IP3R2 regulates apoptosis by Ca2+ transfer through mitochondria-ER contacts in hypoxic photoreceptor injury.

Mitochondria-associated ER membranes (MAMs) are contact sites that enable bidirectional communication between the ER (endoplasmic reticulum) and mitochondria, including the transfer of Ca2+ signals. MAMs are essential for mitochondrial function and cellular energy metabolism. However, unrestrained Ca2+ transfer to the mitochondria can lead to mitochondria-dependent apoptosis. IP3R2 (Inositol 1,4,5-trisphosphate receptor 2) is an important intracellular Ca2+ channel. This study investigated the contribution of IP3R2-MAMs to hypoxia-induced apoptosis in photoreceptor cells. A photoreceptor hypoxia model was established by subretinal injection of hyaluronic acid (1%) in C57BL/6 mice and 1% O2 treatment in 661W cells. Transmission electron microscopy (TEM), ER-mitochondria colocalization, and the MAM reporter were utilized to evaluate MAM alterations. Cell apoptosis and mitochondrial homeostasis were evaluated using immunofluorescence (IF), flow cytometry, western blotting (WB), and ATP assays. SiRNA transfection was employed to silence IP3R2 in 661W cells. Upon hypoxia induction, MAMs were significantly increased in photoreceptors both in vivo and in vitro. This was accompanied by the activation of mitochondrial apoptosis and disruption of mitochondrial homeostasis. Elevated MAM-enriched IP3R2 protein levels induced by hypoxic injury led to mitochondrial calcium overload and subsequent photoreceptor apoptosis. Notably, IP3R2 knockdown not only improved mitochondrial morphology but also restored mitochondrial function in photoreceptors by limiting MAM formation and thereby attenuating mitochondrial calcium overload under hypoxia. Our results suggest that IP3R2-MAM-mediated mitochondrial calcium overload plays a critical role in mitochondrial dyshomeostasis, ultimately contributing to photoreceptor cell death. Targeting MAM constitutive proteins might provide an option for a therapeutic approach to mitigate photoreceptor death in retinal detachment.

RIP3 regulates doxorubicin-induced intestinal mucositis via FUT2-mediated α-1,2-fucosylation.

OBJECTIVE: Intestinal mucositis is one of the common side effects of anti-cancer chemotherapy. However, the molecular mechanisms involved in mucositis development remain incompletely understood. In this study, we investigated the function of receptor-interacting protein kinase 3 (RIP3/RIPK3) in regulating doxorubicin-induced intestinal mucositis and its potential mechanisms. METHODS: Intestinal mucositis animal models were induced in mice for in vivo studies. Rat intestinal cell line IEC-6 was used for in vitro studies. RNA­seq was used to explore the transcriptomic changes in doxorubicin-induced intestinal mucositis. Intact glycopeptide characterization using mass spectrometry was applied to identify α-1,2-fucosylated proteins associated with mucositis. RESULTS: Doxorubicin treatment increased RIP3 expression in the intestine and caused severe intestinal mucositis in the mice, depletion of RIP3 abolished doxorubicin-induced intestinal mucositis. RIP3-mediated doxorubicin-induced mucositis did not depend on mixed lineage kinase domain-like (MLKL) but on α-1,2-fucosyltransferase 2 (FUT2)-catalyzed α-1,2-fucosylation on inflammation-related proteins. Deficiency of MLKL did not affect intestinal mucositis, whereas inhibition of α-1,2-fucosylation by 2-deoxy-D-galactose (2dGal) profoundly attenuated doxorubicin-induced inflammation and mucositis. CONCLUSIONS: RIP3-FUT2 pathway is a central node in doxorubicin-induced intestinal mucositis. Targeting intestinal RIP3 and/or FUT2-mediated α-1,2-fucosylation may provide potential targets for preventing chemotherapy-induced intestinal mucositis.

Lessons learned from a multi-data source research collaboration: The mirabegron post-authorization safety study program.

