293 cells by site-directed knockin of sulfotransferase genes in combination with knockout of genes to eliminate endogenous O-glycan branching (core2 synthase gene GCNT1) and/or sialylation capacities in order to provide simplified substrates (core1 Galß1-3GalNAcα1-O-Ser/Thr) for the introduced sulfotransferases. Expression of the galactose 3-O-sulfotransferase 2 in HEK293 cells resulted in sulfation of core1 and core2 O-glycans, whereas expression of galactose 3-O-sulfotransferase 4 resulted in sulfation of core1 only. We used the engineered cell library to dissect the binding specificity of galectin-4 and confirmed binding to the 3-O-sulfo-core1 O-glycan. This is a first step toward expanding the emerging cell-based glycan arrays with the important sulfation modification for display and production of glycoconjugates with sulfated O-glycans.

MAIT cells confer resistance to Lenvatinib plus anti-PD1 antibodies in hepatocellular carcinoma through TNF-TNFRSF1B pathway.

The combination of antiangiogenic agents and immune checkpoint inhibitors is more efficient than monotherapy in the management of hepatocellular carcinoma (HCC). Lenvatinib plus anti-PD1 antibodies have become the mainstay in HCC treatment. However, more than half the patients with HCC are non-responsive, and the mechanisms underlying drug resistance are unknown. To address this issue, we performed single-cell sequencing on samples from six HCC patients, aiming to explore cellular signals and molecular pathways related to the effect of lenvatinib plus anti-PD1 antibody treatment. GSVA analysis revealed that treatment with lenvatinib plus anti-PD1 antibody led to an increase in the TNF-NFKB pathway across all immune cell types, as compared to the non-treatment group. Mucosal-associated invariant T (MAIT) cells were found to secrete TNF, which activates TNFRSF1B on regulatory T cells, thereby promoting immunosuppression. Additionally, TNFSF9 was highly expressed in anticancer immune cells, including CD8+ effector T cells, MAIT, and γδ T cells in the treatment group. We also detected CD3+ macrophages in both HCC and pan-cancer tissues. Overall, our findings shed light on the potential mechanisms behind the effectiveness of lenvatinib plus anti-PD1 antibody treatment in HCC patients. By understanding these mechanisms better, we may be able to develop more effective treatment strategies for patients who do not respond to current therapies.

Develop and validate a radiomics space-time model to predict the pathological complete response in patients undergoing neoadjuvant treatment of rectal cancer: an artificial intelligence model study based on machine learning.

OBJECTIVE: In this study, we aimed to investigate the predictive efficacy of magnetic resonance imaging (MRI) radiomics features at different time points of neoadjuvant therapy for rectal cancer in patients with pathological complete response (pCR). Furthermore, we aimed to develop and validate a radiomics space-time model (RSTM) using machine learning for artificial intelligence interventions in predicting pCR in patients. METHODS: Clinical and imaging data of 83 rectal cancer patients were retrospectively analyzed, and the patients were classified as pCR and non-pCR patients according to their postoperative pathological results. All patients received one MRI examination before and after neoadjuvant therapy to extract radiomics features, including pre-treatment, post-treatment, and delta features. Delta features were defined by the ratio of the difference between the pre- and the post-treatment features to the pre-treatment feature. After feature dimensionality reduction based on the above three feature types, the RSTM was constructed using machine learning methods, and its performance was evaluated using the area under the curve (AUC). RESULTS: The AUC values of the individual basic models constructed by pre-treatment, post-treatment, and delta features were 0.771, 0.681, and 0.871, respectively. Their sensitivity values were 0.727, 0.864, and 0.909, respectively, and their specificity values were 0.803, 0.492, and 0.656, respectively. The AUC, sensitivity, and specificity values of the combined basic model constructed by combining pre-treatment, post-treatment, and delta features were 0.901, 0.909, and 0.803, respectively. The AUC, sensitivity, and specificity values of the RSTM constructed using the K-Nearest Neighbor (KNN) classifier on the basis of the combined basic model were 0.944, 0.871, and 0.983, respectively. The Delong test showed that the performance of RSTM was significantly different from that of pre-treatment, post-treatment, and delta models (P < 0.05) but not significantly different from the combined basic model of the three (P > 0.05). CONCLUSIONS: The RSTM constructed using the KNN classifier based on the combined features of before and after neoadjuvant therapy and delta features had the best predictive efficacy for pCR of neoadjuvant therapy. It may emerge as a new clinical tool to assist with individualized management of rectal cancer patients.

