Myricitrin promotes osteogenesis and prevents ovariectomy bone mass loss via the PI3K/AKT signalling pathway.

This study aimed to explore the effect of myricitrin on osteoblast differentiation in mice immortalised bone marrow mesenchymal stem cells (imBMSCs). Additionally, ovariectomy (OVX) mice were employed to examine the effect of myricitrin on bone trabecular loss in vivo. The effect of myricitrin on the proliferation of imBMSCs was evaluated using a cell counting kit-8 assay. Alizarin red staining, alkaline phosphatase staining were performed to elucidate osteogenesis. Furthermore, qRT-PCR and western blot determined the expression of osteo-specific genes and proteins. To screen for candidate targets, mRNA transcriptome genes were sequenced using bioinformatics analyses. Western blot and molecular docking analysis were used to examine target signalling markers. Moreover, rescue experiments were used to confirm the effect of myricitrin on the osteogenic differentiation of imBMSCs. OVX mice were also used to estimate the delay capability of myricitrin on bone trabecular loss in vivo using western blot, micro-CT, tartaric acid phosphatase (Trap) staining, haematoxylin and eosin staining, Masson staining and immunochemistry. In vitro, myricitrin significantly enhanced osteo-specific genes and protein expression and calcium deposition. Moreover, mRNA transcriptome gene sequencing and molecular docking analysis revealed that this enhancement was accompanied by an upregulation of the PI3K/AKT signalling pathway. Furthermore, copanlisib, a PI3K inhibitor, partially reversed the osteogenesis promotion induced by myricitrin. In vivo, western blot, micro-CT, hematoxylin and eosin staining, Masson staining, Trap staining and immunochemistry revealed that bone trabecular loss rate was significantly alleviated in the myricitrin low- and high-dose groups, with an increased expression of osteopontin, osteoprotegerin, p-PI3K and p-AKT compared to the OVX group. Myricitrin enhances imBMSC osteoblast differentiation and attenuate bone mass loss partly through the upregulation of the PI3K/AKT signalling pathway. Thus, myricitrin has therapeutic potential as an antiosteoporosis drug.

Selenium deficiency causes oxidative stress and activates inflammation, apoptosis, and necroptosis in the intestine of weaned calves.

Selenium performs a variety of biological functions in organisms, including antioxidant and anti-inflammatory effects. This study investigated how selenium deficiency affects weaned calves' intestines. According to Inductively coupled plasma mass spectrometry (ICP-MS) analysis of intestinal selenium concentrations in calves, the Se-D group had a significantly lower concentration of selenium. Hematoxylin-eosin staining showed that the intestinal epithelial cells were detached, the goblet cells were lost, and the intestinal villi were fragmented and loosely arranged in the Se-D group, along with hyperemia and inflammatory infiltration. Of the 22 selenoprotein genes, 9 were downregulated in response to selenium deficiency in Reverse transcription-PCR (RT-PCR), whereas 6 genes were upregulated. In the Se-D group, oxidative stress was detected by measuring redox levels in the intestines. Furthermore, TdT-mediated dUTP Nick-End Labeling (TUNEL) staining, RT-PCR, and Western blotting (WB) results indicated that both intrinsic and extrinsic apoptosis pathways are activated in the intestine during selenium deficiency. Selenium deficiency also induced necroptosis in the intestine through upregulation of MLKL, RIPK1, and RIPK3 mRNA levels. In addition, according to hematoxylin-eosin staining and ELISA, selenium-deficient calves had severe inflammation in their intestines. As a result of RT-PCR and WB analyses, we found that selenium deficiency was associated with nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. Our study suggested that weaned calves' intestines are affected by selenium deficiency, which causes oxidative stress, inflammation, apoptosis, and necroptosis.

Saffron essential oil ameliorates CUMS-induced depression-like behavior in mice via the MAPK-CREB1-BDNF signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Saffron, the dried stigma of Crocus sativus L., has a long history of use in the treatment of depression in traditional Chinese medicine and Islamic medicine. The unique aroma of saffron, primarily derived from its volatile oil, has been widely used by folk to mitigate anxiety and depression via sniffing because the aroma of saffron has a pleasant and invigorating effect. AIM OF THE STUDY: This study aimed to investigate the antidepressant effect and the underlying mechanism of saffron essential oil (SEO) in mice exposed to chronic unpredictable mild stress (CUMS). MATERIALS AND METHODS: In this study, compounds of SEO were identified using gas chromatography-mass spectrometry analysis, while network pharmacology was used to predict potential active compounds, antidepressant targets, and related signaling pathways of SEO. The CUMS depression model was further used to explore the therapeutic effect and possible mechanism of SEO. During the modeling period, mice were regularly administered fluoxetine (3.6 mg/kg, i.g.) or diluted SEO (2%, 4%, and 6% SEO, inhalation). The antidepressant and neuroprotective effects of SEO were evaluated by behavior tests (the open field test, the sucrose preference test, the tail suspension test, and the forced swimming test), hematoxylin-eosin staining, and Nissl staining. The enzyme-linked immunosorbent assay kits were used to measure dopamine (DA), 5-serotonin (5-HT), brain-derived neurotrophic factor (BDNF), and γ-aminobutyric acid (GABA) levels in serum. The relative abundance of Raf1, MEK1, P-ERK1/2/ERK1/2, P-CREB1/CREB1, BDNF, and P-Trk B/Trk B in the hippocampus was determined using western blot (WB). RESULTS: According to the network pharmacology analysis, seven active SEO compounds mediated 113 targets related to depression treatment, most of which were enriched in the 5-HT synapse, calcium signaling pathway, and cAMP signaling pathway. In vivo experiments indicated that fluoxetine and SEO improved depression-like behaviors in depressed mice. The levels of 5-HT, DA, BDNF, and GABA in serum increased significantly. Histopathological examinations revealed that fluoxetine and SEO ameliorated neuronal damage in the hippocampus. WB analysis showed that the relative expressions of Raf1, MEK1, P-ERK1/2/ERK1/2, P-CREB1/CREB1, BDNF, and P-Trk B/Trk B were significantly higher in the fluoxetine and SEO groups than in the CUMS group. CONCLUSION: Overall, these findings suggest that SEO significantly alleviates the depressive symptoms in CUMS exposed mice and partially restores hippocampal neuronal damage. Meanwhile, the best efficacy was observed in 4% SEO. Furthermore, the antidepressant mechanism of SEO is primarily dependent on the regulation of the MAPK-CREB1-BDNF signaling pathway.