BACKGROUND: Many factors contribute to developing and conducting a successful multi-data source, non-interventional, post-authorization safety study (NI-PASS) for submission to multiple health authorities. Such studies are often large undertakings; evaluating and sharing lessons learned can provide useful insights to others considering similar studies. OBJECTIVES: We discuss challenges and key methodological and organizational factors that led to the delivery of a successful post-marketing requirement (PMR)/PASS program investigating the risk of cardiovascular and cancer events among users of mirabegron, an oral medication for the treatment of overactive bladder. RESULTS: We provide context and share learnings, including sections on research program collaboration, scientific transparency, organizational approach, mitigation of uncertainty around potential delays, validity of study outcomes, selection of data sources and optimizing patient numbers, choice of comparator groups and enhancing precision of estimates of associations, potential confounding and generalizability of study findings, and interpretation of results. CONCLUSIONS: This large PMR/PASS program was a long-term commitment from all parties and benefited from an effective coordinating center and extensive scientific interactions across research partners, scientific advisory board, study sponsor, and health authorities, and delivered useful learnings related to the design and organization of multi-data source NI-PASS.

Anticholinergic exposure and its association with dementia/Alzheimer's disease and mortality in older adults.

BACKGROUND: Use of anticholinergic (ACH) medications is associated with increased risk of cognitive decline in the elderly. However, little is known about this association from a health plan perspective. METHODS: This retrospective cohort study used the Humana Research Database to identify individuals with at least one ACH medication dispensed in 2015. Patients were followed until incidence of dementia/Alzheimer's disease, death, disenrollment or end of December 2019. Multivariate Cox regression models were used to assess the association between ACH exposure and study outcomes, adjusting for demographics and clinical characteristics. RESULTS: A total of 12,209 individuals with no prior ACH use or dementia/Alzheimer's disease diagnosis were included. As ACH polypharmacy increased (i.e., from no ACH exposure, to one, two, three, and four or more ACH medications), there was a stair-step increase in the incidence rate of dementia/Alzheimer's disease (15, 30, 46, 56 and 77 per 1,000 person-years of follow-up) and in the incidence of mortality (19, 37, 80, 115 and 159 per 1,000 person-years of follow-up). After adjusting for confounders, ACH exposure to one, two, three and four or more ACH medications was associated with a 1.6 (95% CI 1.4-1.9), 2.1 (95% CI 1.7-2.8), 2.6 (95% CI 1.5-4.4), and 2.6 (95% CI 1.1-6.3) times, respectively, increased risk of a dementia/Alzheimer's disease diagnosis compared to periods of no ACH exposure. ACH exposure to one, two, three and four or more medications was associated with a 1.4 (95% CI 1.2-1.6), 2.6 (95% CI 2.1-3.3), 3.8 (95% CI 2.6-5.4), and 3.4 (95% CI 1.8-6.4) times, respectively, increased risk of mortality compared to periods of no ACH exposure. CONCLUSIONS: Reducing ACH exposure may potentially minimize long-term adverse effects in older adults. Results suggest populations which may benefit from targeted interventions to reduce ACH polypharmacy.

Dahuang Fuzi Baijiang decoction restricts progenitor to terminally exhausted T cell differentiation in colorectal cancer.

Obesity increases the risk of colorectal cancer (CRC) by 30%. The obese tumor microenvironment compromises antitumor immunity by eliciting exhausted T cells (Tex). Hypothesizing that Dahuang Fuzi Baijiang decoction (DFB) is a combined classical prescription from the "Synopsis of Prescriptions of the Golden Chamber". We first determined that DFB regresses tumor growth in high-fat diet-induced obese mice by expanding the TIM3- subset with intermediate expression of programmed cell death-1 (PD-1int TIM3- ) and restricting the PD-1hi TIM3+ subset. Transcription factor 1 (TCF1) is highly expressed in the PD-1int TIM3- subset but is absent in PD-1hi TIM3+ cells. We next confirmed that progenitor PD-1int TCF+ cells robustly produce tumor necrosis factor-α (TNFα) and interferon-γ, whereas terminally differentiated PD-1int TCF+ cells have defects in generating TNFα. With transgenic ob/ob mice, we found that DFB produces cooperative efficacy with anti-PD-1 (αPD-1) by limiting the PD-1hi Tim3+ subset and amplifying the PD-1int TCF+ population. Finally, we defined the recombinant chemokine C-C-motif receptor 2 (CCR2)+ CD8+ subset as terminal Tex and identified that the differentiation from progenitor to terminal Tex is driven, at least in part, by the chemokine (C-C motif) ligand 2 (CCL2)/CCR2 axis. The CCR2 inhibitor enhances the response to αPD-1 by promoting the counts of progenitor Tex. Altogether, DFB dampens CCL2 and preserves progenitor Tex in the obese microenvironment to restrain CRC progression. These findings provide unambiguous evidence that the traditional Chinese formula DFB can prevent tumor progression by modulating adaptive immunity and establish a strong rationale for further clinical verification.

