Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick-end Research Articles

The Neuroprotective Effect of Sophocarpine against Glutamate-Induced HT22 Cell Cytotoxicity.

Neuronal cell death and dysfunction of the central nervous system can be caused by oxidative stress, which is associated with the development of neurodegenerative diseases. Sophocarpine, an alkaloid compound derived from Sophora moorcroftiana (Benth.) Baker seeds, has a wide range of medicinal value. This study sought to determine how sophocarpine exerts neuroprotective effects by inhibited oxidative stress and apoptosis in mouse hippocampus neuronal (HT22) cells. 20mM glutamate-induced HT22 cells were used to develop an in vitro model of oxidative stress damage. The Cell Counting Kit-8 (CCK-8) assay was used to assess cell viability. According to the instructions on the kits to detect reactive oxygen species (ROS) levels and oxidative stress indicators. HT22 cells were examined using immunofluorescence and Western Blotting to detect Nuclear Factor Erythroid 2-related Factor 2 (Nrf2) expression. The expression of proteins and messenger RNA (mRNA) for heme oxygenase-1 (HO-1) was examined by Western Blotting and Quantitative real time polymerase chain reaction (qRT-PCR). Mitochondrial membrane potential (MMP) and Cell apoptosis were used by 5, 5', 6, 6'-Tetrachloro-1, 1', 3, 3'-tetraethyl-imidacarbocyanine iodide (JC- 1) kit and Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick-End Labeling (TUNEL) apoptosis assay kit, respectively. Finally, the expression of pro-apoptotic proteins was detected by Western Blotting. The result demonstrated that sophocarpine (1.25 μM-10 μM) can significantly inhibit glutamate-induced cytotoxicity and ROS generation, improve the activity of antioxidant enzymes. Sophocarpine increased the expression of HO-1 protein and mRNA and the nuclear translocation of Nrf2 to play a cytoprotective role; however, cells were transfected with small interfering RNA targeting HO-1 (si-HO-1) reversed the above effects of sophocarpine. In addition, sophocarpine significantly inhibited glutamate induced mitochondrial depolarization and further inhibited cell apoptosis by reducing the expression level of caspase-related proteins.

Mango (Mangifera indica L.) seed kernel extract suppresses hyperglycemia by modulating pancreatic β cell apoptosis and dysfunction and hepatic glucose metabolism in diabetic rats.

This study investigated the anti-hyperglycemic action of mango seed kernel extract (MKE) and various mechanisms involved in its actions to improve pancreatic β cells and hepatic carbohydrate metabolism in diabetic rats. An intraperitoneal injection of 60mg/kg of streptozotocin (STZ) followed by 30 consecutive days of treatment with MKE (250, 500, and 1000mg/kg body weight) was used to establish a study group of diabetic rats. Using liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF-MS/MS) for identification, 26 chemical compounds were found in MKE and the high-performance liquid chromatography (HPLC) analysis of the MKE also revealed the existence of mangiferin, gallic acid, and quercetin. The results confirmed that in each diabetes-affected rat, MKE mitigated the heightened levels of fasting blood glucose, diabetic symptoms, glucose intolerance, total cholesterol (TC), and low-density lipoprotein-cholesterol (LDL-C). As demonstrated by a remarkable increment in serum and pancreatic insulin, the diabetic pancreatic β cell function was potentiated by treating with MKE. The effect of MKE on diabetic pancreatic apoptosis clearly reduced the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells, which was related to diminished levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and Bax and an increase in Bcl-xL protein expression. Furthermore, diabetes-induced liver damage was clearly ameliorated along with a notable reduction in serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels and abnormal liver histology. By enhancing anti-oxidant superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities, MKE alleviated diabetes-induced pancreatic and liver oxidative damage, as demonstrated by diminished levels of malondialdehyde. In minimizing the expression levels of glucose 6-phosphatase and phosphoenolpyruvate carboxykinase-1 proteins in the diabetic liver, MKE also enhanced glycogen content and hexokinase activity. Collectively, these findings indicate that by suppressing oxidative and inflammatory processes, MKE exerts a potent anti-hyperglycemic activity in diabetic rats which serve to protect pancreatic β cell apoptosis, enhance their function, and improve hepatic glucose metabolism.

Hederagenin protects against myocardial ischemia-reperfusion injury via attenuating ALOX5-mediated ferroptosis.