Fufang-zhenzhu-tiaozhi formula protects islet against injury and promotes ß cell regeneration in diabetic mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Fufang-zhenzhu-tiaozhi formula (FTZ) is a patented preparation of traditional Chinese medicine that has been used to treat hyperglycemia and hyperlipidemia in the clinic for almost 10 years. Our previous study had demonstrated that FTZ can protect islet ß cell injury in vitro. However, the efficacy of FTZ on ß cell regeneration in vivo and the involved anti-diabetic mechanism remains unknown. AIM OF THE STUDY: We aim to investigate the effects of FTZ as a good remedy for islet protection and ß cell regeneration, and to reveal the underlying mechanism. MATERIALS AND METHODS: C57BL/6 mice were fed with high-fat diet for 3 weeks and then intraperitoneally injected with streptozotocin (90 mg/kg/d × 1 d) to establish type 2 diabetes (T2D) models. Mice in each group were divided into three batches that sacrificed after 3, 7 and 28 days of FTZ administration. Body weight, blood glucose, and oral glucose tolerance test were measured at indicated time points. Fasting insulin was determined by enzyme-linked immunosorbent assay (ELISA) kit. Neonatal ß cell was assessed by insulin & PCNA double immunofluorescence staining, and the underlying mechanisms related to ß cell regeneration were further performed by hematoxylin-eosin staining, insulin & glucagon double immunofluorescence staining and Western blot. RESULTS: FTZ and metformin can significantly help with the symptoms of DM, such as alleviating weight loss, reducing blood glucose, improving the level of insulin in vivo, and relieving insulin resistance, suggesting FTZ and metformin treatment maintained the normal morphological function of islet. Notably, ß cell regeneration, which is indicated by insulin and PCNA double-positive cells, was promoted by FTZ, whereas few neonatal ß cells were observed in metformin group. Hematoxylin-eosin staining, and its quantification results showed that FTZ effectively prevented the invasion of inflammatory cells into the islets in diabetic mice. Most ß cells in the islets of diabetic model mice were devoid, and the islets were almost all α cells, while the diabetic mice administered FTZ could still maintain about half of the ß cells in the islet. Furthermore, FTZ upregulated the expression of critical transcription factors during ß cell development and maturation (such as PDX-1, MAFA and NGN3) in diabetic mice. CONCLUSIONS: FTZ can alleviate diabetes symptoms and promote ß cell regeneration in diabetic mice. Moreover, FTZ promotes ß cell regeneration by preserving islet (resisting inflammatory cells invading islets), maintaining the number of ß cells in islets, and increasing the expression of PDX-1, MAFA and NGN3.

Swimming exercise and nano-l-arginine supplementation improve oxidative capacity and some autophagy-related genes in the soleus muscle of aging rats.

The present research aims to evaluate the effect of swimming exercise and chitosan-coated l-arginine on mitochondrial oxidation, BCL2 Interacting Protein 3 (Bnip3), NIP-like protein × (Nix), B-cell lymphoma-extra-large (Bcl-xL) and autophagy-related protein light chain 3(LC3) expression in soleus muscle of aging rats. In this experimental research, 25 male Wistar rats were assigned into five groups randomly: young, old, old + Nano l-arginine (Nano L-a), old + exercise (Ex), and old + Nano l-arginine (Nano L-a) + exercise (Ex) (n = 5 in each). They performed a swimming exercise program five days a week for six weeks. To determine the relative strength for rats before and after performing these interventions, the 1repetition maximum (1RM) test was done as a pre and post-test. The exercise program started with 20 min and after four sessions, gradually increased to 60 min and this time was maintained until the completion of the training period. l-arginine coated with chitosan nanoparticles was given to the rats in the l-arginine-supplemented group via gavage at a dosage of 500 mg/kg/day, five days a week, for six weeks. Additionally, the rats in all groups were fed a normal diet (2.87 kcal/g and 15 % energy from fat). Upon the completion of the protocol implementation, the rats were sacrificed and the soleus muscle was fixed and frozen to determine hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), gene expression analysis, levels of reactive oxygen species (ROS), and total antioxidant capacity (TAC). The results from the present research indicated that swimming exercise and Nano l-arginine improve the strength and histology of muscle tissue in old rats (p < 0.05). Aging significantly increased the expression of Nix and Bnip3 (p < 0.05) and reduced the Bcl-xL gene expression (p < 0.05). The expression of LC3 protein also increased with aging (p < 0.05). Therapeutic interventions, such as combined treatment (old + Nano L-a + Ex) for old animals, reduced the amount of this protein in soleus muscle (p < 0.05). The ROS values also showed a significant reduction only in the old + Nano L-a + Ex group compared to the old group. Moreover, TAC values show a significant decrease in the old and old + Ex groups in comparison to the young group. The use of arginine supplement, especially in nano form, along with swimming exercise seems to reduce the oxidative damage to the elderly muscle tissue, which has a positive effect on the structure and function of the soleus muscle. Since these interventions only had a significant effect on LC3 protein, further studies with more diverse measurement methods for autophagy are suggested.

