Kaempferol 3-O-Rutinoside, a Flavone Derived from Tetrastigma hemsleyanum Diels et Gilg, Reduces Body Temperature through Accelerating the Elimination of IL-6 and TNF-α in a Mouse Fever Model.

Fever is a serious condition that can lead to various consequences ranging from prolonged illness to death. Tetrastigma hemsleyanum Diels et Gilg (T. hemsleyanum) has been used for centuries to treat fever, but the specific chemicals responsible for its antipyretic effects are not well understood. This study aimed to isolate and identify the chemicals with antipyretic bioactivity in T. hemsleyanum extracts and to provide an explanation for the use of T. hemsleyanum as a Chinese herbal medicine for fever treatment. Our results demonstrate that kaempferol 3-rutinoside (K3OR) could be successfully isolated and purified from the roots of T. hemsleyanum. Furthermore, K3OR exhibited a significant reduction in rectal temperature in a mouse model of fever. Notably, a 4 µM concentration of K3OR showed more effective antipyretic effects than ibuprofen and acetaminophen. To explore the underlying mechanism, we conducted an RNA sequencing analysis, which revealed that PXN may act as a key regulator in the fever process induced by lipopolysaccharide (LPS). In the mouse model of fever, K3OR significantly promoted the secretion of IL-6 and TNF-α during the early stage in the LPS-treated group. However, during the middle to late stages, K3OR facilitated the elimination of IL-6 and TNF-α in the LPS-treated group. Overall, our study successfully identified the chemicals responsible for the antipyretic bioactivity in T. hemsleyanum extracts, and it answered the question as to why T. hemsleyanum is used as a traditional Chinese herbal medicine for treating fever. These findings contribute to a better understanding of the therapeutic potential of T. hemsleyanum in managing fever, and they provide a basis for further research and development in this field.

Promotion of neurite outgrowth by 3,5,7,3',4'-pentamethoxyflavone is mediated through ERK signaling pathway in Neuro2a cells.

In this study, the effects of 3,5,7,3',4'-pentamethoxyflavone (KP1), a major bioactive ingredient isolated from the Kaempferia parviflora rhizomes, on a neurite outgrowth in Neuro2a cells and its mechanism have been investigated. KP1 increased concentration-dependently the percentage of neurite-bearing cells. KP1 showed a remarkable capability to elicit neurite outgrowth in Neuro2a cells, as evidenced by morphological alterations and immunostaining using anti-class III ß-tubulin and anti-NeuN antibodies. KP1 also displayed a higher neurogenic activity than retinoic acid (RA), a promoter of neurite outgrowth in Neuro2a cells. KP1 treatment caused significant elevation in phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38 MAPK) and glycogen synthase kinase-3ß (GSK-3ß). However, KP1-triggered neurite outgrowth was markedly inhibited by treatment with the ERK inhibitor U0126, whereas p38 MAPK inhibitor SB203580 and GSK-3ß inhibitor SB216763 did not influence KP1-induced neurite outgrowth. These results demonstrate that KP1 elicits neurite outgrowth and triggers cell differentiation of Neuro2a cells through ERK signal pathway.

A study on the anti-senescent effects of flavones derived from Prinsepia utilis Royle seed residue.

ETHNOPHARMACOLOGICAL RELEVANCE: Prinsepia utilis Royle, also known as the Anas fruit, is a unique perennial woody oil plant from Yunnan Province, China. In the ancient texts of Dongba sutras and Yunnan Southern Materia Medica, it has been documented that the local Naxi, Tibetan, and Mosuo communities extensively utilize the root and leaf fruits of green thorns for various purposes. These include treating mild-to-moderate specific dermatitis, moisturising the skin, providing protection against UV damage, aiding childbirth in pregnant women, safeguarding stomach health, reducing the risk of arteriosclerosis, and delaying aging. AIM OF THE STUDY: In this study, leftover residues from oil extraction were efficiently reused, and flavonoids were identified during subsequent extraction and separation processes. The anti-senescent effects of flavonoids in P. utilis Royle have not been systematically studied. Therefore, the objective of this study was to explore the anti-senescent properties of the flavonoids obtained from P. utilis Royle. METHODS: First, HPLC and other analytical techniques were used to identify the components of the P. utilis Royle flavonoid (PURF). Next, DPPH, hydroxyl radicals, superoxide anion O2-, collagenase, and elastase were initially detected using in vitro biochemical assays. To examine its antioxidant properties, a zebrafish model was used, and to confirm its anti-senescent effects, a d-galactose-induced mouse aging model was employed. The anti-senescent mechanism of PURF was examined using a natural senescence HFF model. Furthermore, the anti-senescent target was confirmed using a 3D full T-Skin™ model. RESULTS: In vitro biochemical assays demonstrated that flavones exhibited potent antioxidant activity and anti-senescent potential by inhibiting DPPH, hydroxyl radicals, superoxide anion O2-, collagenase, and elastase. It significantly enhanced the antioxidant effect on zebrafish while suppressing ROS and inflammatory injury, up-regulating COL1A1, COL3A1, AMPK, and mTOR gene expression and down-regulating MMP-9, TGF-ß, p21, and p16 gene expression suggesting its potential anti-senescent ability. Findings from the D-galactose-induced aging mouse model showed that PURF greatly increased SOD levels, while simultaneously decreasing HYP and MDA levels. In addition, when PURF was given to the HFF cell and 3D full T-Skin™ model, consistent trends were observed in gene and protein expression, with up-regulation of COL1A1, COL3A1, AMPK, and mTOR genes and down-regulation of TGF-ß, MMP-1, MMP-9, p21, and p16 genes. Therefore, these preliminary findings indicate that flavones can modulate AMPK/mTOR/TGF-ß signalling pathways to exert its influence. CONCLUSION: The kernel residue of natural P. utilis Royle oil extracted from Yunnan province was previously considered agricultural waste, but we successfully extracted and isolated its flavonoid components. Our preliminary studies demonstrated its potential as an environmentally friendly anti-senescent raw material.