Hederagenin (HDG), a medical herb, is known for its beneficial activities against diverse diseases. The cardioprotective effect of HDG has been preliminarily disclosed, but the efficacy and underlying mechanism by which HDG protects against myocardial ischemia-reperfusion (MI/R) injury have not been elucidated yet. To simulate MI/R injury, the left anterior descending artery was occluded for 30min and then reperfusion for 120min in a rat model, and the cellular model of hypoxia-reoxygenation (H/R) injury was constructed in H9c2 cardiomyocytes. Hematoxylin-eosin, Prussian blue, and 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) staining were conducted to assess the histological injury, iron deposition, and myocardial infarction. Myocardial enzymes and oxidative stress-related factors were detected using their commercial kits. Lipid peroxidation was measured using BODIPY581/591 probe, and iron content was detected. Cell counting kit (CCK)-8, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), and flow cytometry assays were performed to assess cell viability and apoptosis. Protein levels were investigated by western blot. The interaction between HDG and 5-lipoxygenase (ALOX5) was verified using molecular docking. Our findings indicated that HDG significantly attenuated myocardial dysfunction by reducing infarction and myocardial injury. HDG significantly attenuated myocardial apoptosis in vitro and in vivo, as well as alleviating oxidative stress via reducing reactive oxygen species (ROS) and maintaining the balance between antioxidant and oxidant enzymes. Meanwhile, HDG inhibited I/R-induced ferroptosis in myocardium and cardiomyocytes, including reducing lipid peroxidation and iron level. Moreover, the binding relationship between HDG and ALOX5 was verified, and HDG could concentration dependently downregulate ALOX5. Furthermore, ALOX5 overexpression eliminated the inhibition of HDG on H/R-induced apoptosis, oxidative stress, and ferroptosis in H9c2 cardiomyocytes. HDG ameliorated myocardial dysfunction and cardiomyocyte injury by reducing apoptosis, oxidative stress, and ferroptosis through inhibiting ALOX5, providing a new perspective on the prevention and treatment of MI/R injury.

Mechanism of protective effect of resveratrol on poor ovarian response in mice

This study aims to investigate the therapeutic effects and potential mechanisms of resveratrol(Res) on poor ovarian response(POR) in mice. The common target genes shared by Res and POR were predicted by network pharmacology, used for Gene Ontology(GO) annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment, and then validated by animal experiments. The mice with regular estrous cycle after screening were randomized into normal, POR, and low-and high-dose(20 and 40 mg·kg~(-1), respectively) Res groups. The normal group was administrated with an equal volume of 0.9% sodium chloride solution by gavage, and the mice in other groups with tripterygium glycosides suspension(50 mg·kg~(-1)) by gavage for 2 weeks. After the modeling, the mice in low-and high-dose Res groups were treated with Res by gavage for 2 weeks, and the mice in normal and POR groups with an equal volume of 0.9% sodium chloride solution by gavage. Ovulation induction and sample collection were carried out on the day following the end of treatment. Vaginal smears were collected for observation of the changes in the estrous cycle, the counting of retrieved oocytes, and the measurement of ovarian wet weight and ovarian index. The enzyme-linked immunosorbent assay(ELISA) was employed to measure the levels of anti-mullerian hormone(AMH), follicle-stimulating hormone(FSH), estradiol(E_2), and luteinizing hormone(LH) in the serum. The ovarian tissue morphology and granulosa cell apoptosis were observed by hematoxylin-eosin(HE) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL), respectively. Western blot was employed to determine the protein levels of phosphatidylinositol 3-kinase(PI3K), protein kinase B(AKT), forkhead box O(FOXO) 3a, hypoxia-inducible factor(HIF)-1α, B-cell lymphoma-2(Bcl-2), and Bcl-2-associated X protein(Bax). A total of 222 common targets shared by Res and POR were collected. GO annotation indicated that these targets were mainly involved in oxidative stress response. KEGG enrichment analysis revealed that Res can intervene in POR via PI3K/AKT, HIF-1, and FOXO signaling pathways. Animal experiments showed that the model group had higher rate of estrous cycle disorders, lower number and poorer morphology of normally developed follicles at all levels, more atretic follicles, higher apoptosis of ovarian granulosa cells, lower number of retrieved oocytes, lower ovarian wet weight and ovarian index, higher serum levels of FSH and LH, lower levels of AMH and E_2, higher expression levels of HIF-1α, FOXO3a and Bax, and lower expression levels of PI3K, AKT, and Bcl-2 in the ovarian tissue than the normal group. Compared with the POR group, low-and high-dose Res decreased the rate of estrous cycle disorders, improved the follicle number and morphology, reduced atretic follicles, promoted the apoptosis of ovarian granulosa cells, increased retrieved oocytes, ovarian wet weight and ovarian index, and lowered serum FSH and LH levels. Moreover, Res down-regulated the expression levels of HIF-1α, FOXO3a and Bax, and up-regulated the expression levels of PI3K, AKT and Bcl-2 in the ovarian tissue. In summary, Res can inhibit apoptosis and mitigate poor ovarian response in mice by regulating the PI3K/AKT/FOXO3a and HIF-1α pathways.

SHED-exos promote saliva secretion by suppressing p-ERK1/2-mediated apoptosis in glandular cells.

Confirm that stem cells from human exfoliated deciduous teeth-derived exosomes (SHED-exos) can limit inflammation-triggered epithelial cell apoptosis and explore the molecular mechanism. SHED-exos were injected into the submandibular glands (SMGs) of non-obese diabetic (NOD) mice, an animal model of Sjögren's syndrome (SS). Cell death was evaluated by western blotting and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling staining. SHED-exos treatment promoted the saliva flow rates of NOD mice, accompanied by decreased cleaved caspase-3 levels and apoptotic cell numbers in SMGs. SHED-exos inhibited autophagy, pyroptosis, NETosis, ferroptosis, necroptosis and oxeiptosis marker expression in SS-damaged glands. Mechanistically, Kyoto Encyclopedia of Genes and Genomes analysis of exosomal miRNAs suggested that the rat sarcoma virus (RAS)/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway might play an important role. Invivo, the expression of Kirsten RAS, Harvey RAS, MEK1/2 and p-ERK1/2 was upregulated in SMGs, and this change was blocked by SHED-exos treatment. Invitro, SHED-exos suppressed p-ERK1/2 activation and increased cleaved caspase-3 and apoptotic cell numbers, which were induced by IFN-γ. SHED-exos suppress epithelial cell death, which is responsible for promoting salivary secretion. SHED-exos inhibited inflammation-triggered epithelial cell apoptosis by suppressing p-ERK1/2 activation, which is involved in these effects.