Xianglian Pill attenuates ulcerative colitis through TLR4/MyD88/NF-κB signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Xianglian Pill (XLP) is a classical Chinese medicine prescription applied for controlling ulcerative colitis (UC). Whereas, the underlying mechanism remains unclear. AIM OF THE STUDY: The present work was aimed to investigate the mechanism of XLP in dextran sulfate sodium (DSS)-induced UC via the Toll Like Receptor 4 (TLR4)/Myeloid Differentiation factor 88 (MyD88)/Nuclear Factor kappa-B (NF-κB) signaling in mice. MATERIALS AND METHODS: The major components of XLP were detected by high-performance liquid chromatography-diode array detection (HPLC-DAD). The ulcerative colitis model was induced by DSS in mice. 5-Amino Salicylic Acid (5-ASA) group and XLP group were intragastrically treated. Disease activity index (DAI) and colon length were monitored and hematoxylin-eosin (HE) staining was conducted. Gasdermin D (GSDMD)-N and TLR4 expressions in colon tissues were visualized by immunofluorescence. TLR4 mRNA was measured by Real Time Quantitative PCR (RT-qPCR). The expressions of NOD-like receptor thermal protein domain associated protein 3 (NLRP3), active-caspase-1, GSDMD-N, TLR4, MYD88, NF-κB, p-NF-κB, and the ubiquitination of TLR4 in colon tissues were detected by Western blot. Myeloperoxidase (MPO) enzyme activity was examined and serum inflammatory factors Interleukin (IL)-1ß, IL-6, Tumor Necrosis Factor-α (TNF-α), and IL-18 were determined by Enzyme-linked Immunosorbent Assay (ELISA). TLR4-/- mice were applied for verifying the mechanism of XLP attenuated DSS symptoms. RESULTS: The XLP treatment extended colon length, reduced DAI, and attenuated histopathological alteration in DSS-induced mice. XLP administration suppressed MPO activity and reduced the content of IL-1ß, IL-6, TNF-α and IL-18 in serum. XLP also inhibited the expression levels of GSDMD-N, TLR4, NLRP3, active-caspase-1, MyD88, p-NF-κB/NF-κB in colon tissues of DSS-induced mice. TLR4-/- mice proved that TLR4 was involved in XLP-mediated beneficial effect on DSS-induced ulcerative colitis. CONCLUSIONS: XLP might treat ulcerative colitis by regulating the TLR4/MyD88/NF-κB signaling pathway.

Danhong injection alleviates cerebral ischemia-reperfusion injury by inhibiting autophagy through miRNA-132-3p/ATG12 signal axis.

ETHNOPHARMACOLOGICAL RELEVANCE: Danhong injection (DHI) is a renowned traditional Chinese medicine often used clinically to treat cardiovascular and cerebrovascular diseases. Studies have shown that DHI can significantly alter microRNA (miRNA) expression in the brain tissue. Therefore, exploring specific miRNAs' regulatory mechanisms during treatment with DHI is essential. AIM OF THE STUDY: To investigate DHI's regulatory mechanism on cerebral autophagy in rats with cerebral ischemia-reperfusion injury (CIRI). MATERIAL AND METHODS: Rats were randomly divided into the sham, middle cerebral artery occlusion (MCAO) model, and DHI-treatment groups. The extent of brain damage was evaluated using triphenyl tetrazolium chloride and hematoxylin-eosin staining. Hippocampal cell autophagy was observed using transmission electron microscopy. Autophagy-related proteins were analyzed using western blotting. Differentially expressed miRNAs were screened using high-throughput and real-time quantitative reverse transcription PCR. The relationship between miR-132-3p and ATG12 was confirmed using a dual-luciferase assay. The miR-132-3p mimics and inhibitors were transfected into PC12 cells subjected to oxygen-glucose deprivation (OGD) in vitro and MCAO model rats in vivo. RESULTS: DHI significantly altered the miRNA expression profile in rat brain tissues. The pathological changes in the brain tissues were improved, and the autophagic hippocampal cell vehicles were significantly reduced after DHI treatment. miRNA-132-3p, one of the miRNAs with a significantly different expression, was screened. Kyoto Encyclopedia of Genes and Genomes signal pathway analysis showed that its target genes were closely related to autophagy. Western blotting revealed that the p-PI3K, p-AKT, and mTOR expression increased significantly; AMPK, ULK1, ATG12, ATG16L1, and LC3II/I were downregulated in the DHI group. Dual-luciferase reporter gene experiments showed that miRNA-132-3p could target the ATG12 3'-UTR region directly. In vitro, miRNA-132-3p had a protective effect on OGD/R-induced oxidative stress injury in PC12 cells, improving cell viability, and affecting the expression of autophagy pathway-related proteins. In vivo transfection experiments showed that miR-132-3p could regulate ATG12 expression in CIRI rats' lateral brain tissue, affecting the autophagy signaling pathway. miR-132-3p overexpression reduces CIRI-induced autophagy and protects neurons. CONCLUSION: This study showed that DHI inhibits neuronal autophagy after cerebral ischemia-reperfusion. This may have resulted from miR-132-3p targeting ATG12 and regulating the autophagy signaling pathway protein expression.