Recent advances in the therapeutic potential of nobiletin against respiratory diseases.

BACKGROUND: Nobiletin is a natural polymethoxylated flavonoid widely present in citrus fruit peels. It has been demonstrated to exert the effects of anti-tumor, anti-inflammation, anti-oxidative, anti-apoptotic and improve cardiovascular function. Increasing evidences suggest that nobiletin plays an important role in respiratory diseases (RDs) treatment. OBJECTIVE: This review aimed to investigate the therapeutic potential of nobiletin against RDs, such as lung cancer, COPD, pulmonary fibrosis, asthma, pulmonary infection, acute lung injury, coronavirus disease 2019, and pulmonary arterial hypertension. METHODS: We retrieved extensive literature of relevant literatures in English until June 26, 2023 from the database of PubMed, Web of Science, and Scopus databases. The keywords of "nobiletin and lung", "nobiletin and respiratory disease", "nobiletin and chronic respiratory diseases", "nobiletin and metabolites", "nobiletin and pharmacokinetics", "nobiletin and toxicity" were searched in pairs. A total of 298 literatures were retrieved from the above database. After excluding the duplicates and reviews, 53 were included in the current review. RESULTS: We found that the therapeutic mechanisms are based on different signaling pathways. Firstly, nobiletin inhibited the proliferation and suppressed the invasion and migration of cancer cells by regulating the related pathway or key target, like Bcl-2, PD-L1, PARP, and Akt/GSK3ß/ß-catenin in lung cancer treatment. Secondly, nobiletin treats COPD and ALI by targeting classical signaling pathway mediating inflammation. Besides, the available findings show that nobiletin exerts the effect of PF treatment via regulating mTOR pathway. CONCLUSIONS: With the wide range of pharmacological activities, high efficiency and low toxicity, nobiletin can be used as a potential agent for preventing and treating RDs. These findings will contribute to further research on the molecular mechanisms of nobiletin and facilitate in-depth studies on nobiletin at both preclinical and clinical levels for the treatment of RDs.

Nobiletin targets SREBP1/ACLY to induce autophagy-dependent cell death of gastric cancer cells through PI3K/Akt/mTOR signaling pathway.

BACKGROUND: Autophagy could sense metabolic conditions and safeguard cells against nutrient deprivation, ultimately supporting the survival of cancer cells. Nobiletin (NOB) is a kind of bioactive component of the traditional Chinese medicine Citri Reticulatae Pericarpium and has been proven to induce GC cell death by reducing de novo fatty acid synthesis in our previous study. Nevertheless, the precise mechanisms by which NOB induces cell death in GC cells still need further elucidation. OBJECTIVES: To examine the mechanism by which NOB inhibits gastric cancer progression through the regulation of autophagy under the condition of lipid metabolism inhibition. METHODS/ STUDY DESIGN: Proliferation was detected by the CCK-8 assay. RNA sequencing (RNA-seq) was used to examine signaling pathway changes. Electron microscopy and mRFP-GFP-LC3 lentiviral transfection were performed to observe autophagy in vitro. Western blot, plasmid transfection, immunofluorescence staining, and CUT & Tag-qPCR techniques were utilized to explore the mechanisms by which NOB affects GC cells. Molecular docking and molecular dynamics simulations were conducted to predict the binding mode of NOB and SREBP1. CETSA was adopted to verify the predicted of binding model. A patient-derived xenograft (PDX) model was employed to verify the therapeutic efficacy of NOB in vivo. RESULTS: We conducted functional studies and discovered that NOB inhibited the protective effect of autophagy via the PI3K/Akt/mTOR axis in GC cells. Based on previous research, we found that the overexpression of ACLY abrogated the NOB-induced autophagy-dependent cell death. In silico analysis predicted the formation of a stable complex between NOB and SREBP1. In vitro assays confirmed that NOB treatment increased the thermal stability of SREBP1 at the same temperature conditions. Moreover, CUT&TAG-qPCR analysis revealed that NOB could inhibit SREBP1 binding to the ACLY promoter. In the PDX model, NOB suppressed tumor growth, causing SREBP1 nuclear translocation inhibition, PI3K/Akt/mTOR inactivation, and autophagy-dependent cell death. CONCLUSION: NOB demonstrated the ability to directly bind to SREBP1, inhibiting its nuclear translocation and binding to the ACLY promoter, thereby inducing autophagy-dependent cell death via PI3K/Akt/mTOR pathway.