Sevoflurane improves renal ischemia-reperfusion injury in rats through RISK signaling pathway

Purpose: To investigate the effect of sevoflurane on renal ischemia-reperfusion injury (IRI) in rats and its regulatory effect on reperfusion injury salvage kinase (RISK) signaling pathway.Methods: A total of thirty (30) Sprague-Dawley rats were randomly divided into sham, model and sevoflurane groups with 10 animals per group. Renal IRI models were created in model and sevoflurane groups, while sham group had no ligation. Renal injury was determined using hematoxylin and eosin (HE) staining. Blood urea nitrogen (BUN) and serum creatinine (Scr) levels were assayed while apoptosis was determined via terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. Enzyme-linked immunosorbent assay (ELISA) was used to assess malonaldehyde (MDA) content and inflammatory factors in kidney tissues and peripheral blood, respectively. Reactive oxygen species (ROS) level was determined using 2,7-dichlorodi-hydro fluorescein diacetate (DCFHDA) while Western blotting was used to determine the expression of apoptosis- and RISK signaling pathway-related proteins in kidney tissues.Results: Compared to model group, renal injury in sevoflurane group rats was significantly alleviated (p < 0.01). The levels of BUN and Scr in peripheral blood, apoptosis level in kidney tissues, MDA content and ROS level in kidney tissues, interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α) and IL-6 content, and content of caspase-3 protein in kidney tissues were significantly reduced (p < 0.01), whereas IL-10 content, Bcl-2/Bax ratio and expression levels of p-ERK1/2, p-Akt and phosphorylated glycogen synthase kinase 3β (p-GSK-3β) were significantly increased (p < 0.01) in the sevoflurane group.Conclusion: Sevoflurane represses the release of inflammatory factors, lowers ROS level and apoptosis of renal cells and improves renal function through activation of RISK signaling pathway in kidney tissues of rats with renal IRI. Thus, sevoflurane is a potential agent for the treatment of IRI.

3-Bromopyruvate Inhibits the Growth and Glucose Metabolism of TNBC Xenografts in Nude Mice by Targeting c-Myc.

Due to the lack of effective drug treatment, triple-negative breast cancer (TNBC) is prone to recurrence and metastasis after an operation. As a glycolytic inhibitor, 3-bromopyruvic acid (3-BrPA) can inhibit the proliferation and induce apoptosis of TNBC cells. However, whether it has similar effects in animal models remains unclear. To observe the effect of 3-BrPA on the growth and glucose metabolism of human TNBC transplanted tumors in nude mice and to investigate the mechanism. We constructed subcutaneous xenografts of human TNBC in nude mice and treated them with low, medium and high concentrations of 3-BrPA. After 15 days, nude mice were sacrificed to detect hexokinase (HK) activity and adenosine triphosphate (ATP) content in tumor tissues. Hematoxylin-eosin (HE) staining was used to detect the damage of transplanted tumors and liver and kidney in nude mice, which 3-BrPA caused. The expression of c-Myc in tumor tissues was detected by Immunohistochemistry (IHC). Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining was used to detect the apoptosis of tumor tissues. Besides, the expressions of Cytc, Bax, Bcl-2 and Caspase-9 were detected by Western blotting. Compared with the control group, intraperitoneal injection of 3-BrPA inhibited the growth of human TNBC transplant tumors, decreased HK activity and ATP production in tumor tissues, disrupted the tissue structure of transplant tumors, and did not significantly damage liver and kidney tissues. IHC staining and Western blotting showed that 3-BrPA could decrease the expression of c-Myc and Bcl-2, increase the expression of Cyt -c, Bax and Caspase-9 expression and promote apoptosis in tumor tissues. The above data indicate that 3-BrPA inhibits the growth of human TNBC transplanted tumors and promotes their apoptosis. Its anti-cancer mechanism might reduce HK activity by down-regulating c-Myc expression, eventually leading to decreased glycolytic pathway energy production and promoting apoptosis of transplanted tumors.