Relaxin-3 Ameliorates Diabetic Cardiomyopathy by Inhibiting Endoplasmic Reticulum Stress.

Background: This study is aimed at investigating whether relaxin-3 exhibits protective effects against cardiomyopathy in diabetic rats by suppressing ERS. Methods: Eighty male SD rats were randomly divided into two groups: controls (n = 20) and diabetes (n = 60). The streptozotocin-treated rats were randomly divided into three groups: diabetic group (DM), low-dose relaxin-3 group (0.2 µg/kg/d), and high-dose relaxin-3 group (2 µg/kg/d). The myocardial tissues and collagen fiber were observed by hematoxylin and eosin (H&E) and Masson staining. Serum brain natriuretic peptide (BNP), troponin (TNI), myoglobin, interleukin (IL-17), interleukin (IL)-1α, and tumor necrosis factor (TNF)-α were determined by ELISA. The protein expression of glucose regulatory protein 78 (GRP78) and C/EBP homologous protein (CHOP) in the heart tissue of each group was detected by Western blot analysis. Results: (1) HE and Masson staining indicated that relaxin-3 could attenuate myocardial lesions and myocardial collagen volume fraction. (2) BNP, TnI, and myoglobin in the DM group at four and eight weeks were significantly higher than in the controls (P < 0.01). The relaxin-3-treated groups showed significantly reduced serum BNP, TnI, and myoglobin levels compared with the DM group (P < 0.05). (3) IL-17, IL-1α, and TNF-α levels in the DM rats at 4 weeks were higher than in the controls (P < 0.05). Low or high dose of relaxin-3-treated groups showed reduced serum IL-17 and TNF-α levels compared with the DM group at four and eight weeks (P < 0.05). (4) CHOP and GRP78 protein expression was increased in the DM group at four and eight weeks compared with the controls (P < 0.01), and small and large doses of relaxin-3 significantly reduced GRP78 and CHOP protein expression. Conclusions: Exogenous relaxin-3 ameliorates diabetic cardiomyopathy by inhibiting ERS in diabetic rats.

Healing Potential of Propolis Extract-Passiflora edulis Seed Oil Emulgel Against Excisional Wound: Biochemical, Histopathological, and Cytokines Level Evidence.

Propolis is rich in natural bioactive compounds, and considering its importance for many skin therapies, emulgel was prepared. This study examines how a propolis extract (PE) and Passiflora edulis seed (PS) oil emulgel affect rat deep skin wound healing. Based on preset criteria of maximum drug content and optimum drug permeation through the stratum corneum along with drug retention in the skin layers, an optimized emulgel formula based on Box-Behnken factorial design was prepared and used for subsequent in vitro and in vivo evaluations. In vivo wound-healing activities of emulgel and control treatments were investigated in a rat model. The optimized emulgel formula exhibited superior healing activity compared with plain PE suspension-treated rats on day 14 of wounding. Histopathological investigations of hematoxylin and eosin and Masson's Trichrome-stained skin sections supported this effect. Emulgel promotes cutaneous wound healing through a variety of mechanisms, including anti-inflammatory through modulation of cytokines tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6 production, and promotion of collagen fiber formation, all of which contribute to tissue remodeling. Furthermore, when compared with propolis suspension, emulgel showed significant antioxidant and anti-inflammatory effects. Emulgel significantly increased the skin's hydroxyproline level, antioxidant potential, wound contraction, increased penetration, and localized propolis deposition across the skin. Incorporation of PS oil into the emulgel accelerates the tissue regeneration process. The findings suggest that 5% propolis emulgel could be used as an alternative to treat wounds.

Triptolide improves chondrocyte proliferation and secretion via down-regulation of miR-221 in synovial cell exosomes.

BACKGROUND: Rheumatoid arthritis (RA), the most common type of inflammatory arthritis, can cause bone damage and disability. Triptolide, a prominent treatment for RA, has satisfactory anti-inflammatory effects. However, the mechanism of action of triptolide in RA remains unknown. PURPOSE: This study aimed to explore the molecular mechanisms underlying triptolide-mediated improvements in RA and identify the miRNA pathway responsible for these effects. METHODS: We identified various dysregulated miRNAs associated with RA by mining previously described microarray data and verified and screened these candidates using RT-qPCR. Hematoxylin-eosin staining was then applied to identify pathological changes in the affected joints, and cell counting kit-8 analysis and flow cytometry were employed to examine cell proliferation and apoptosis, respectively. Extracted exosomes were verified using transmission electron microscopy. RESULTS: Our results revealed that the legs of rats with collagen-induced arthritis presented with obvious swelling and bone damage, a high degree of inflammatory cell infiltration into the synovium, and structural changes to the cartilage. Data mining identified 39 dysregulated miRNAs in these tissues, and RT-qPCR further refined these observations to highlight miR-221 as a potential RA biomarker. Subsequent evaluations revealed that fibroblast-like synovial (FLS) cells secrete Exs carrying dysregulated miR-221 in vitro. These Exs mediate miR-221 levels, inflammation, and TLR4/MyD88 signaling via their fusion with chondrocytes, leading to changes in chondrocyte growth and metabolic factor levels. Additionally, the addition of triptolide impaired miR-221 expression, cell proliferation, inflammatory factors, and the protein levels of TLR4/MyD88 in RA-FLS and promoted the apoptosis of FLS. The therapeutic effect of triptolide on miR-221 Exs was reversed by miR-221 inhibitor in both normal and RA FLS. CONCLUSION: Our research shows that effective treatment with triptolide is mediated by its regulation of growth and secretory functions of chondrocytes via the inhibition of miR-221 secretion by FLS, providing a new target and natural medicinal candidate for future RA treatments.