Mechanism insights into the pleiotropic effects of nobiletin as a potential therapeutic agent on non-alcoholic fatty liver disease (NAFLD).

Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases and is emerging as one of the fastest-growing causes of liver-related deaths worldwide. It is necessary to find strategies to effectively prevent and treat NAFLD, as no definitive drug has been approved. Nobiletin (NOB) is the critical active ingredient of Chinese herbal medicines such as Citrus aurantium and Citri Reticulatae Pericarpium, which have anti-inflammatory, antioxidant, lipid regulating, and insulin resistance regulating effects. Numerous studies have demonstrated that NOB can prevent and treat the onset and progression of NAFLD. In this review, the mechanisms of NOB for treating NAFLD have been summarized, hoping to provide a basis for subsequent studies of NOB and to provide a research ground for the development of therapeutic drugs for NAFLD.

Phytochemical constituents from the rhizomes of Kaempferia parviflora Wall. ex Baker and their acetylcholinesterase inhibitory activity.

Phytochemical study on the rhizomes of Kaempferia parviflora led to the isolation of twenty-three compounds including six phenolic glycosides (1-6), thirteen flavones (7-19), and five phenolic compounds (20-23). Of these, the new compounds were determined to be 2,4-dihydroxy-6-methoxyacetophenone-2-ß-D-apiofuranosyl-(1→6)-ß-D-glucopyranoside (1), 2-hydroxy-4-propionyl-phenyl O-ß-D-glucopyranoside (2), and 4-hydroxy-3,5-dimethoxyacetophenone 8-O-α-L-rhamnopyranosyl-(1→6)-ß-D-glucopyranoside (3) and named as kaempanosides A-C, respectively. Their chemical structures were established based on HR-ESI-MS, 1D and 2D NMR spectra. All compounds 1-23 exhibited acetylcholinesterase inhibitory activity with IC50 values ranging from 57.76 to 253.31 µM.

Luteolin, a flavone ingredient: Anticancer mechanisms, combined medication strategy, pharmacokinetics, clinical trials, and pharmaceutical researches.

Current pharmaceutical research is energetically excavating the pharmacotherapeutic role of herb-derived ingredients in multiple malignancies' targeting. Luteolin is one of the major phytochemical components that exist in various traditional Chinese medicine or medical herbs. Mounting evidence reveals that this phytoconstituent endows prominent therapeutic actions on diverse malignancies, with the underlying mechanisms, combined medication strategy, and pharmacokinetics elusive. Additionally, the clinical trial and pharmaceutical investigation of luteolin remain to be systematically delineated. The present review aimed to comprehensively summarize the updated information with regard to the anticancer mechanism, combined medication strategies, pharmacokinetics, clinical trials, and pharmaceutical researches of luteolin. The survey corroborates that luteolin executes multiple anticancer effects mainly by dampening proliferation and invasion, spurring apoptosis, intercepting cell cycle, regulating autophagy and immune, inhibiting inflammatory response, inducing ferroptosis, and pyroptosis, as well as epigenetic modification, and so on. Luteolin can be applied in combination with numerous clinical anticarcinogens and natural ingredients to synergistically enhance the therapeutic efficacy of malignancies while reducing adverse reactions. For pharmacokinetics, luteolin has an unfavorable oral bioavailability, it mainly persists in plasma as glucuronides and sulfate-conjugates after being metabolized, and is regarded as potent inhibitors of OATP1B1 and OATP2B1, which may be messed with the pharmacokinetic interactions of miscellaneous bioactive substances in vivo. Besides, pharmaceutical innovation of luteolin with leading-edge drug delivery systems such as host-guest complexes, nanoparticles, liposomes, nanoemulsion, microspheres, and hydrogels are beneficial to the exploitation of luteolin-based products. Moreover, some registered clinical trials on luteolin are being carried out, yet clinical research on anticancer effects should be continuously promoted.

Effects of total flavones of Abelmoschus manihot (L.) on the treatment of diabetic nephropathy via the activation of solute carriers in renal tubular epithelial cells.