Knockdown of Small Nucleolar RNA Host Gene 14 Alleviates Sepsis-Induced Acute Kidney Injury by Targeting miR-145-5p In Vitro and In Vivo

Acute kidney injury (AKI) manly occurred after sepsis and led to severe damage of the kidney. The present study was designed to explore the underlying mechanism of long non-coding RNA (lncRNA) SNHG14 in sepsis-induced AKI in In Vitro and In Vivo. The cecal ligation and puncture (CLP) rats was used as sepsis-induced AKI model for In Vivo studies and HK-2 cells with lipopolysaccharide (LPS) administration were used as cell model for In Vitro studies. Kidney tissues were stained by Hematoxylin and Eosin (H&E) for histological examination. The relative levels of SNHG14 and miR-145-5p was detected using qRT-PCR. The levels of serum cytokines, inflammatory factors, oxidative stress factors, apoptosis-related proteins were evaluated by ELISA and western blot. Apoptosis was detected using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Compared with the sham group, the rats in the CLP group showed injured pathological morphology of kidney, high levels of serum Scr, and BUN, elevated contents of TNF-α, IL-6 and IL-1β, malondialdehyde (MDA) and apoptosis, but low SOD level in kidney tissues. However, all these changes were reversed by shRNA-SNHG14 administration or transfection In Vivo and In Vitro. SNHG14 level was upregulated, while miR-145-5p significantly decreased in kidney tissues of CLP rats and LPS-induced HK-2 cells. The target gene of miR-145-5p was SNHG14, verifying through luciferase reporter analysis. Further, miR-145-5p knockdown transfection significantly reversed the inhibition of SNHG14 interference on the secretion of inflammatory cytokines and apoptosis in LPS-induced HK-2 cells. SNHG14 silence can alleviate AKI in sepsis by upregulating the miR-145-5p expression. SNHG14 might be used as a potential therapeutic target for sepsis-induced AKI.

Medicinal and edible plant Allium macrostemon Bunge for the treatment of testosterone-induced androgenetic alopecia in mice

Ethnopharmacological relevanceAllium macrostemon Bunge (AMB), a widely distributed wild garlic plant, possesses a variety of health-promoting properties. Androgenetic alopecia (AGA) is a common disorder that affects quality of life. Aim of the studyWe sought to investigate whether AMB stimulates hair regrowth in AGA mouse model, and clarify the underlying molecular mechanisms. Materials and methodsThe chemical constituents of AMB water extract were identified by ultra-high performance liquid chromatography-quadrupole-time of flight-mass spectrometry (UPLC-Q/TOF-MS) analysis. Cell viability assay and Ki-67 immunostaining were undertaken to evaluate the impacts of AMB on human hair dermal papilla cell (HDPC) proliferation. Wound-healing assay was undertaken to assess cell migration. Flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay were performed to examine cell apoptosis. Western blotting, real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and immunostaining assays were undertaken to determine the impacts of AMB on Wnt/β-catenin signaling and growth factors expression in HDPC cells. AGA mouse model was induced by testosterone treatment. The effects of AMB on hair regeneration in AGA mice were demonstrated by hair growth measuring and histological scoring. The levels of β-catenin, p-GSK-3β, and Cyclin D1 in dorsal skin were measured. ResultsAMB promoted proliferation and migration, as well as the expression of growth factors in cultured HDPC cells. Meanwhile, AMB restrained apoptosis of HDPC cells by increasing the ratio of anti-apoptotic Bcl-2/pro-apoptotic Bax. Besides, AMB activated Wnt/β-catenin signaling and thereby enhancing growth factors expression as well as proliferation of HDPC cells, which was abolished by Wnt signaling inhibitor ICG-001. In addition, an increase of hair shaft elongation was observed in mice suffering from testosterone-induced AGA upon the treatment of AMB extract (1% and 3%). Consistent with the in vitro assays, AMB upregulated the Wnt/β-catenin signaling molecules in dorsal skin of AGA mice. ConclusionThis study demonstrated that AMB promoted HDPC cell proliferation and stimulated hair regrowth in AGA mice. Wnt/β-catenin signaling activation, which induced production of growth factors in hair follicles and, eventually, contributed to the influence of AMB on the hair regrowth. Our findings may contribute to effective utilization of AMB in alopecia treatment.

Effect of miR-499-5p/SOX6 axis on atrial fibrosis in rats with atrial fibrillation.

Atrial fibrosis is involved in the progression of atrial fibrillation (AF). miR-499-5p is the most downregulated microRNA in arrhythmogenic cardiomyopathy hearts. Sry-related high-mobility-group box 6 (SOX6) is associated with apoptosis, inflammatory responses, and fibrosis. This study investigated the mechanism of miR-499-5p in ameliorating AF rats by regulating SOX6. AF rat models were established by injecting Ach-CaCl2 mixture, and the rats were treated with Lv-miR-499-5p/oe-SOX6/si-SOX6 before modeling. AF duration was recorded using electrocardiogram. miR-499-5p and SOX6 expression levels in the myocardium were determined by reverse transcription-quantitative polymerase chain reaction. The binding of miR-499-5p and SOX6 was validated. The atrial fibrosis degree and cardiomyocyte apoptosis were assessed using the Masson and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining methods. Levels of SOX6, atrial fibrosis markers (collage I/α-SMA/TGFβ1), cell cycle-related proteins (p21/CDC25/Cyclin B1), and cell senescence markers (SA-β-gal/γ-H2AX) were measured using Western blotting and immunohistochemistry. miR-499-5p was downregulated and SOX6 was upregulated in AF rats. miR-499-5p overexpression shortened the AF duration, alleviated atrial fibrosis, and decreased collage I/α-SMA/TGFβ1. miR-499-5p targeted SOX6 to ameliorate atrial fibrosis. AF rats exhibited increased p21/CDC25/Cyclin B1/SA-β-gal/γ-H2AX levels and raised cardiomyocyte apoptosis. SOX6 silencing downregulated p21 and alleviated cardiomyocyte cycle arrest, cell senescence, and apoptosis in AF rats. Shortly, miR-499-5p suppresses atrial fibrosis and cardiomyocyte senescence by targeting SOX6 and downregulating p21, thus mitigating AF in rats.