Danshen injection induces autophagy in podocytes to alleviate nephrotic syndrome via the PI3K/AKT/mTOR pathway.

BACKGROUND: Danshen injection (DSI) is an agent extracted from the Salvia miltiorrhiza Bunge, a natural drug commonly used to alleviate kidney diseases. However, the material basis and therapeutic effects of DSI on nephrotic syndrome (NS) remain unclear. PURPOSE: To investigate the material basis of DSI and the therapeutic effects and underlying mechanisms of NS. METHODS: NS models were established using adriamycin-induced BALB/c mice and lipopolysaccharide-induced mouse podocytes (MPC-5). Following DSI and prednisone administration, kidney coefficients, 24 h urine protein, blood urea nitrogen, and serum creatinine levels were tested. Histomorphology was observed by periodic acid-Schiff staining and hematoxylin and eosin staining of the kidney sections. The glomerular basement membrane and autophagosomes of the kidneys were observed using transmission electron microscopy. Nephrin and desmin levels in the glomeruli were tested using immunohistochemistry. The viability of MPC-5 cells was tested using cell counting kit-8 after chloroquine and rapamycin administration in combination with DSI. The in vivo and in vitro protein levels of phosphatidylinositol 3-kinase (PI3K), AKT, phosphorylated AKT (Ser473), mammalian target of rapamycin (mTOR), microtubule-associated protein light chain 3 (LC3), beclin1, cleaved caspase-3, and caspase-3 were detected using western blotting. RESULTS: Our results showed that DSI contained nine main components: caffeic acid, danshensu, lithospermic acid, rosmarinic acid, salvianolic acid A, salvianolic acid B, salvianolic acid C, salvianolic acid D, and 3, 4-Dihydroxybenzaldehyde. In in vivo studies, the NS mice showed renal function and pathological impairment. Podocytes were damaged, with decreased levels of autophagy and apoptosis, accompanied by inhibition of the PI3K/AKT/mTOR signaling. DSI administration resulted in improved renal function and pathology in NS mice, with the activation of autophagy and PI3K/AKT/mTOR signaling in the kidneys. Additionally, podocytes were less damaged and intracellular autophagosomes were markedly increased. In vitro studies have shown that DSI activated MPC-5 autophagy and reduced apoptosis via the PI3K/AKT/mTOR pathway. CONCLUSION: Collectively, this study demonstrated that DSI activated podocyte autophagy and reduced apoptosis via the PI3K/AKT/mTOR signaling, ultimately attenuating NS. Our study clarified the main components of DSI and elucidated its therapeutic effects and potential mechanisms for NS, providing new targets and agents for the clinical treatment of NS.

Curcumin treatment attenuates cisplatin-induced gastric mucosal inflammation and apoptosis through the NF- κ B and MAPKs signaling pathway.

To investigate the protective effects of curcumin (Cur) on gastric mucosal injury induced by cisplatin (DDP), and explore possible molecular mechanisms. A mouse of gastric mucosal injury was established by intraperitoneal injection of DDP (27 mg/kg). Thirty mice were randomly divided into control group, DDP group and DDP + Cur group. Serum and gastric mucosal samples were collected on the 7th day after Cur treatment. The index of gastric mucosa injury was calculated, and the expression levels of inflammation, apoptosis and signaling pathway proteins were evaluated using hematoxylin and eosin staining, ELISA and western blotting analysis. These data showed that Cur treatment significantly attenuated DDP-induced decrease in body weight, food intake, fat and muscle ratios, and improved the gross gastric injury, scores of ulcer index, and histopathology changes triggered by DDP (p < .05). Meanwhile, Cur significantly decreased serum IL-23 and IL-17 proteins, reduced the expression levels of gastric mucosal IL-1ß, TNF- α and MPO, and restored the level of IL-10 protein (p < .05). Moreover, Cur treatment significantly inhibited the expression levels of Caspase-3, PARP and Bax, and increased the expression of Bcl-2 protein. Furthermore, Cur treatment significantly decreased the expression levels of IL-1R, MyD88 and TAK1, and also repressed the activation of NF-κB and nuclear translocation of NF-κB p65. And more importantly, Cur treatment significantly inhibited DDP-induced gastric mucosal JNK1/2, ASK1, P38 and JUN phosphorylation, and promoted the phosphorylation of ERK1/2 and C-Myc proteins. Our data suggest that Cur treatment alleviates DDP-induced gastric mucosal inflammation and apoptosis, which may be mediated through the NF- κ B and MAPKs signaling pathway.

Design, Synthesis, Characterization, Catalytic, Fluorometric Sensing, Antimicrobial and Antioxidant Activities of Schiff Base Ligand Capped AgNPs.