As a traditional Chinese medicine, Huangkui capsule (HKC) has been used to treat patients with kidney diseases, including diabetic nephropathy (DN). We have recently demonstrated that HKC could re-regulate the activities of solute carriers (SLC)s in proximal and distal convoluted tubules of kidneys in regression of the development of DN. The main active chemical constituents of HKC are the flavones of Abelmoschus manihot (L.). The current study aims to further evaluate the efficacy of total flavones of A. manihot (TFA) in the regression of DN by analyzing SLC activities in proximal and distal convoluted tubules of kidneys. TFA (0.076 g/kg/d) or vehicle was administered in db/db mice, the animal model of type 2 diabetes and DN, daily via oral gavage for four weeks. Blood glucose levels and urinary albumin-to-creatinine ratio (UACR) were measured and used for the determination of T2D and DN. Ten SLCs, including slc2a2, slc4A1, slc5a2, slc5A3, slc5a8, slc6a20, slc27a2, slc12a3, slc34a1 and slc38a2 were highly expressed in proximal and distinct convoluted tubules of kidneys. Their expression at mRNA and protein levels before and after TFA treatment were analyzed with real-time RT-PCR and immunohistochemistry. Data showed that UACR in the db/db mice after TFA treatment was significantly decreased. Compared with the group of non-diabetic control, slc2a2, slc4A1, slc5a2, slc5A3, slc5a8, slc6a20, slc27a2, slc12a3, slc34a1 and slc38a2 in the group of DN were down-regulated but up-regulated after TFA treatment. Further analyses of whole kidney sections indicated that the numbers and structures of the nephron in db/db mice was increased and improved after TFA treatment. Thereby, the current study provides further evidence that the flavones in A. manihot have pharmacological effects on the treatment of DN by improving the biological function of SLCs in kidneys.

[Effects and mechanisms of total flavones of Abelmoschus manihot in inhibiting podocyte necroptosis and renal fibrosis in diabetic kidney disease].

Previous studies have shown that high blood glucose-induced chronic microinflammation can cause inflammatory podocyte injury in patients with diabetic kidney disease(DKD). Therein, necroptosis is a new form of podocyte death that is closely associated with renal fibrosis(RF). To explore the effects and mechanisms in vivo of total flavones of Abelmoschus manihot(TFA), an extract from traditional Chinese herbal medicine Abelmoschus manihot for treating kidney diseases, on podocyte necroptosis and RF in DKD, and to further reveal its scientific connotation with multi-pathway and multi-target, the authors randomly divided all rats into four groups: a namely normal group, a model group, a TFA group and a rapamycin(RAP) group. After the modified DKD rat models were successfully established, four group rats were given double-distilled water, TFA suspension and RAP suspension, respectively by gavage every day. At the end of the 4th week of drug treatment, all rats were sacrificed, and the samples of their urine, blood and kidneys were collected. And then, the various indicators related to podocyte necroptosis and RF in the DKD model rats were observed, detected and analyzed, respectively. The results indicated that, general condition, body weight(BW), serum creatinine(Scr), urinary albumin(UAlb), and kidney hypertrophy index(KHI) in these modified DKD model rats were both improved by TFA and RAP. Indicators of RF, including glomerular histomorphological characteristics, fibronectin(FN) and collagen type Ⅰ(collagen Ⅰ) staining extent in glomeruli, as well as the protein expression levels of FN, collagen Ⅰ, transforming growth factor-ß1(TGF-ß1) and Smad2/3 in the kidneys were improved respectively by TFA and RAP. Podocyte damage, including foot process form and the protein expression levels of podocin and CD2AP in the kidneys was improved by TFA and RAP. In addition, tumor necrosis factor-α(TNF-α)-mediated podocyte necroptosis in the kidneys, including the morphological characteristics of podocyte necroptosis, the extent and levels of the protein expression of TNF-α and phosphorylated mixed lineage kinase domain like pseudokinase(p-MLKL) was improved respectively by TFA and RAP. Among them, RAP had the better effect on p-MLKL. More importantly, the activation of the receptor interacting serine/threonine protein kinase 1(RIPK1)/RIPK3/MLKL signaling axis in the kidneys, including the expression levels of its key signaling molecules, such as phosphorylated receptor interacting serine/threonine protein kinase 1(p-RIPK1), p-RIPK3, p-MLKL and cysteinyl aspartate specific proteinase-8(caspase-8) was improved respectively by TFA and RAP. Among them, the effect of TFA on p-RIPK1 was superior. On the whole, in this study, the authors demonstrated that TFA alleviates podocyte necroptosis and RF in DKD through inhibiting the activation of the TNF-α-mediated RIPK1/RIPK3/MLKL signaling axis in diabetic kidneys. The authors' findings provide new pharmacological evidence to reveal the scientific connotation of TFA in treating RF in DKD in more depth.

Total syntheses and antibacterial evaluations of cudraflavones A-C and related Flavones.