Profilin1 Regulates Trophoblast Invasion and Macrophage Differentiation

Unexplained recurrent spontaneous abortion (URSA) has been associated with the dysfunction of trophoblasts and decidual macrophages. Current evidence suggests that profilin1 (PFN1) plays an important role in many biological processes. However, little is known about whether PFN1 is related to URSA. Herein, the location of PFN1 was detected by immunohistochemistry, and the level of PFN1 was detected by quantitative real-time PCR, Western blot analysis, and immunohistochemistry. The proliferation of trophoblasts was detected by CCK8 and 5-ethynyl-2'-deoxyuridine assays, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays were used to detect apoptosis of trophoblasts. The migration and invasion ability of trophoblasts was assessed by using the wound-healing test and transwell test. Polarization of macrophages was detected in macrophages cultured in trophoblast conditioned medium. PFN1 expression was observed in cytotrophoblasts, syncytiotrophoblasts, and extravillous trophoblasts and was decreased in the villous tissue of patients with URSA. The migration and invasion ability and cell viability of trophoblastic cell lines that underwent PFN1 knockdown significantly decreased, and apoptosis increased. Opposite findings were observed after the overexpression of PFN1 in trophoblastic cells. In addition, PFN1 could regulate trophoblast function through phosphatidylinositol 3-kinase/AKT signal transduction rather than mitogen-activated proteinkinasesignaling pathways. Finally, knockdown of PFN1 in trophoblasts promoted tumor necrosis factor-α secretion to induce macrophage polarization to M1 phenotype, mediated by the NF-κB signaling pathway. These findings indicate that PFN1 has a broad therapeutic potential for patients with URSA.

Aspirin attenuates morphine antinociceptive tolerance in rats with diabetic neuropathy by inhibiting apoptosis in the dorsal root ganglia.

Morphine is a drug used in chronic pain such as diabetic neuropathy, but the development of tolerance to its antinociceptive effect is an important clinical problem. Aspirin is an analgesic and antiapoptotic drug used in combination with morphine as an adjuvant in diabetic neuropathy. Our aim in this study was to investigate the effects of aspirin on morphine-induced neuronal apoptosis and analgesic tolerance in rats with diabetic neuropathy. The antinociceptive effects of aspirin (50mg/kg) and morphine (5mg/kg) were evaluated by thermal pain tests. Streptozotocin (65mg/kg) was injected intraperitoneally to induce diabetic neuropathy. To evaluate apoptosis, ELISA kits were used to measure caspase-3, Bax and Bcl-2 levels. Apoptotic cells were detected histologically by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) method. Study results indicate that prior administration of aspirin to diabetic rats significantly increased the antinociceptive efficacy of morphine compared to morphine alone. Thermal pain tests showed that aspirin significantly reduced morphine tolerance in rats with diabetic neuropathy. Biochemical analysis revealed that aspirin significantly decreased the levels of pro-apoptotic proteins, caspase-3 and Bax, while increasing the anti-apoptotic Bcl-2 in DRG neurons. Semiquantitative scoring demonstrated that aspirin provided a significant reduction in apoptotic cell counts in diabetic rats. In conclusion, these data suggested that aspirin attenuated morphine antinociceptive tolerance through anti-apoptotic activity in diabetic rat DRG neurons.

Rice tapetum differentiation is sensitive to downregulation of OsUCH3, a ubiquitin C-terminal hydrolase.

