AMPK x NF-κB x PGC-1α x SIRT1

This study explores the role of PFKFB3 in pulpitis and its potential as a therapeutic target by modulating glycolytic reprogramming in dental pulp stem cells (DPSCs). Pulpitis, a common inflammatory disease, causes long‐term damage to dental structures. Recent evidence suggests that metabolic reprogramming can modulate inflammatory responses and promote tissue repair. This study aims to investigate the anti‐inflammatory and reparative effects of glycolysis inhibitors on inflamed dental pulp through in vivo and in vitro experiments.

In vitro, hDPSCs were stimulated with lipopolysaccharide (LPS) (1 μg/mL, 3 h) to mimic pulpitis. The effects of the PFKFB3 inhibitor 3PO (10 μM) and siRNA targeting PFKFB3 (50 nM) on glycolysis were assessed using Seahorse analysis, while their impacts on inflammation were evaluated via ELISA and qRT‐PCR. A co‐culture of DPSCs and macrophages was used to study 3PO's effects on inflammation interactions and glycolytic reprogramming of the inflammatory microenvironment. The influences of 3PO on odontogenic differentiation were examined through qRT‐PCR, Western blotting, ALP staining and ARS staining. The related signalling pathways were validated through Western blot (WB) experiments. An SD rat model was employed to validate the in vivo efficacy of 3PO@GelMA pulp capping, and HE staining and immunohistochemistry were used to evaluate the degree of pulp inflammation and reparative dentine formation.

In vitro, LPS elevated glycolytic activity and inflammatory factors (IL‐6, IL‐1β, TNF‐α) in DPSCs. 3PO and si‐PFKFB3 mitigated these factors and promoted odontogenic differentiation, evidenced by increased DSPP and ALP expression. Metabolic reprogramming mediated by 3PO and si‐PFKFB3, as assessed by Seahorse XF analysis, indicated a shift from glycolysis to oxidative phosphorylation. Additionally, 3PO induced metabolic reprogramming of the co‐culture system, reduced the levels of pro‐inflammatory cytokines, and promoted the polarisation of macrophages towards the M2 phenotype. PFKFB3 inhibition activated the AMPK/SIRT1/PGC‐1α/NF‐κB and AMPK/mTOR/NF‐κB signalling pathways. In vivo, in the rat pulpitis model, 3PO@GelMA hydrogel application resulted in significantly decreased IL‐6 and increased DSPP expression compared to the LPS group ( p  < 0.001), accompanied by reduced inflammation and enhanced reparative dentine formation.

The PFKFB3 inhibitor 3PO reduces inflammation and promotes reparative dentine formation in pulpitis via metabolic reprogramming and specific signalling pathways, offering a new therapeutic strategy.

Obesity is typically identified by a high body mass index. Chronic inflammation is a central characteristic of obesity, contributing to many complications. The unrelenting rise in obesity and inflammation has been a global crisis. Due to the weight loss effects of black tea and anti-inflammatory effect of polyphenols, Theaflavins (TFs), the major bioactive polyphenols of black tea, may provide new strategies against obesity and inflammation.

This study aimed to investigate the potential regulatory roles and the underlying molecular mechanism of TFs on obesity and obesity-related inflammation.

Animal experiments were performed using high-fat diet induced obese SD rats. TFs were orally administered at doses of 25, 50, and 100 mg·kg⁻¹ for 8 consecutive weeks. The rats' weight and length were recorded. The blood, liver, epididymal fat pads, colon, interscapular adipose tissue, and feces were collected and stored.

TFs' effects on obesity and inflammation were evaluated by body weight (daily observation) and inflammatory cytokines (ELISA). The lipid deposition and hepatic function was examined using biochemical assessment and histopathological analysis. AMPK-mediated metabolism assessed by Western blotting (WB), immunohistochemistry, and qRT-PCR. The intestinal barrier integrity was evaluated by biochemical assessment, histopathological analysis, WB, qRT-PCR, and immunofluorescence staining. Gut microbiota and fecal metabolites were using 16S rRNA sequencing and LC-MS/MS analysis, respectively.

TFs exerted protective effects by mitigating body weight, lipid deposition, and immune response in the whole body, adipose tissues, and colon. The underlying mechanism was associated with the gut microbiota, AMPK-mediated metabolism, and NF-κB pathway. Specifically, TFs promoted the potential probiotics (Alistipes, Akkermansia, Coprococcus, NK4A214_group, Collinsella, and Rikenellaceae_RC9_gut_group) and regulated metabolic pathway (starch and sucrose metabolism, α-linolenic acid metabolism, and glycine‑serine-threonine metabolism), thereby inhibiting TRL4/MyD88/NF-κB pathway and protecting the intestinal barrier. Moreover, TFs activated the AMPK/SREBP-1 pathway and AMPK/SIRT1/PGC-1α/UCP1 pathway to mitigate the lipid metabolism and energy metabolism, respectively.

We have innovatively demonstrated for the first time that TFs could alleviate the obesity and systemic inflammation via the modulation of gut microbiota and AMPK-mediated metabolism in HFD-fed rats. The impacts on white adipose tissue browning and thermogenic activity of brown adipose tissue of theaflavins firstly proposed a potential mechanism underlying the protection of theaflavins through the gut microbiota-liver/adipose tissue axis. These findings provide new sights on the bioactive ingredients through which black tea modulates obesity and the underlying mechanism.

The reno-protective potential of canagliflozin (Cana), an inhibitor of the sodium glucose–linked co-transporter-2 (SGLT-2), has been demonstrated in different models of kidney injury. However, its potential role in preventing glycerol (Gly)-induced acute kidney injury (AKI) remains to be divulged. Therefore, the aim of this study is to investigate the potential reno-protective effect of Cana and its underlying mechanism in a rat model of Gly-induced AKI. Rats were randomly allocated into five groups: normal, Gly, Gly pretreated with 10 mg/kg Cana, Gly pretreated with Cana 25 mg/kg, and normal pretreated with Cana 25 mg/kg for 14 consecutive days. Pretreatment with Cana improved renal structure and enhanced kidney functions manifested by reducing serum creatinine and blood urea nitrogen, as well as renal contents of neutrophil gelatinase-associated lipocalin and kidney injury molecule. Moreover, Cana signified its anti-inflammatory effect by reducing the Gly-induced elevation in renal contents of nuclear factor-κB and interleuκin-6. Additionally, Cana augmented the defense enzymatic antioxidants superoxide dismutase (SOD), manganese-SOD, and heme oxygenase-1, besides increasing the protein expression of the antioxidant transcription factor nuclear factor erythroid 2–related factor 2 to point for its ability to correct redox balance. Cana also upregulated the protein expression of the 5′ adenosine monophosphate-activated protein kinase (AMPK), Sirtuin1 (SIRT1), Forkhead box protein O3 (FOXO-3a), and peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α), as well as the transcriptional activity of growth arrest and DNA damage–inducible protein alpha (GAAD45a). In conclusion, Cana demonstrated potentially novel reno-protective mechanisms and mitigated the consequences of AKI through its antioxidant and anti-inflammatory properties, partially by activating the AMPK/SIRT1/FOXO-3a/PGC-1α pathway.