The total syntheses of the natural prenylated flavones cudraflavones A-C (1-3), artoheterophyllin D (28) and artelasticin (29) are reported, along with the evaluations of their antibacterial activities. The key steps of the synthesis involved a Baker-Venkataraman rearrangement and an intramolecular cyclization for the construction of the flavone core and the regioselective formation of the pyran and isopentenyl scaffolds. The tested natural flavones 1-3 and 27-29 exhibited potent activity against S. aureus ATCC 29213, S. epidermidis ATCC 14990, E. faecalis ATCC 29212 and B. subtilis ATCC 6633 with MIC values ranging from 0.125 µg/mL to 16 µg/mL. Compound 3 displayed the strongest potency, with MIC values in the range between 0.125 and 1 µg/mL, as a potential candidate to combat G+ bacterial infections. Preliminary mechanism of action studies suggested that this compound killed bacteria by disrupting bacterial membrane integrity.

Garcinia buchananii stem bark extract and its bioactive constituents manniflavanone, GB-2 and buchananiflavanone attenuate intestinal inhibitory neuromuscular transmission.

Garcinia buchananii stem bark extract (GBB), commonly used for treating diarrhea in Africa, triggers ectopic aboral contractions, causing inhibition of propulsive motility in the colon ex vivo. To determine whether or not these effects were associated with decreased inhibitory neuromuscular transmission, the responsible constituent compounds, and mechanisms of action, we studied the effects of GBB and specific fractions and flavanones isolated from GBB on intestinal motility using pellet propulsion assays in guinea pig distal colons. In addition, microelectrode recordings were used to measure the effects on the inhibitory junction potentials (IJPs) in the porcine ileum and descending colon smooth muscle. Psychoactive Drug Screening Program secondary receptor functional assays were used to determine whether or not GBB and its constituent compounds act via purinergic (P2Y) and muscarinic receptors. GBB inhibited propulsive motility, but (2R,3S,2″R,3″R)-manniflavanone (MNF), (2R,3S,2″R,3″R)-GB-2 (GB-2) and (2R,3S,2″S)-buchananiflavanone (BNF), the main ingredients of GBB, did not affect motility. We discovered that, in the porcine descending colon, IJPs contained purinergic, nitrergic, and nonpurinergic nonnitrergic components. Furthermore, ileal IJPs were purely purinergic. GBB blocked all components of IJPs, while MNF and GB-2 inhibited purinergic IJPs only. BNF inhibited the purinergic and nonpurinergic components of IJPs. MRS2365, a Y1 (P2Y) agonist, did not evoke sustained membrane hyperpolarization in the presence of GBB. However, GBB, MNF, GB-2 and BNF did not affect P2Y or muscarinic receptors. In conclusion, inhibitory neuromuscular transmission in the porcine descending colon involves all components of IJPs. GBB decreases inhibitory neuromuscular transmission, likely by the actions of MNF, GB-2 and BNF. These effects do not involve P2Y or muscarinic receptors.

Genkwanin: An emerging natural compound with multifaceted pharmacological effects.

Plant bioactive molecules could play key preventive and therapeutic roles in chronological aging and the pathogenesis of many chronic diseases, often accompanied by increased oxidative stress and low-grade inflammation. Dietary antioxidants, including genkwanin, could decrease oxidative stress and the expression of pro-inflammatory cytokines or pathways. The present study is the first comprehensive review of genkwanin, a methoxyflavone found in several plant species. Indeed, natural sources, and pharmacokinetics of genkwanin, the biological properties were discussed and highlighted in detail. This review analyzed and considered all original studies related to identification, isolation, quantification, investigation of the biological and pharmacological properties of genkwanin. We consulted all published papers in peer-reviewed journals in the English language from the inception of each database to 12 May 2023. Different phytochemical demonstrated that genkwanin is a non-glycosylated flavone found and isolated from several medicinal plants such as Genkwa Flos, Rosmarinus officinalis, Salvia officinalis, and Leonurus sibiricus. In vitro and in vivo biological and pharmacological investigations showed that Genkwanin exhibits remarkable antioxidant and anti-inflammatory activities, genkwanin, via activation of glucokinase, has shown antihyperglycemic activity with a potential role against metabolic syndrome and diabetes. Additionally, it revealed cardioprotective and neuroprotective properties, thus reducing the risk of cardiovascular diseases and assisting against neurodegenerative diseases. Furthermore, genkwanin showed other biological properties like antitumor capability, antibacterial, antiviral, and dermato-protective effects. The involved mechanisms include sub-cellular, cellular and molecular actions at different levels such as inducing apoptosis and inhibiting the growth and proliferation of cancer cells. Despite the findings from preclinical studies that have demonstrated the effects of genkwanin and its diverse mechanisms of action, additional research is required to comprehensively explore its therapeutic potential. Primarily, extensive studies should be carried out to enhance our understanding of the molecule's pharmacodynamic actions and pharmacokinetic pathways. Moreover, toxicological and clinical investigations should be undertaken to assess the safety and clinical efficacy of genkwanin. These forthcoming studies are of utmost importance in fully unlocking the potential of this molecule in the realm of therapeutic applications.

[Effects and mechanisms of total flavones of Abelmoschus manihot in improving insulin resistance and podocyte epithelial-mesenchymal transition in diabetic kidney disease based on IRS1/PI3K/Akt pathway].