The tapetum is a special tissue within the stamen that supplies nutrients to support microspore differentiation. Many genes have been identified to function in tapetum differentiation (Astrand et al., 2021). Ubiquitin C-terminal hydrolase (UCH) is a type of cysteine protease involved in protein deubiquitination process (Mevissen and Komander, 2017) and plays roles in gonadal transformation and spermatogenesis in animals (Luo et al., 2009; Kwon et al,. 2004; Wang et al., 2006). OsUCH3 (Os02g43760) was highly expressed in early rice stamen cells (Chen et al., 2015), the OsUCH3 expression was first observed in all cells of anther primordium and then gradually concentrated into the germ cells and tapetum cells, and OsUCH3 protein exhibits stronger ubiquitin C-terminal hydrolase enzymatic activity (Wang et al., 2018). These data suggested that OsUCH3 is likely associated with stamen development. To explore whether OsUCH3 plays roles in rice stamen development, we constructed transgenic lines to down-regulate OsUCH3 expression through RNAi vector as described previously with primers (ccACTAGTATGGAGGATGCTCATTCC and TcGGATCCCACAACTTTCGAAAGAGC) (Liu et al., 2005) (Figure 1a–g) and the OsUCH3-targeting CRISPR-Cas9 mutant lines (Figure 1h). CRISPR-Cas9 osuch3 mutant lines exhibited the same male-sterile phenotype as OsUCH3R. In observation of the overall morphology, there was no significant difference at vegetable development (Figure 1a). However, low seed setting rate was observed in the RNAi lines (Figure 1b) and CRISPR-Cas9 osuch3 mutant (Figure 1h). Alexander staining showed that OsUCH3R lines had abnormal pollen (Figure 1d) with the latter being indicated by the significant reduction in pollen grain number (Figure 1e, g). The RT-qPCR results revealed that OsUCH3 expression levels were significantly reduced in OsUCH3R transgenic rice (Figure 1f). To examine whether OsUCH3 affected stamen development, we pollinated OsUCH3R pistils with wild-type pollen grains, which rescued the sterility of OsUCH3R. The result suggested that the low seed setting rate resulted mainly from down-expression of OsUCH3 (Figure 1i). Morphological analysis revealed abnormal tapetum differentiation and locule collapse were observed in the OsUCH3R lines from stage 5 (S5) through 12 (S12) (Figure 1j). This observation suggested that the downregulation of OsUCH3 caused a defect in the tapetum differentiation and stamen development. In contrast to the degeneration of the tapetum that was observed in the WT from stages 7 (S7) to 9 (S9) (Figure 1k–m), the tapetum in the OsUCH3R lines was not properly degenerated during these stages (Figure 1n–p) as revealed by transmission electron microscopy (TEM) analysis. In addition, TEM observations revealed that the secondary cell walls of the endothecium cells were thinner in the OsUCH3R lines than in the wild type (Figure 1p, red arrowhead). We found green fluorescence in wild-type tapetal cells from stages 7 to 9 (Figure 1q-t) using the terminal deoxynucleotidyl transferase-mediated TdT-mediated dUTP Nick-End Labeling (TUNEL) assay (Chen et al., 2018). No such TUNEL signals were detected in OsUCH3R tapetal cells during the same developmental period (Figure 1u–x). These results suggested that DNA degradation during tapetum differentiation after stage 7 was abnormal in OsUCH3R. Numerous genes were known being involved in tapetum differentiation (Chen et al., 2018). GAMYB4 was up-regulated while the expression of YY1, OsCP1 and UDT1 exhibited no change in the OsUCH3R lines (Figure 1y). OsUCH3 is lower expressed in shoot tips and leaf primordia. While no differential expression of SAM genes OsWOX3, the expression of OSH1 and OsWUS was significantly decreased in the OsUCH3R lines (Figure 1y). These changes demonstrated the link between abnormal tapetum differentiation in the OsUCH3R lines with the changes in the expression levels of known tapetum genes. The phenotypes we found in OsUCH3R lines and CRISPR mutants demonstrated that tapetal differentiation is sensitive to the change of expression level of OsUCH3 and protein deubiquitination is an importance factor for proper tapetum differentiation. This finding provided a new opportunity not only in deciphering the regulatory network of tapetum differentiation at protein level, but in manipulating male sterility for agricultural applications. Sequence data from this article can be found in the GenBank/EMBL data library. This work was supported by grants from the Chinese Transgenic Project (grant no. CARS-01-06, 2016ZX08010001 and 2016ZX08009-003) and the National Natural Science Foundation of China (grant no. 31630006). The authors declare no competing interests. D.H.W., Z.H.X. and S.N.B. designed the research and wrote the manuscript. D.H.W. performed most of the experiments. N.L. and S.D.Y. provided an assistance for mutant genotyping and plant material fixation. Z.S.C and Y.N.L provided technical assistance for transgenic plant analyse. Z.H.L helped the semithin sectioning.

Retinoic acid inhibits the pyroptosis of degenerated nucleus pulposus cells by activating Sirt1-SOD2 signaling

ABSTRACT Aim Intervertebral disc (IVD) degeneration is a common disease initiated by the degeneration of the nucleus pulposus (NP). The pyroptosis of degenerated NP cells (dNPCs) plays an important role in NP degeneration. The purpose of this study is to identify a feasible solution that can inhibit NP cell pyroptosis to therapy the degeneration of the intervertebral disc. Methods Cell viability and proliferation were quantified by Cell Counting Kit-8 assay. The measurement of cellular reactive oxygen species (ROS) was detected by 2,7-Dichlorodi-hydrofluorescein diacetate. The death of cells was analyzed by the Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick-End Labeling (TUNEL) method of fluorescence analysis. The pyroptosis of cells was assessed by flow cytometry analyses. The contents of sulfate glycosaminoglycans were detected by a blyscan assay kit. Result In this study, we determined the effects of retinoic acid (RA) on dNPCs and investigated the underlying mechanism of RA-mediated pyroptosis in dNPCs. We also verified the effects of RA on IVD degeneration in vivo. Our results demonstrated that RA significantly increased the proliferation and the protein expression of sox9, aggrecan, and collagen II of dNPCs. Pyroptosis-related proteins and the pyroptosis rate of dNPCs were significantly decreased by RA. We found that Sirt1-SOD2 signaling was activated, while ROS generation and TXNIP/NLRP3 signaling in dNPCs were inhibited after the addition of RA. Furthermore, RA also recovered the structure of NP and increased the contents of sulfated glycosaminoglycans and collagen in vivo. Conclusion Our study demonstrated that RA could inhibit the pyroptosis and increase the extracellular matrix synthesis function of dNPCs and verified that RA has a protective effect on IVD degeneration.