Pretreatment Serum Lactate Dehydrogenase and Metastases Numbers as Potential Determinants of Anti-PD-1 Therapy Outcome in Nasopharyngeal Carcinoma.

BACKGROUND: We aimed to investigate the determinant factors of anti-PD-1 therapy outcome in nasopharyngeal carcinoma (NPC). METHODS: In this retrospective study, we included 64 patients with recurrent/metastatic NPC. The association of patients' characteristics, C-reactive protein (CRP), neutrophil to lymphocyte ratio (NLR), and lactate dehydrogenase (LDH) with survival benefit of anti-PD-1 therapy were analyzed using Cox regression models and Kaplan-Meier analyses. Patients were divided based on the median value of CRP, NLR or LDH into different subgroups. RESULTS: At a median follow-up time of 11.4 months (range: 1-28 months), median progression-free survival (PFS) and overall survival (OS) were 1.9 months (95% CI, .18-3.6) and 15 months (95% CI, 10.9-19.1) months, respectively. Pretreatment metastases numbers was significant predictor of PFS (HR = 1.99; 95% CI 1.10-3.63; P = .024) and OS (HR = 2.77; 95% CI 1.36-5.61; P = .005). Baseline LDH level was independent predictor of OS (HR = 7.01; 95% CI 3.09-15.88; P < .001). Patients with LDH level >435 U/L at the baseline had significantly shorter PFS and OS compared to patients with LDH level ≤435 U/L (median PFS: 1.7 vs 3.5 months, P = .040; median OS: 3.7 vs 18.5 months, P < .001). Patients with non-durable clinical benefit (NDB) had significantly higher LDH level at the baseline compared to patients who achieved durable clinical benefit (DCB) (P = .025). Post-treatment levels of CRP, LDH, and NLR were decreased compared to baseline in patients with DCB (P = .030, P = .088, and P = .066, respectively), whereas, there was a significant increase in post-treatment level of LDH compared with baseline in patients with NDB (P = .024). CONCLUSIONS: LDH level at the baseline was an independent predictor of OS and pretreatment metastases numbers was a significant predictor of PFS and OS.

Integrative analyses identify CD73 as a prognostic biomarker and immunotherapeutic target in intrahepatic cholangiocarcinoma.

BACKGROUND: CD73 promotes progression in several malignancies and is considered as a novel immune checkpoint. However, the function of CD73 in intrahepatic cholangiocarcinoma (ICC) remains uncertain. In this study, we aim to investigate the role of CD73 in ICC. METHODS: Multi-omics data of 262 ICC patients from the FU-iCCA cohort were analyzed. Two single-cell datasets were downloaded to examine the expression of CD73 at baseline and in response to immunotherapy. Functional experiments were performed to explore the biological functions of CD73 in ICC. The expression of CD73 and HHLA2 and infiltrations of CD8 + , Foxp3 + , CD68 + , and CD163 + immune cells were evaluated by immunohistochemistry in 259 resected ICC samples from Zhongshan Hospital. The prognostic value of CD73 was assessed by Cox regression analysis. RESULTS: CD73 correlated with poor prognosis in two ICC cohorts. Single-cell atlas of ICC indicated high expression of CD73 on malignant cells. TP53 and KRAS gene mutations were more frequent in patients with high CD73 expression. CD73 promoted ICC proliferation, migration, invasion, and epithelial-mesenchymal transition. High CD73 expression was associated with a higher ratio of Foxp3 + /CD8 + tumor-infiltrating lymphocytes (TILs) and CD163 + /CD68 + tumor-associated macrophages (TAMs). A positive correlation between CD73 and CD44 was observed, and patients with high CD73 expression showed elevated expression of HHLA2. CD73 expression in malignant cells was significantly upregulated in response to immunotherapy. CONCLUSIONS: High expression of CD73 is associated with poor prognosis and a suppressive tumor immune microenvironment in ICC. CD73 could potentially be a novel biomarker for prognosis and immunotherapy in ICC.

Study on Synthesizing Isobornyl Acetate/Isoborneol from Camphene Using α-Hydroxyl Carboxylic Acid Composite Catalyst.