An in vitro cell model to study microglia activation in diabetic retinopathy.

Microglial activation has been studied extensively in diabetic retinopathy. We have previously detected activation and migration of microglia in 8-week-old diabetic rat retinas. It is widely acknowledged that microglia-mediated inflammation contributes to the progression of diabetic retinopathy. However, existing cell models do not explore the role of activated microglia in vitro. In this study, microglia were subject to various conditions mimicking diabetic retinopathy, including high glucose, glyoxal, and hypoxia. Under high glucose or glyoxal treatment, microglia demonstrated only partially functional changes, while under hypoxia, microglia became fully activated showing enlarged cell bodies, enhanced migration and phagocytosis as well as increased production of pro-inflammatory factors such as cyclooxygenase-2 (COX-2), interleukin-1ß (IL-1ß), and inducible nitric oxide synthase (iNOS). The data indicate that hypoxia-treated microglia is an optimal in vitro model for exploration of microglia activation in diabetic retinopathy.

Genipin improves lipid metabolism and sperm parametersin obese mice via regulation of miR-132 expression.

Obesity has now surpassed malnutrition and infectious diseases as the most significant contributor to health problems worldwide. In particular, obesity is associated with several metabolic disorders, including hyperlipidemia, hepatic steatosis, and subfertility. Genipin (GNP), the aglycone of geniposide, is isolated from the extract of the traditional Chinese medicine Gardenia jasminoides Ellis and has been used in traditional oriental medicine against several inflammation-driven diseases. However, the effect and molecular mechanism of GNP on obesity-associated dyslipidemia and sperm dysfunction still need to be explored. In this study, we detect the effects of GNP on hyperlipidemia, hepatic lipid accumulation and sperm function using a high-fat diet (HFD)-induced obese mouse model. We find that obese mice treated with GNP show an improvement in body weight, serum triglyceride levels, serum hormone levels, serum inflammatory cytokines, hepatic steatosis and sperm function. At the molecular level, HFD/GNP diversely regulates the expression of miR-132 in a tissue-specific manner. miR-132 further targets and regulates the expression of SREBP-1c in liver cells, as well as the expressions of SREBP-1c and StAR in Leydig cells in the testis, thus modifying lipogenesis and steroidogenesis, respectively. Collectively, our data demonstrate that GNP shows a broad effect on the improvement of HFD-induced metabolic disorder and sperm dysfunction in male mice by tissue-specific regulation of miR-132. Our findings reveal the function GNP in ameliorating hepatic lipid metabolism and sperm function and suggest that this compound is a versatile drug to treat metabolic disorders.

Developmental toxicity of bromoacetamide via the thyroid hormone receptors-mediated disruption of thyroid hormone homeostasis in zebrafish embryos.

Bromoacetamide (BAcAm) is a nitrogenous disinfection by-product. We previously found that BAcAm induced developmental toxicity in zebrafish embryos, but the underlying mechanisms remain to be elucidated. Since thyroid hormones (THs) homeostasis is crucial to development, we hypothesized that disruption of THs homeostasis may play a role in the developmental toxicity of BAcAm. In this study, we found BAcAm exposure significantly increased mortality and malformation rate, decreased hatching rate and body length, inhibited the locomotor capacity in zebrafish embryos. BAcAm elevated TSH, T3 and T4 levels, down-regulated T3/T4 ratios, and up-regulated mRNA expression changes of THs related genes (trh, tsh, tg, nis, tpo, dio1, dio2, ugt1ab，klf9 and rho), but down-regulated mRNA expression changes of TH receptors (tr α and tr ß). Up-regulated tr α and tr ß mRNAs by rescue treatment confirmed that both tr α and tr ß were involved in the developmental toxicity of BAcAm. In conclusion, our study indicates disruption of THs homeostasis via the thyroid hormone receptors was responsible for the developmental toxicity of BAcAm.

Genipin improves reproductive health problems caused by circadian disruption in male mice.