HIF‐1α‐induced upregulated miR‐322 forms a feedback loop by targeting Smurf2 and Smad7 to activate Smad3/β‐catenin/HIF‐1α, thereby improving myocardial ischemia‐reperfusion injury

Myocardial ischemia/reperfusion injury (MIRI) is a major cause of heart failure after myocardial infarction. It has been reported that miR-322 is involved in MIRI progression, while the molecular mechanism of miR-322 in regulating MIRI progression needs to be further probed. MIRI cell model was established by oxygen-glucose deprivation/reoxygenation (OGD/R). Cell viability was assessed using MTS assay. Flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining were employed to analyze cell apoptosis. In addition, the interactions between miR-322, Smad7/Smurf2, hypoxia-inducible factor alpha (HIF-1α), and β-catenin were verified by dual-luciferase reporter gene assay. Our results displayed that miR-322 was significantly downregulated in OGD/R-treated H9c2 cells, and its overexpression resulted in increased cell viability and reduced the apoptosis. Smurf2 and Smad7 were identified as the direct targets of miR-322. Smad7 knockdown or Smurf2 knockdown increased OGD/R-treated H9c2 cell viability and suppressed the apoptosis. Meanwhile, miR-322 mimics abolished the mitigating effect of Smad7 or Smurf2 overexpression on MIRI. In addition, the Smad3/β-catenin pathway was identified as the downstream pathway of Smurf2/Smad7. Moreover, it was found that HIF-1α interacted with the miR-322 promoter, and β-catenin interacted with the HIF-1α promoter to form a loop. HIF-1α-induced upregulated miR-322 activated the Smad3/β-catenin pathway by targeting Smurf2 and Smad7 to improve MIRI; meanwhile, β-catenin/HIF-1α formed a positive feedback loop to continuously improve MIRI.

Honokiol attenuates mitochondrial fission and cell apoptosis by activating Sirt3 in intracerebral hemorrhage.

Sirtuin-3 (Sirt3) has been documented to protect against mitochondrial dysfunction and apoptosis. Honokiol (HKL) is a Sirt3 pharmacological activator with reported neuroprotective effects in multiple neurological disorders. The present study aimed to explore the neuroprotective effects of HKL and the role of Sirt3 following intracerebral hemorrhage (ICH). An in vivo ICH model in rats was established by injecting autologous blood into the right basal ganglia. PC12 cells were stimulated with hemin. For the in vivo investigation, the modified Neurological Severity Scores and the Morris water maze test were performed to assess neurological deficits. Hematoxylin-Eosin and Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining were employed to evaluate the histopathology and apoptosis. Immunohistochemical staining was used to investigate the expression of Sirt3. Adenosine triphosphate (ATP) levels were quantified to assess mitochondrial dysfunction. Cell counting kit-8, lactate dehydrogenase assay, and flow cytometry were used to analyze cell vitality and apoptosis in vitro. Immunofluorescence staining was performed to observe mitochondrial morphology and dynamin-related protein 1 (Drp1) localization to mitochondria. Western blot was applied to quantify the expression of Sirt3, Bax, Bcl-2, cleaved-caspase-3, Drp1, phosphorylation of Drp1 at serine-616, and phosphorylation of Drp1 at serine-637 in vivo and in vitro. HKL treatment alleviated neurological deficits, attenuated the histopathological damage and cell apoptosis, and restored the decreased ATP levels in ICH rats. HKL improved cell survival rate, reduced cell apoptosis, and inhibited mitochondrial fission in PC12 cells. Moreover, both in vivo and in vitro models showed increased phosphorylation of Drp1 at Ser616, and reduced phosphorylation of Drp1 at Ser637. Meanwhile, immunofluorescence co-localization analysis revealed that hemin increased the overlap of Drp1 and mitochondria in PC12 cells. The phosphorylation and mitochondrial translocation of Drp1 were effectively reversed by HKL treatment. Importantly, the selective Sirt3 inhibitor 3-(1H-1,2,3-triazol-4-yl) pyridine suppressed these effects. Our findings demonstrated that HKL ameliorated ICH-induced apoptosis and mitochondrial fission by Sirt3, suggesting that HKL has immense prospects for the treatment of ICH.

Mechanism Repositioning Based on Integrative Pharmacology: Anti-Inflammatory Effect of Safflower in Myocardial Ischemia–Reperfusion Injury

Safflower (Carthamus tinctorius. L) possesses anti-tumor, anti-thrombotic, anti-oxidative, immunoregulatory, and cardio-cerebral protective effects. It is used clinically for the treatment of cardio-cerebrovascular disease in China. This study aimed to investigate the effects and mechanisms of action of safflower extract on myocardial ischemia-reperfusion (MIR) injury in a left anterior descending (LAD)-ligated model based on integrative pharmacology study and ultra-performance liquid chromatography-quadrupole time-of-flight-tandem mass spectrometer (UPLC-QTOF-MS/MS). Safflower (62.5, 125, 250 mg/kg) was administered immediately before reperfusion. Triphenyl tetrazolium chloride (TTC)/Evans blue, echocardiography, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, lactate dehydrogenase (LDH) ability, and superoxide dismutase (SOD) levels were determined after 24 h of reperfusion. Chemical components were obtained using UPLC-QTOF-MS/MS. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to analyze mRNA and protein levels, respectively. Safflower dose-dependently reduced myocardial infarct size, improved cardiac function, decreased LDH levels, and increased SOD levels in C57/BL6 mice. A total of 11 key components and 31 hub targets were filtered based on the network analysis. Comprehensive analysis indicated that safflower alleviated inflammatory effects by downregulating the expression of NFκB1, IL-6, IL-1β, IL-18, TNFα, and MCP-1 and upregulating NFκBia, and markedly increased the expression of phosphorylated PI3K, AKT, PKC, and ERK/2, HIF1α, VEGFA, and BCL2, and decreased the level of BAX and phosphorylated p65. Safflower shows a significant cardioprotective effect by activating multiple inflammation-related signaling pathways, including the NFκB, HIF-1α, MAPK, TNF, and PI3K/AKT signaling pathways. These findings provide valuable insights into the clinical applications of safflower.