This study aimed to explore the effects and mechanisms of total flavones of Abelmoschus manihot(TFA), the extracts from traditional Chinese medicine indicated for kidney diseases, on insulin resistance(IR) and podocyte epithelial-mesenchymal transition(EMT) in diabetic kidney disease(DKD), and further to reveal the scientific connotation. Thirty-two rats were randomly divided into a normal group, a model group, a TFA group, and a rosiglitazone(ROS) group. The modified DKD model was induced in rats by methods including high-fat diet feeding, unilateral nephrectomy, and streptozotocin(STZ) intraperitoneal injection. After modeling, the rats in the four groups were given double-distilled water, TFA suspension, and ROS suspension correspondingly by gavage every day. At the end of the 8th week of drug administration, all rats were sacrificed, and the samples of urine, blood, and kidney tissues were collected. The parameters and indicators related to IR and podocyte EMT in the DKD model rats were examined and observed, including the general condition, body weight(BW) and kidney weight(KW), the biochemical parameters and IR indicators, the protein expression levels of the key signaling molecules and structural molecules of slit diaphragm in the renal insulin receptor substrate(IRS) 1/phosphatidylinositol 3-kinase(PI3K)/serine-threonine kinase(Akt) pathway, foot process form and glomerular basement membrane(GBM) thickness, the expression of the marked molecules and structural molecules of slit diaphragm in podocyte EMT, and glomerular histomorphological characteristics. The results showed that for the DKD model rats, both TFA and ROS could improve the general condition, some biochemical parameters, renal appearance, and KW. The ameliorative effects of TFA and ROS were equivalent on BW, urinary albumin(UAlb)/urinary creatinine(UCr), serum creatinine(Scr), triglyceride(TG), and KW. Secondly, they could both improve IR indicators, and ROS was superior to TFA in improving fast insulin(FIN) and homeostasis model assessment of insulin resistance(HOMA-IR). Thirdly, they could both improve the protein expression levels of the key signaling molecules in the IRS1/PI3K/Akt pathway and glomerulosclerosis in varying degrees, and their ameliorative effects were similar. Finally, both could improve podocyte injury and EMT, and TFA was superior to ROS. In conclusion, this study suggested that podocyte EMT and glomerulosclerosis could be induced by IR and the decreased activation of the IRS1/PI3K/Akt pathway in the kidney in DKD. Similar to ROS, the effects of TFA in inhibiting podocyte EMT in DKD were related to inducing the activation of the IRS1/PI3K/Akt pathway and improving IR, which could be one of the scientific connotations of TFA against DKD. This study provides preliminary pharmacological evidence for the development and application of TFA in the field of diabetic complications.

[Effects and mechanisms of total flavones of Abelmoschus manihot in attenuating diabetic tubulopathy by targeting endoplasmic reticulum stress-induced cell apoptosis].

Renal tubular injury in patients with diabetic kidney disease(DKD) may be accompanied by glomerular and microvascular diseases. It plays a critical role in the progression of renal damage in DKD, and is now known as diabetic tubulopathy(DT). To explore the multi-targeted therapeutic effects and pharmacological mechanisms in vivo of total flavones of Abelmoschus manihot(TFA), an extract from traditional Chinese medicine for treating kidney disease, in attenuating DT, the authors randomly divided all rats into four groups: a normal control group(normal group), a DT model group(model group), a DT model+TFA-treated group(TFA group) and a DT model+rosiglitazone(ROS)-treated group(ROS group). The DT rat model was established based on the DKD rat model by means of integrated measures. After successful modeling, the rats in the four groups were continuously given double-distilled water, TFA suspension, and ROS suspension, respectively by gavage every day. After 6 weeks of treatment, all rats were sacrificed, and the samples of their urine, blood, and kidneys were collected. The effects of TFA and ROS on various indicators related to urine and blood biochemistry, renal tubular injury, renal tubular epithelial cell apoptosis and endoplasmic reticulum stress(ERS), as well as the activation of the protein kinase R-like endoplasmic reticulum kinase(PERK)-eukaryotic translation initiation factor 2α(eIF2α)-activating transcription factor 4(ATF4)-C/EBP homologous protein(CHOP) signaling pathway in the kidney of the DT model rats were investigated. The results indicated that hypertrophy of renal tubular epithelial cells, renal tubular hyperplasia and occlusion, as well as interstitial extracellular matrix and collagen deposition occurred in the DT model rats. Moreover, significant changes were found in the expression degree and the protein expression level of renal tubular injury markers. In addition, there was an abnormal increase in tubular urine proteins. After TFA or ROS treatment, urine protein, the characteristics of renal tubular injury, renal tubular epithelial cell apoptosis and ERS, as well as the activation of the PERK-eIF2α-ATF4-CHOP signaling pathway in the kidney of the DT model rats were improved to varying degrees. Therein, TFA was superior to ROS in affecting the pathological changes in renal tubule/interstitium. In short, with the DT model rats, this study demonstrated that TFA could attenuate DT by multiple targets through inhibiting renal tubular ERS-induced cell apoptosis in vivo, and its effect and mechanism were related to suppressing the activation of the PERK-eIF2α-ATF4-CHOP signaling pathway in the kidney. These findings provided preliminary pharmacological evidence for the application of TFA in the clinical treatment of DT.