BACKGROUND: Circadian rhythm disruption impacts a wide range of physiological processes, including fertility. However, the effect of circadian disruption on male spermatogenesis and fertility, and treatments for these effects have been largely unexplored at the molecular level. METHODS: In this study, we examined the effects of genipin on improving the reproductive health problems caused by circadian disruption. Three groups of animals were fed under different conditions: control group (normal T cycle with saline), group of shortened T cycles (Light/Dark = 4 hours/4 hours) with saline, and a group of shortened T cycles with genipin by oral gavage. The male fertility was evaluated by fertility study and pups parameters analysis after successful sexual behavior and mating with female mice. We sacrificed the treated animals after 5 or 10 weeks and collected the testis, sperm and serum for histological analysis, sperm motility assay, and serum hormone detection, respectively. Furthermore, the effect of genipin was assessed by detection of progesterone secretion and steroidogenic key proteins expression, including StAR and CYP11A1, in mouse Leydig tumor MLTC-1 cells. RESULTS: Male mice exposed to shortened light-dark cycles, much shorter than 24 hours, had reduced fertility with decreased sperm concentrations and sperm motility. Male mice under circadian disruption have reduced testis size and abnormal morphology, leading to lower fertility rates, reduced litter size and pup body weight. Treatment with exogenous genipin, a natural plant-derived compound, alleviated circadian disruption-induced damage to fertility and spermatogenesis and normalized testosterone, dihydrotestosterone (DHT), and androstenedione (ASD) levels in the male mice. The levels of key proteins involved in steroidogenesis, StAR and CYP11A1, were reduced in mouse testes after the circadian disruption, but genipin treatment restored the reduction. The mRNA expression of SRD5A1, which encodes an androgen synthesis enzyme, was also upregulated by genipin treatment. Furthermore, genipin treatment showed a positive effect on steroidogenesis in MLTC-1 cells, resulting in an increase in hormone secretion and the upregulation of StAR and CYP11A1. CONCLUSIONS: Our results showed an association between circadian disruption and reproductive health problems in male mice and indicated that treatments with genipin have positive effects on the reproductive health of male mice with circadian rhythm disorders.

In Vivo Structural and Functional Abnormalities of the Striatums Is Related to Decreased Astrocytic BDNF in Itpr2-/- Mice Exhibiting Depressive-Like Behavior.

Background: Previous researches indicate that Itpr2 -/- mice (inositol 1,4,5-trisphosphate receptor type 2 knockout mice) show depressive-like symptoms; however, little is known regarding the in vivo neurobiological effect of Itpr2 as well as the specific pattern of brain abnormalities in Itpr2 -/- mice. Methods/Materials. First, behavioral tests, structural magnetic resonance imaging (MRI), and resting-state functional MRI were performed on Itpr2 -/- mice and matched healthy controls. Voxel-based morphometry and seed-based voxel-wise functional connectivity (FC) were, respectively, calculated to assess the gray matter volume and the functional activities of the brain in vivo. Second, the sample of relevant changed brain regions was extracted to detect the expression of BDNF. Finally, to further validate the relationship between Itpr2 deficiency and the observed brain abnormalities, we performed Western blotting to detect the expression of pro-BDNF and mBDNF in Itpr2 -/- C8-D1A (a type of astrocyte). Results: Compared with controls, Itpr2 -/- mice showed depressive-like behaviors as well as significantly lower gray matter volume in striatums mainly, periaqueductal GM, and the right frontoparietal cortices as well as lower striatal-hippocampal and striatal-right parietal cortex (mainly for the primary and secondary somatosensory cortex) FC. Moreover, decreased expression of mBDNF was found in both sample tissues of the striatum in Itpr2 -/- mice and Itpr2 -/- C8-D1A. Conclusion: By combining biochemistry and MR analyses, this study provides evidences to support that the Itpr2-related neuropathological effect is possibly mediated by the striatal abnormality associated with dysfunctional astrocytes in Itpr2 -/- mice in vivo, thus may help us better understand underlying mechanisms of Itpr2 deficiency as well as its relation to depressive-like behavior.

TrkB agonistic antibodies superior to BDNF: Utility in treating motoneuron degeneration.