Bavachinin protects the liver in NAFLD by promoting regeneration via targeting PCNA

IntroductionNonalcoholic fatty liver disease (NAFLD) is the most common liver disease all over the world, and no drug is approved for the treatment of NAFLD. Bavachinin (BVC) is proven to possess liver-protecting effect against NAFLD, but its mechanism is still blurry. ObjectivesWith the use of Click Chemistry-Activity‐Based Protein Profiling (CC-ABPP) technology, this study aims to identify the target of BVC, and investigate the mechanism by which BVC exerts its liver-protecting effect. MethodsThe high fat diet induced hamster NAFLD model is introduced to investigate BVC’s lipid-lowering and liver-protecting effects. Then, a small molecular probe ofBVC is designed and synthesized based on theCC-ABPP technology, and BVC’s target is fished out. A series of experiments are performed to identify the target, including competitive inhibition assay, surface-plasmon resonance (SPR), cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS) assay, and co-immunoprecipitation (Co-IP). Afterward, the pro-regeneration effects of BVC are validated in vitro and in vivo through flow cytometry, immunofluorescence, and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). ResultIn the hamster NAFLD model, BVC shows lipid-lowing effect and improvement on the histology. PCNA is identified as the target of BVC with the method mentioned above, and BVC facilitates the interaction between PCNA and DNA polymerase delta. BVC promotes HepG2 cells proliferation which is inhibited by T2AA, an inhibitor suppresses the interaction between PCNA and DNA polymerase delta. In NAFLD hamsters, BVC enhances PCNA expression and liver regeneration, reduces hepatocyte apoptosis. ConclusionThis study suggests that, besides the anti-lipemic effect, BVC binds to the pocket of PCNA facilitating its interaction with DNA polymerase delta and pro-regeneration effect, thereby exerts the protective effect against HFD induced liver injury.

Zhao’s Weitiao No 3 inhibits tumor growth in gastric cancer-bearing nude mice

Purpose: To investigate the effect of Zhao’s Weitiao No. 3 (ZW3) on the progression of gastric cancer (GC) in mice, and to determine its effects on the proliferation and apoptosis of gastric cancer tissues.
 Methods: Tumor growth in vivo was used to determine the effect of ZW3 (15, 30, and 60 mg/kg) on GC growth in mice. Immunohistochemical and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays were conducted to evaluate the effects of ZW3 on the proliferation and apoptosis of mouse GC tissues, while quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunoblot assays were conducted to determine its effects on GC cell apoptosis.
 Results: ZW3 inhibited tumor growth in tumor-bearing mice, suppressed the expression of proliferation-related proteins (p < 0.001), increased the apoptosis of tumor cells in tumor-bearing mice (p < 0.001), and suppressed the expression of apoptosis-related proteins in mouse tumor tissues (p < 0.001).
 Conclusion: Thus, ZW3 is a potential drug for the management of GC. However, further investigations are required to determine its full potential

Comprehensive Analysis of RNA-Binding Protein-Related lncRNA in Breast Invasive Carcinoma.

RNA-binding proteins (RBPs), which form complexes or single/multiple RNA-binding domains, have a functional role in regulating and determining the function or stability of the bound RNAs in various cancers, including breast invasive carcinoma (BRCA). However, the biological functions and clinical implications of RBP-related long noncoding RNAs (lncRNAs) in BRCA remain largely unknown. Herein, we first identified and characterized RBP-related lncRNAs in BRCA. Then we built an RBP-related lncRNA signature (RBPLSig) and explored the clinical evaluation and prediction performance of the RBPLSig by bioinformatic analysis. In addition, to optimize treatment plans, prediction online tools was developed to predict the patient survival rate. Lastly, to verify the function of lncRNA WAC antisense RNA 1 (WAC-AS1), the experiments such as Quantitative real-time PCR (qRT-PCR), lncRNA knockdown, CCK-8, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining were performed. We also gained the potential mechanisms of the druggable compounds of the WAC-AS1 related RBP gene, putative NSUN6, using molecular docking. The results showed that RBPLSig, as an independent prognostic factor for BRCA patients, was involved in numerous malignancy-associated immunoregulatory pathways. We found different immune statuses and responses to immunotherapy, chemotherapy, and targeted therapy between the high- and low-risk groups stratified by RBPLSig. Our data broaden the comprehensive understanding of the biological functions of RBP-related lncRNAs, and demonstrate a novel and independent RBPLSig to assess prognosis and the immune microenvironment, thus helping to guide treatment decisions for BRCA.