This study examined the preparation of isobornyl acetate/isoborneol from camphene using an α-hydroxyl carboxylic acid (HCA) composite catalyst. Through the study of the influencing factors, it was found that HCA and boric acid exhibited significant synergistic catalysis. Under optimal conditions, when tartaric acid-boric acid was used as the catalyst, the conversion of camphene and the gas chromatography (GC) content and selectivity of isobornyl acetate were 92.9%, 88.5%, and 95.3%, respectively. With the increase in the ratio of water to acetic acid, the GC content and selectivity of isobornol in the product increased, but the conversion of camphene decreased. The yield of isobornol was increased by adding ethyl acetate or titanium sulfate/zirconium sulfate to form a ternary composite catalyst. When a ternary complex of titanium sulfate, tartaric acid, and boric acid was used as the catalyst, the GC content of isobornol in the product reached 55.6%. Under solvent-free conditions, mandelic acid-boric acid could catalyze the hydration reaction of camphene, the GC content of isoborneol in the product reached 26.1%, and the selectivity of isoborneol was 55.9%. The HCA-boric acid composite catalyst can use aqueous acetic acid as a raw material, which is also beneficial for the reuse of the catalyst.

Enhanced Rhodamine B degradation by GAC/Mn-Sn particles electrodes.

Rhodamine B (RhB) wastewater could be degraded by a three-dimensional electrolytic reactor with surface-modified titanium anodes, and a variety of materials had been tried to prepare for particles electrodes to enhance its removal effects, among them, granular activated carbon (GAC) with large specific surface areas and stable chemical properties was selected as particles materials and coated by manganese oxidation (Mn) as the main active ingredient. The experimental results showed that 98.3% of RhB and 60.7% of chemical oxygen demand were removed respectively, and the RhB wastewater's biodegradability was improved either. On the superficial sites of GAC/Mn-Sn particles, hydroxyl radicals were generated, and some absorbed RhB molecular was initially decolored by hypochlorite removing the two ethyl groups on both sides of the molecular, then oxidized by hydroxyl, and continually decomposed by these strong oxidants into a variety of intermediates.

Genetic glycoengineering in mammalian cells.

Advances in nuclease-based gene-editing technologies have enabled precise, stable, and systematic genetic engineering of glycosylation capacities in mammalian cells, opening up a plethora of opportunities for studying the glycome and exploiting glycans in biomedicine. Glycoengineering using chemical, enzymatic, and genetic approaches has a long history, and precise gene editing provides a nearly unlimited playground for stable engineering of glycosylation in mammalian cells to explore and dissect the glycome and its many biological functions. Genetic engineering of glycosylation in cells also brings studies of the glycome to the single cell level and opens up wider use and integration of data in traditional omics workflows in cell biology. The last few years have seen new applications of glycoengineering in mammalian cells with perspectives for wider use in basic and applied glycosciences, and these have already led to discoveries of functions of glycans and improved designs of glycoprotein therapeutics. Here, we review the current state of the art of genetic glycoengineering in mammalian cells and highlight emerging opportunities.

Abdominal-B contributes to abdominal identity in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae).

The homeotic complex gene Abdominal-B (Abd-B) is involved in regulating the development of posterior abdomens and has been extensively studied in holometabolous insects. However, the function of Abd-B in hemimetabolous insects is not fully understood. Here, we functionally characterize an Abd-B homologue in the brown planthopper (BPH), Nilaparvata lugens. The full-length cDNA of the N. lugens Abd-B homologue (NlAbd-B) is 2334 nt, with an open reading frame of 1113 bp. NlAbd-B has the highest expression level at the egg stage relative to the nymphal and adult stages and is mainly expressed in the fourth to the ninth abdominal segment of embryos. RNA interference (RNAi)-mediated knockdown of NlAbd-B in nymphs disrupted the development of genitalia both in females and males and caused a genitalia-to-leg transformation. Parental RNAi of NlAbd-B in both female and male adults caused an extra abdominal segment in offspring nymphs, while parental RNAi of the N. lugens abdominal-A homologue in both female and males adults led to embryos with leg-like appendages on the second to the eighth abdominal segment. These findings suggest that NlAbd-B plays a pivotal role in genital development and posterior abdominal patterning and thus highlight the conservational role of Abd-B in holometabolous and hemimetabolous insects.

Lactobacillus plantarum-derived extracellular vesicles protect against ischemic brain injury via the microRNA-101a-3p/c-Fos/TGF-ß axis.