While Brain-derived Neurotrophic Factor (BDNF) has long been implicated in treating neurological diseases, recombinant BDNF protein has failed in multiple clinical trials. In addition to its unstable and adhesive nature, BDNF can activate p75NTR, a receptor mediating cellular functions opposite to those of TrkB. We have now identified TrkB agonistic antibodies (TrkB-agoAbs) with several properties superior to BDNF: They exhibit blood half-life of days instead of hours, diffuse centimeters in neural tissues instead millimeters, and bind and activate TrkB, but not p75NTR. In addition, TrkB-agoAbs elicit much longer TrkB activation, reduced TrkB internalization and less intracellular degradation, compared with BDNF. More importantly, some of these TrkB-agoAbs bind TrkB epitopes distinct from that by BDNF, and work cooperatively with endogenous BDNF. Unlike BDNF, the TrkB-agoAbs exhibit a half-life of days/weeks and diffused readily in nerve tissues. We tested one of TrkB-agoAbs further and showed that it enhanced motoneuron survival in the spinal-root avulsion model for motoneuron degeneration in vivo. Thus, TrkB-agoAbs are promising drug candidates for the treatment of neural injury.

Cinobufagin suppresses colorectal cancer angiogenesis by disrupting the endothelial mammalian target of rapamycin/hypoxia-inducible factor 1α axis.

Inducing angiogenesis is a hallmark of cancers that sustains tumor growth and metastasis. Neovascularization is a surprisingly early event during the multistage progression of cancer. Cinobufagin, an important bufadienolide originating from Chan Su, has been clinically used to treat cancer in China since the Tang dynasty. Here, we show that cinobufagin suppresses colorectal cancer (CRC) growth in vivo by downregulating angiogenesis. The hierarchized neovasculature is significantly decreased and the vascular network formation is disrupted in HUVEC by cinobufagin in a dose-dependent way. Endothelial apoptosis is observed by inducing reactive oxygen species (ROS) accumulation and mitochondrial dysfunction which can be neutralized by N-acetyl-l-cysteine (NAC). Expression of hypoxia-inducible factor 1α (HIF-1α) is reduced and phosphorylation of mTOR at Ser2481 and Akt at Ser473 is downregulated in HUVEC. Endothelial apoptosis is triggered by cinobufagin by stimulation of Bax and cascade activation of caspase 9 and caspase 3. Increased endothelial apoptosis rate and alterations in the HIF-1α/mTOR pathway are recapitulated in tumor-bearing mice in vivo. Further, the anti-angiogenesis function of cinobufagin is consolidated based on its pro-apoptotic effects on an EOMA-derived hemangioendothelioma model. In conclusion, cinobufagin suppresses tumor neovascularization by disrupting the endothelial mTOR/HIF-1α pathway to trigger ROS-mediated vascular endothelial cell apoptosis. Cinobufagin is a promising natural anti-angiogenetic drug that has clinical translation potential and practical application value.

LY303511 displays antiproliferation potential against oral cancer cells in vitro and in vivo.

LY303511 was developed as a negative control of LY294002 without pan-phosphoinositide 3-kinase (PI3K) inhibition. We hypothesize LY303511 generate reactive oxygen species (ROS) to induce apoptosis for killing oral cancer cells. In MTS assay, LY303511 dose-responsively decreases survival in three kinds of oral cancer cells but little damage to normal oral cells (HGF-1). Two oral cancer cells (CAL 27 and SCC-9) with highly sensitivity to LY303511 were used. In 7-aminoactinomycin D (7AAD) assay, LY303511 slightly increases subG1 population in oral cancer cells. In annexin V/7AAD and/or pancaspase assays, LY303511 induces apoptosis in oral cancer cells but HGF-1 cells remains in basal level. In oxidative stress, LY303511 induces ROS and mitochondrial superoxide in oral cancer cells. In 8-oxo-2'-deoxyguanosine assay, LY303511 induces oxidative DNA damage in oral cancer cells. In zebrafish model, LY303511 inhibits CAL 27-xenografted tumor growth. Therefore, LY303511 displays antiproliferation potential against oral cancer cells in vitro and in vivo.

Ulcerative Colitis Treatment Patterns and Cost of Care.