In recent days, the usage of biological and non-biological pollutants increased which poses a significant threat to environmental and biological systems. Therefore, the present aim is to develop effective methods to treat such pollutants by using highly stable and small-sized Schiff base ligand capped silver nanoparticles (AgNPs) with a face-centered cubic (fcc) crystalline structure and the size range is 5-10 nm. The potent role of the resulting synthesized AgNPs was found to be on multiple platforms such as catalyst, sensor, antioxidant, and antimicrobial disinfectant. The synthesized AgNPs were characterized through UV-vis spectroscopy, PL, FTIR, XRD, SEM, and TEM. The FTIR spectrum of AgNPs exhibited the interacted functional groups of Schiff base and size was estimated by XRD and TEM. AgNPs were able to catalytically degrade approximately 95% of methylene blue (MB), rhodamine B (RhB), and eosin Y (EY) dyes within 80 min of reaction time using NaBH4. The fluorometric sensor studies of synthesized AgNPs showed selective sensing of the potentially hazardous Fe2+ ion in water. As an antimicrobial agent, the AgNPs are effective against both Gram-positive and Gram-negative bacteria; as well as fungi, with the zones of clearance as approximately compatible with standard drugs. The AgNPs displayed a greater ability to scavenge free radicals, especially DPPH when compared with AgNPs and ascorbic acid. Thus, the results of this study validate the triple role of AgNPs derived via a simple synthesis as a catalyst, sensor, antioxidant, and antimicrobial agent for effective environmental remediation.

Study on the mechanism of Tong-Qiao-Huo-Xue decoction regulating apoptosis via ASK1/MKK4/JNK pathway in MCAO/R rats.

BACKGROUND: Activation of blood stasis is a crucial aspect of stroke treatment, and the Tong-Qiao-Huo-Xue-Decoction (TQHXD) formula is commonly utilized for this purpose. However, the mechanism underlying the protective effects of TQHXD against cerebral ischemia-reperfusion (I/R) injury is unclear. PURPOSE: Identification of the TQHXD components responsible for its protective effects and determination of their mode of action against cerebral I/R injury. METHODS: Gas chromatography (GC) and high-performance liquid chromatography (HPLC) were carried out to determine the active aspects of TQHXD. The active components and targets of TQHXD were looked up in the TCMSP and HERB databases; the Genecards, OMIM, TTD, and DrugBank databases were used to identify targets related to cerebral infarction; and the intersecting targets were obtained. The drug-ingredient-target-disease network and PPI network were subsequently built using Cytoscape 3.7.1 and STRING websites. Autodock VINA was used to perform molecular docking between the core target ASK1 and the active components of TQHXD detected by HPLC and GC. After successfully creating a rat model of middle cerebral artery occlusion (MCAO), the therapeutic effect of TQHXD was observed using triphenyltetrazolium and hematoxylin-eosin staining. We used Tunel-NeuN staining and transmission electron microscopy (TEM) to quantify hippocampal apoptosis. RT-qPCR and western blotting were used to detect protein and mRNA expression, respectively. RESULTS: HPLC and GC identified six active ingredients. Network pharmacology analyses were performed to test 66 intersection targets, including ASK1, MKK4, and JNK. Ferulic acid, HSYA, ligustilide, paeoniflorin, and muscone all displayed high binding affinity with ASK1 in molecular docking studies. The neuroprotective effects of TQHXD in I/R rats were demonstrated in the experimental models. In comparison with the model group, TQHXD decreased the apoptosis rate and reduced the protein levels of p-ASK1, caspase 3, p-MKK4, CytC, p-c-Jun, Bax/Bcl-2, and p-JNK, while considerably increasing the mRNA levels of Bcl-2 and decreasing those of Bax. CONCLUSION: By controlling the ASK1/MKK4/JNK pathway, TQHXD protects neurons from I/R damage and prevents apoptosis. Thus, TQHXD may be effective for the treatment of ischemic stroke. And the mechanism behind these therapeutic actions of TQHXD is supported by this research.

Anti-tumour activity and toxicological studies of combination treatment of Orthosiphon stamineus and gemcitabine on pancreatic xenograft model.

BACKGROUND: Pancreatic cancer is the most aggressive cancer type. Gemcitabine is the first line chemo-drug used for pancreatic cancer but exerts a broad spectrum of organ toxicities and adverse effects in patients. AIM: To evaluate the anti-tumour activity and toxicological effects of Orthosiphon stamineus extract formulation (ID: C5EOSEW5050ESA trademarked as Nuva-staticTM), and gemcitabine combination on pancreatic xenograft model. METHODS: Mice were randomly divided into six groups of 6 mice each (n = 6) and given different treatments for 28 d. The study design consisted of a 2 x 3 factorial treatment structure, with gemcitabine (yes/no) by oral (at 1200 and 400 mg/kg per day). Human pancreatic cancer cells were injected subcutaneously into the flanks of athymic nude mice. C5EOSEW5050ESA (200 or 400 mg/kg per day) was administered orally, while gemcitabine (10 mg/kg per 3 d) was given intraperitoneally either alone or in combination treatment. Histopathological analyses of vital organs, tumour tissues, and incidence of lethality were analysed. Analyses of tumour necrosis and proliferation were determined by haematoxylin-eosin staining and immunohistochemistry for Ki-67, respectively. RESULTS: No signs of toxicity or damage to vital organs were observed in all treatment groups compared to the untreated group. C5EOSEW5050ESA at 200 mg/kg and gemcitabine combination had no additive antitumor effects compared to a single treatment. Remarkably, a comparably greater response in a reduction in tumour growth, Ki-67 protein expression, and necrosis was demonstrated by 400 mg/kg of C5EOSEW5050ESA and gemcitabine combination than that of the individual agents. CONCLUSION: These results highlighted the synergistic activity of C5EOSEW5050ESA with gemcitabine to reduce pancreatic tumour growth in mice compared to a single treatment. Thus, this study provides valuable insights into using C5EOSEW5050ESA as a complementary treatment with gemcitabine for pancreatic cancer.