The neurotrophic activities of brain-derived neurotrophic factor are potentiated by binding with apigenin, a common flavone in vegetables, in stimulating the receptor signaling.

AIMS: We aimed to identify the neurotrophic activities of apigenin (4',5,7-trihydroxyflavone) via its coordination with brain-derived neurotrophic factor (BNDF) and an elevated signaling of tyrosine kinase receptor B (Trk B receptor). METHODS: The direct binding of apigenin to BDNF was validated by ultrafiltration and biacore assay. Neurogenesis, triggered by apigenin and/or BDNF, was determined in cultured SH-SY5Y cells and rat cortical neurons. The amyloid-beta (Aß)25-35 -induced cellular stress was revealed by propidium iodide staining, mitochondrial membrane potential, bioenergetic analysis, and formation of reactive oxygen species levels. Activation of Trk B signaling was tested by western blotting. RESULTS: Apigenin and BDNF synergistically maintained the cell viability and promoted neurite outgrowth of cultured neurons. In addition, the BDNF-induced neurogenesis of cultured neurons was markedly potentiated by applied apigenin, including the induced expressions of neurofilaments, PSD-95 and synaptotagmin. Moreover, the synergy of apigenin and BDNF alleviated the (Aß)25-35 -induced cytotoxicity and mitochondrial dysfunction. The synergy could be accounted by phosphorylation of Trk B receptor, and which was fully blocked by a Trk inhibitor K252a. CONCLUSION: Apigenin potentiates the neurotrophic activities of BDNF through direct binding, which may serve as a possible treatment for its curative efficiency in neurodegenerative diseases and depression.

An investigation into the mechanism of nobiletin's inhibition of papillary thyroid cancer using network pharmacology analysis and experimental pharmacology.

OBJECTIVE: Surgery and radioactive iodine therapy are the main treatments for papillary thyroid carcinoma (PTC), and effective drugs are lacking. As a promising natural product, nobiletin (NOB) has a wealth of pharmacological activities like anti-tumor, antivirus, and other effects. In this research, bioinformatics methods and cellular assays were combined to explore how NOB inhibited PTC. MATERIALS AND METHODS: Our NOB targets were derived from three databases, including the SwissTargetPrediction database, Traditional Chinese Medicine System Pharmacology Database, and the TargetNet server. Four databases were used to identify disease-related targets: GeneCards, PharmGkb, Online Mendelian Inheritance in Man, and DisGeNET. Finally, cross-targets of disease and drug were deemed as pharmacological targets, and they were used for GO and KEGG enrichment analysis. STRING and Cytoscape were applied for PPI Network and core Targets Ranking. Molecular docking analysis validated binding affinity values for NOB and core targets. By using cell proliferation and migration assays, NOB was assessed for its effects on PTC proliferation and migration phenotype. Western blot validated the downregulation of the PI3K/Akt pathway. RESULTS: (1) Preliminarily, 85 NOB targets were predicted for NOB intervention in PTC. (2) Our core target screening identified TNF, TP53, and EGFR, and our molecular docking results confirmed good binding between NOB and protein receptors. (3) NOB inhibited proliferation and migration of PTC cells. PI3K/AKT pathway target proteins were downregulated. CONCLUSIONS: (1) Bioinformatics analyses revealed that NOB may inhibit PTC by regulating TNF, TP53, EGFR and PI3K/AKT signalling pathway. (2) As evidenced by cell experiments, there was an inhibition of proliferating and migrating PTCs by NOB via the PI3K/AKT signalling pathway.

JFD, a Novel Natural Inhibitor of Keap1 Alkylation, Suppresses Intracellular Mycobacterium Tuberculosis Growth through Keap1/Nrf2/SOD2-Mediated ROS Accumulation.