OBJECTIVES: To examine treatment patterns, dosing, health care resource utilization, and cost of tumor necrosis factor inhibitors (TNFi), adalimumab (ADA) and infliximab (IFX), among patients enrolled in US Humana insurance plans who have been diagnosed with ulcerative colitis (UC). METHODS: This retrospective cohort study identified the first pharmacy or medical claim for ADA or IFX (from January 1, 2007, to December 31, 2014) in patients with continuous enrollment for 6 months or more preindex and 12 months or more postindex, with one or more UC diagnosis claim 6 months pre- or postindex. TNFi discontinuation was defined as a therapy gap of 56 days or more for ADA and 112 days or more for IFX. TNFi switch was defined as nonindex TNFi initiation. Health care resource utilization and costs were characterized quarterly according to treatment patterns. RESULTS: The study population comprised 295 patients: mean age 50.9 years, 50.5% females, and 61.7% in southern United States. At the index date, 17% of patients received ADA and 83% received IFX. Treatment discontinuation was observed in 52% of ADA and 45% of IFX users through 12 months postindex (mean time 19 and 22 weeks, respectively). Among discontinuers, 46% of ADA and 68% of IFX users did not restart/switch TNFi. ADA and IFX showed mean times to switch of 18 and 30 weeks, respectively. TNFi discontinuers had the lowest mean quarterly total health care cost ($3,935) versus patients who initiated/switched TNFi ($15,004). Nevertheless, discontinuers had higher UC-related hospitalization versus patients receiving therapy. CONCLUSIONS: Approximately half of ADA and IFX users discontinued, with approximately half of discontinuers not restarting/switching therapies. Further investigation of treatment patterns and outcomes after TNFi discontinuation is required.

Multifactorial theory applied to the neurotoxicity of paraquat and paraquat-induced mechanisms of developing Parkinson's disease.

Laboratory studies involving repeated exposure to paraquat (PQ) in different animal models can induce many of the pathological features of Parkinson's disease (PD), such as the loss of dopaminergic neurons in the nigrostriatal dopamine system. Epidemiological studies identify an increased risk of developing PD in human populations living in areas where PQ exposure is likely to occur and among workers lacking appropriate protective equipment. The mechanisms involved in developing PD may not be due to any single cause, but rather a multifactorial situation may exist where PQ exposure may cause PD in some circumstances. Multifactorial theory is adopted into this review that includes a number of sub-cellular mechanisms to explain the pathogenesis of PD. The theory is placed into an environmental context of chronic low-dose exposure to PQ that consequently acts as an oxidative stress inducer. Oxidative stress and the metabolic processes of PQ-inducing excitotoxicity, α-synuclein aggregate formation, autophagy, alteration of dopamine catabolism, and inactivation of tyrosine hydroxylase are positioned as causes for the loss of dopaminergic cells. The environmental context and biochemistry of PQ in soils, water, and organisms is also reviewed to identify potential routes that can lead to chronic rates of low-dose exposure that would replicate the type of response that is observed in animal models, epidemiological studies, and other types of laboratory investigations involving PQ exposure. The purpose of this review is to synthesize key relations and summarize hypotheses linking PD to PQ exposure by using the multifactorial approach. Recommendations are given to integrate laboratory methods to the environmental context as a means to improve on experimental design. The multifactorial approach is necessary for conducting valid tests of causal relations, for understanding of potential relations between PD and PQ exposure, and may prevent further delay in solving what has proven to be an evasive etiological problem.