Wumei pills attenuates 5-fluorouracil-induced intestinal mucositis through Toll-like receptor 4/myeloid differentiation factor 88/nuclear factor-κB pathway and microbiota regulation.

BACKGROUND: Radiotherapy and chemotherapy can kill tumor cells and improve the survival rate of cancer patients. However, they can also damage normal cells and cause serious intestinal toxicity, leading to gastrointestinal mucositis[1]. Traditional Chinese medicine is effective in improving the side effects of chemotherapy. Wumei pills (WMP) was originally documented in the Treatise on Exogenous Febrile Diseases. It has a significant effect on chronic diarrhea and other gastrointestinal diseases, but it is not clear whether it affects chemotherapy-induced intestinal mucositis (CIM). AIM: To explore the potential mechanism of WMP in the treatment of CIM through experimental research. METHODS: We used an intraperitoneal injection of 5-fluorouracil (5-Fu) to establish a CIM mouse model and an oral gavage of WMP decoction (11325 and 22650 mg/kg) to evaluate the efficacy of WMP in CIM. We evaluated the effect of WMP on CIM by observing the general conditions of the mice (body weight, food intake, spleen weight, diarrhea score, and hematoxylin and eosin stained tissues). The expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-1ß, and myeloperoxidase (MPO), as well as the Toll-like receptor 4/myeloid differentiation factor 88/nuclear factor-κB (TLR4/MyD88/NF-κB) signaling pathway proteins and tight junction proteins (zonula occludens-1, claudin-1, E-cadherin, and mucin-2) was determined. Furthermore, intestinal permeability, intestinal flora, and the levels of short-chain fatty acids (SCFA) were also assessed. RESULTS: WMP effectively improved the body weight, spleen weight, food intake, diarrhea score, and inflammatory status of the mice with intestinal mucositis, which preliminarily confirmed the efficacy of WMP in CIM. Further experiments showed that in addition to reducing the levels of TNF-α, IL-1ß, IL-6, and MPO and inhibiting the expression of the TLR4/MyD88/NF-κB pathway proteins, WMP also repaired the integrity of the mucosal barrier of mice, regulated the intestinal flora, and increased the levels of SCFA (such as butyric acid). CONCLUSION: WMP can play a therapeutic role in CIM by alleviating inflammation, restoring the mucosal barrier, and regulating gut microbiota.

The combination of EGCG with warfarin reduces deep vein thrombosis in rabbits through modulating HIF-1α and VEGF via the PI3K/AKT and ERK1/2 signaling pathways.

Deep venous thrombosis (DVT) poses a major challenge to public health worldwide. Endothelial cell injury evokes inflammatory and oxidative responses that contribute to thrombus formation. Tea polyphenol (TP) in the form of epigallocatechin-3-gallate (EGCG) has anti-inflammatory and oxidative effect that may ameliorate DVT. However, the precise mechanism remains incompletely understood. The current study was designed to investigate the anti-DVT mechanism of EGCG in combination with warfarin (an oral anticoagulant). Rabbits were randomly divided into five groups. A DVT model of rats was established through ligation of the inferior vena cava (IVC) and left common iliac vein, and the animals were orally administered with EGCG, warfarin, or vehicle for seven days. In vitro studies included pretreatment of human umbilical vein endothelial cells (HUVECs) with different concentrations of EGCG for 2 h before exposure to hydrogen peroxide. Thrombus weight and length were examined. Histopathological changes were observed by hematoxylin-eosin staining. Blood samples were collected for detecting coagulation function, including thrombin and prothrombin times, activated partial thromboplastin time, and fibrinogen levels. Protein expression in thrombosed IVCs and HUVECs was evaluated by Western blot, immunohistochemical analysis, and/or immunofluorescence staining. RT-qPCR was used to determine the levels of AGTR-1 and VEGF mRNA in IVCs and HUVECs. The viability of HUVECs was examined by CCK-8 assay. Flow cytometry was performed to detect cell apoptosis and ROS generation was assessed by 2',7'-dichlorofluorescein diacetate reagent. In vitro and invivo studies showed that EGCG combined with warfarin significantly reduced thrombus weight and length, and apoptosis in HUVECs. Our findings indicated that the combination of EGCG and warfarin protects HUVECs from oxidative stress and prevents apoptosis. However, HIF-1α silencing weakened these effects, which indicated that HIF-1α may participate in DVT. Furthermore, HIF-1α silencing significantly up-regulated cell apoptosis and ROS generation, and enhanced VEGF expression and the activation of the PI3K/AKT and ERK1/2 signaling pathways. In conclusion, our results indicate that EGCG combined with warfarin modifies HIF-1α and VEGF to prevent DVT in rabbits through anti-inflammation via the PI3K/AKT and ERK1/2 signaling pathways.

[Effect of Rehmanniae Radix on depression-like behavior and hippocampal monoamine neurotransmitters of chronic unpredictable mild stress model rats].