It is an effective strategy to treat tuberculosis by enhancing reactive oxygen species- (ROS-) mediated killing of Mycobacterium tuberculosis in macrophages, but there are no current therapeutic agents targeting this pathway. Honeysuckle has been used as the traditional medicine for tuberculosis treatment for 1500 years. Japoflavone D (JFD) is a novel biflavonoid isolated from Honeysuckle promoting ROS accumulation by Nrf2 pathway in hepatocarcinoma cells. However, its activity to kill M. tuberculosis in macrophages and molecular mechanism has not been reported. Our results showed that JFD enhances the M. tuberculosis elimination by boosting ROS levels in THP-1 cells. Moreover, the massive ROS accumulation activates p38 to induce apoptosis. Notably, the mechanism revealed that JFD suppresses the nuclear transport of Nrf2, thereby inhibiting SOD2 transcription, leading to a large ROS accumulation. Further studies showed that JFD disrupts the Keap1 alkylation at specific residues Cys14, Cys257, and Cys319, which is crucial for Nrf2 activation, thereby interrupts the nuclear transport of Nrf2. In pharmacokinetic study, JFD can stay as the prototype for 24 h in mice and can be excreted in feces without any toxicity. Our data reveal for the first time that a novel biflavonoid JFD as a potent inhibitor of Keap1 alkylation can suppress the nuclear transport of Nrf2. And it is the first research of the inhibitor of Keap1 alkylation. Furthermore, JFD robustly promotes M. tuberculosis elimination from macrophages by inhibiting Keap1/Nrf2/SOD2 pathway, resulting in the ROS accumulation. This work identified Keap1 alkylation as a new drug target for tuberculosis and provides a preliminary basis for the development of antituberculosis lead compounds based on JFD.

Alpinetin inhibits neuroinflammation and neuronal apoptosis via targeting the JAK2/STAT3 signaling pathway in spinal cord injury.

BACKGROUND: A growing body of research shows that drug monomers from traditional Chinese herbal medicines have antineuroinflammatory and neuroprotective effects that can significantly improve the recovery of motor function after spinal cord injury (SCI). Here, we explore the role and molecular mechanisms of Alpinetin on activating microglia-mediated neuroinflammation and neuronal apoptosis after SCI. METHODS: Stimulation of microglia with lipopolysaccharide (LPS) to simulate neuroinflammation models in vitro, the effect of Alpinetin on the release of pro-inflammatory mediators in LPS-induced microglia and its mechanism were detected. In addition, a co-culture system of microglia and neuronal cells was constructed to assess the effect of Alpinetin on activating microglia-mediated neuronal apoptosis. Finally, rat spinal cord injury models were used to study the effects on inflammation, neuronal apoptosis, axonal regeneration, and motor function recovery in Alpinetin. RESULTS: Alpinetin inhibits microglia-mediated neuroinflammation and activity of the JAK2/STAT3 pathway. Alpinetin can also reverse activated microglia-mediated reactive oxygen species (ROS) production and decrease of mitochondrial membrane potential (MMP) in PC12 neuronal cells. In addition, in vivo Alpinetin significantly inhibits the inflammatory response and neuronal apoptosis, improves axonal regeneration, and recovery of motor function. CONCLUSION: Alpinetin can be used to treat neurodegenerative diseases and is a novel drug candidate for the treatment of microglia-mediated neuroinflammation.

Total flavones of Rhododendron induce the transformation of A1/A2 astrocytes via promoting the release of CBS-produced H2S.

BACKGROUND: We previously found that total flavones of Rhododendron (TFR) protected against the cerebral ischemia/reperfusion (I/R) injury. But the detailed mechanism is not clear. Recent research revealed that reactive astrocytes were divided into A1 and A2 phenotypes for their morphological and functional remodeling and neurotoxic- vs-neuroprotective effect on the injury of the central nervous system (CNS). PURPOSE: The present study was undertaken to explore the role and mechanism of TFR on the phenotypic change of astrocytes following cerebral I/R in vivo and oxygen glucose deprivation/re-oxygenation (OGD/R) in vitro. STUDY DESIGN AND METHODS: We tested the expression of astrocytes marker glial fibrillary acidic protein (GFAP), A1 astrocytes marker C3 protein and A2 astrocytes marker S100a10, as well as the BrdU/GFAP-positive cells, GFAP/S100a10-positive cells and GFAP/C3-positive cells in mice hippocampal tissues to evaluate the phenotypic change of astrocytes. Besides, we assessed the change of astrocyte phenotypes following OGD/R in vitro. RESULTS: We found that mice cerebral I/R promoted the astrocytes proliferation of both A1 and A2 phenotypes in hippocampal tissues. While treatment with TFR could promote the proliferation of A2 astrocytes but inhibit the A1 astrocytes proliferation in mice hippocampal tissues, suggesting that TFR could accelerate the astrocytes transformation into A2 subtype following cerebral I/R. Whereas, in OGD/R model of astrocytes, we found that TFR inhibited the proliferation of both A1 and A2 astrocytes. Besides, we found that TFR could up-regulate the release of cystathionine ß-synthase (CBS)-produced hydrogen sulfide (H2S) and inhibit RhoA/Rho kinase pathway, and revealed that the inhibitory effect of TFR on astrocytes proliferation could be blocked by aminooxyacetic acid (AOAA), an CBS inhibitor. Furthermore, TFR could ameliorate the mice cerebral I/R injury and the OGD/R-induced astrocytic damage. CONCLUSION: These findings suggested that TFR could affect the transformation of astrocytes subtypes following cerebral I/R, which may be related to up-regulation of CBS-produced H2S and subsequent inhibition of RhoA/ROCK pathway.