Currently, the reported source of extracellular vesicles (EVs) for the treatment of ischemic stroke（IS）is limited to mammals. Moreover, these EVs are restricted to clinical translation by the high cost of cell culture. In this respect, Lactobacillus plantarum culture is advantaged by low cost and high yield. However, it is poorly understood whether Lactobacillus plantarum-derived EVs (LEVs) are applicable for the treatment of IS. Here, our results demonstrated that LEVs reduced apoptosis in ischemic neuron both in vivo and in vitro. As revealed by high-throughput sequencing, miR-101a-3p expression was significantly elevated by LEV treatment in OGD/R-induced neurons, as confirmed in the tMCAO mice treated with LEVs. Mechanistically, c-Fos was directly targeted by miR-101a-3p. In addition, c-Fos determined ischemia-induced neuron apoptosis in vivo and in vitro through the TGF-ß1 pathway, miR-101a-3p inhibition aggravated ischemia-induced neuron apoptosis in vitro and in vivo, and miR-101a-3p overexpression produced the opposite results. Hsa-miR-101-3p was downregulated in the plasma of patients with IS but upregulated in the patients with neurological recovery after rt-PA intravenous thrombolysis. In conclusion, Our results demonstrated for the first time that LEVs might inhibit neuron apoptosis via the miR-101a-3p/c-Fos/TGF-ß axis, and has-miR-101-3p is a potential marker of neurological recovery in IS patients.

Cardioprotective effect of NRG-4 gene expression on spontaneous hypertension rats and its mechanism through mediating the activation of ErbB signaling pathway.

To explore the myocardial protective effect of Neuregulin-4 (NRG-4) gene expression on spontaneous hypertension (SHR) rats and its mechanism through mediating the activation of the ErbB signaling pathway, this study was conducted. For this purpose, forty 24-week-old male SPF SHR rats were selected as the experimental group, and 10 age and sex-matched Wistar Kyoto (WKY) rats were selected as the control group. Cardiac tissues were collected for hematoxylin-eosin (HE) staining, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining, Masson staining, and immunohistochemical staining. Following tail vein injection of recombinant lentiviral vectors, the experimental groups were constructed as the control group (SHR rats without any treatment), Empty vector group (Empty Vector transfection), shRNA negative control (NC) group (LV-shRNA-NRG-4 NC transfection to silence the expression of NRG-4), shRNA group (LV-shRNA-NRG-4 transfection), pcDNA3.1(-) NC group (pcDNA3.1(-)-NRG-4 empty vector transfection) and pcDNA3.1(-) group (pcDNA3.1(-)-NRG-4 transfection to overexpress NRG-4). Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to detect the expression levels In addition, methyl thiazolyl tetrazolium salt (MTT) assay and flow cytometry were performed to detect the proliferation and apoptosis of cardiomyocytes, respectively. Results showed that in the SHR group, the cardiomyocytes showed hypertrophy, disordered arrangement, hyperchromatic nucleus, irregular shape, obvious rupture of myocardial fibers, and obvious proliferation of fibrous stroma; obvious myocardial fibrosis, and there were more blue collagen fibers around cardiomyocytes and myocardial arterioles; cardiomyocytes were swollen, muscle fibers arranged disorderly, collagen around the coronary artery and myocardial interstitium increased significantly with a cross-linking appearance; besides, compared with WKY group, the apoptosis index of cardiomyocytes in SHR group was significantly increased (P<0.05). The expression of NRG-4 protein was decreased in the SHR group compared with the WKY group (P<0.05). In vitro, there was no difference in the mRNA expression of NRG-4, ErbB2 and ErbB4, MMP2, TGFß1 and α-SMA, as well as Caspase3, Bax and Bcl-2 among the control group, Empty vector group, shRNA NC group and pcDNA3.1(-) NC group (P>0.05). While shRNA group showed decreased expressions of NRG-4, ErbB2, ErbB4, MMP2, TGFß1, α-SMA and Bcl-2, while increased Caspase3 and Bax expressions, as well as promoted cell proliferation and cell apoptosis when compared with the shRNA NC group (P<0.05); while compared with pcDNA3.1(-) NC group, pcDNA3.1(-) group had highly increased expressions of NRG-4, ErbB2, ErbB4, MMP2, TGFß1, α-SMA and Bcl-2, while decreased Caspase3 and Bax expressions, inhibited cell proliferation and cell apoptosis (all P<0.05). It is concluded Upregulation of NRG-4 gene expression can promote the activation of the ErbB signaling pathway, thus inhibiting the proliferation and apoptosis of cardiomyocytes in SHR rats, reversing myocardial fibrosis, and playing its cardioprotective role.