To investigate the effect of Rehmanniae Radix on depression-like behavior and monoamine neurotransmitters of chronic unpredictable mild stress(CUMS) model rats. CUMS combined with isolated feeding was used to induce the depression model of rats. The depression-like behavior of rats was evaluated by sucrose preference test, open field test, and forced swim test. Hematoxylin-Eosin(HE) staining was used to investigate the pathological changes of neurons in the CA1 and CA3 area of hippocampus. Ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS) was used to detect the contents of 5-hydroxytryptamine(5-HT), 5-hydroxyindoleacetic acid(5-HIAA), dopamine(DA), 3,4-dihydroxyphenylacetic acid(DOPAC), homovanillic acid(HVA), norepinephrine(NE), and 3-methoxy-4-hydroxyphenyl glycol(MHPG) in rats. Western blot was used to detect the protein expressions of tryptophan hydroxylase 2(TPH2), serotonin transporter(SERT), and monoamine oxidase A(MAO-A) in the hippocampus of rats. Compared with the normal group, depressive-like behavior of rats was obvious in the model group. The arrangements of neurons in the CA1 and CA3 area of hippocampus were loose and disorderly. The levels of 5-HT, 5-HIAA, and 5-HT/5-HIAA in the hippocampal area were decreased(P<0.01). The protein expression of TPH2 was decreased(P<0.01), but those of SERT and MAO-A were increased(P<0.01). In the Rehmanniae Radix groups with 1.8 g·kg~(-1) and 7.2 g·kg~(-1), the depression-like behavior of CUMS rats and pathological changes of neurons in CA1, CA3 area of hippocampus were improved. The protein expression of TPH2(P<0.05, P<0.01) was increased, and those of SERT and MAO-A were down-regulated(P<0.05, P<0.01). The levels of 5-HT, 5-HIAA, and 5-HT/5-HIAA in hippocampus were increased(P<0.05, P<0.01). The changes in DA, DOPAC, HVA, DA/(DOPAC +HVA), NE, DHPG, and NE/DHPG were not statistically significant. The results suggested that Rehmanniae Radix improved depression-like behavior of CUMS rats, and the mechanism might be related to the regulation of synthesis, transportation, and metabolism of 5-HT neurotransmitter in the hippocampus.

Adansonia digitata L. fruit shell extract alleviates lead-induced neurotoxicity in mice via modulation of oxidative stress and a possible chelating activity.

BACKGROUND: Lead is a ubiquitous environmental heavy metal known to induce neurotoxicity. It has been postulated that substance with high antioxidant capacity could alleviate lead-induced neurotoxicity. Adansonia digitata fruit shell extract (ADFS) has been reported to have high phenolic contents and exerts antioxidant activity. This study investigated the effects of Adansonia digitata fruit shell extract on lead-induced neurotoxicity in mice. METHODS: Male balb/c mice (n = 7) were administered with Pb-acetate (50 mg/kg) 30 mins before ADFS (250 mg/kg and 500 mg/kg) or succimer (50 mg/kg) per orally for 28 days. Motor activities were evaluated on days 29 and 30 through horizontal bar and open field tests respectively. Further, spectrophotometry, atomic absorption spectrophotometry and haematoxylin and eosin staining were carried-out to determine the expression of oxidative stress biomarkers, level of lead concentration in the brain and histology of the cerebellum respectively. RESULTS: Lead acetate exposure significantly (p < 0.05) induced motor deficits in horizontal bar test and open field test, caused oxidative stress, high concentration of lead in the brain as well as histological aberration in the cerebellum. ADFS significantly (p < 0.05) reversed the motor deficits evident by increased muscle strength and number of lines crossed. Further, ADFS significantly reversed oxidative stress evident by increased levels of SOD, CAT and GSH and decreased level of MDA. There was also significant (p < 0.05) decrease in brain lead concentration as well as reduced cerebellar cells death. CONCLUSION: Findings suggest that ADFS attenuated motor deficits via inhibition of oxidative stress and chelating activity which is comparable to succimer. Hence, ADFS should be explored for possible development of chelating agent against lead and other heavy metals toxicity.

Independent and combined effects of liraglutide and aerobic interval training on glycemic control and cardiac protection in diabetic cardiomyopathy rats.

OBJECTIVE: This study intended to explore the hypoglycemic and cardioprotective effects of 8-week aerobic interval training combined with liraglutide and elucidate the underlying mechanisms. METHOD: Male Wistar rats were randomly divided into 5 groups - normal control group (CON), diabetic cardiomyopathy group (DCM), high-dose liraglutide group (DH), low-dose liraglutide group (DL), and aerobic interval training combined with liraglutide group (DLE). High-fat diet and streptozotocin (STZ) were used to induce the DCM model, and both the liraglutide administration group and combination therapy group allocated to 8 weeks of either liraglutide or liraglutide and exercise intervention. Cardiac functions were analyzed by electrocardiography. Blood biochemical parameters were measured to judge glycemic control conditions. Hematoxylin and eosin (HE) staining and Sirus red staining was used to identify cardiac morphology and collagen accumulation, respectively. Advanced glycation end products (AGEs) were determined by enzymatic methods. The mRNA expression of myocardial remodeling genes (BNP, GSK3ß, α-MHC, ß-MHC and PPARα) and the protein expression of GLP-1, GLP-1R were analyzed. RESULTS: DCM rats developed hyperglycemia, impaired cardiac function with accumulation of AGEs and collagen (P < 0.05). The development of hyperglycemia and cardiac dysfunction was significantly attenuated with all interventions, as reduced cardiac fibrosis and improved cardiac function (P < 0.05). Cardiac remodeling genes were normalized after all interventions, these positive modifications were due to increased GLP-1 and GLP-1R expression in DCM heart (P < 0.05). Liraglutide combined with AIT significantly increased the diameters of cardiomyocytes, increased the α-MHC expressionx, reduced PPARαexpression and reduced the fluctuation of blood glucose level, which showed the safety and effective of medicine combined with exercise. CONCLUSION: Liraglutide combined with AIT intervention normalized blood glucose alleviates myocardial fibrosis and improves cardiac contractile function in DCM rats, supporting the efficacy and safety of the combination therapy.