Jiawei guomin decoction regulates the degranulation of mast cells in atopic dermatitis mice via the HIS/PAR-2 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Guomin decoction (GMD) is a traditional Chinese medicine commonly used in clinical practice. It has traditionally been used to treat all allergic diseases. Currently, Jiawei Guomin Decoction (JWGMD) is used to treat sensitive skin after initial therapy. Although it has a significant clinical therapeutic effect, the exact role of mast cell degranulation in treating atopic dermatitis (AD) is still unclear. AIM OF THE STUDY: GMD and JWGMD can both treat allergic diseases, while JWGMD focuses on skin allergies. This study aims to explore the potential effect of JWGMD on the degranulation of mast cells in an AD mouse model induced by 2,4-dinitrofluorobenzene (DNFB) and investigate the effectiveness of JWGMD in alleviating disease progression to further provide specific therapeutic targets for treating AD. MATERIALS AND METHODS: The scratching times and skin lesions of model mice induced by DNFB were observed, and skin tissues were collected for subsequent measurement. Histopathological changes in the back skin of mice were observed by haematoxylin eosin (H&E) staining, Toluidine blue staining was used to detect the degranulation of mouse skin mast cells, and the relationship between the expression of histamine (HIS), mast cell tryptase (MCT) and mast cell degranulation was analysed by enzyme-linked immunosorbent assay (ELISA). The expression of protease-activated receptor-2 (PAR-2), histamine 1 receptor (H1R), H2R, H4R and MCT proteins in AD mice was detected by Western blot (WB). Immunofluorescence assay (IFA) further confirmed the localization of PAR-2, H1R, H2R, H4R, and MCT proteins in the skin. Quantitative real-time PCR (qPCR) was used to determine PAR-2, H1R, H2R and H4R mRNA levels in skin lesions to further clarify the mechanism by which JWGMD amplifies mast cell degranulation in AD. In addition, a reliable ultrahigh-performance liquid chromatography-quadrupole electrostatic field orbitrap mass spectrometry (UPLC-QE-MS) nontargeted metabolomics analysis was performed to analyse the differences in metabolite abundance between GMD and JWGMD, and these results were used to identify the active components in JWGMD that may have antipruritic and anti-inflammatory properties and inhibit mast cell degranulation. RESULTS: After intermittent stimulation with DNFB, the skin lesions showed extensive desquamation, dryness, scabbing, skin thickening, and slight bleeding. Both treatments alleviated this phenomenon and reduced the number of scratches, with JWGMD being the most effective. JWGMD can significantly reduce inflammatory cell infiltration, oedema, and some capillary neogenesis in mice and reduce the degranulation of mast cells. The ELISA results showed that JWGMD can increase the levels of MCT and HIS proteins. The WB and IFA results demonstrated that JWGMD reduced the expression levels of PAR-2, H1R, H4R, and MCT proteins in skin lesions, with protein localization mainly in the epidermal layer, while H2R protein levels were increased and mainly localized in the dermis. In addition, JWGMD downregulates the mRNA expression of PAR-2, H1R, H2R, and H4R. Interestingly, through UPLC-QE-MS nontargeted metabolomic analysis, we detected the anti-inflammatory and antiallergy active substances in JWGMD, such as methyl eugenol, dictamnine and sinapine. CONCLUSIONS: JWGMD may alleviate itching through methyl syringol, dictamnine, sinapine and other substances, and its mechanism may be related to inhibiting the HIS/PAR-2 pathway in AD model mice and further regulating the self-amplification of mast cell degranulation. JWGMD is a potential drug for treating AD. Therefore, it deserves continuous attention and research.

Recent advances in anti-inflammation via AMPK activation.

Inflammation is a complex physiological phenomenon, which is the body's defensive response, but abnormal inflammation can have adverse effects, and many diseases are related to the inflammatory response. AMPK, as a key sensor of cellular energy status, plays a crucial role in regulating cellular energy homeostasis and glycolipid metabolism. In recent years, the anti-inflammation effect of AMPK and related signalling cascade has begun to enter everyone's field of vision - not least the impact on metabolic diseases. A great number of studies have shown that anti-inflammatory drugs work through AMPK and related pathways. Herein, this article summarises recent advances in compounds that show anti-inflammatory effects by activating AMPK and attempts to comment on them.

Palliation of malignant esophageal stent esophagorespiratory fistula with rigid bronchoscope and Y silicone trachea stent: experience with seven patients.

Background: Malignant esophageal stent esophagorespiratory fistula (ERF) is an abnormal communication between esophagus and airway among advanced tumor patients with indwelling esophageal stent, which is devastating and life-threatening. This study aims to provide a new feasible treatment scheme for malignant esophageal stent ERF and report its potential advantage compared with double stenting, which was recommended by European Society of Gastrointestinal Endoscopy Guideline. Methods: We retrospectively analyzed the medical data of malignant esophageal stent ERF patients between January 2018 to May 2023 at the First Affiliated Hospital of Guangzhou Medical University and divided them into two groups. Group 1 consisted of patients treated with rigid bronchoscopy to remove the esophageal stent and implant Y silicone trachea stent, while group 2 consisted of patients treated with additional airway stenting without removing the esophageal stent. Demographic parameters, disease diagnoses and treatment, radiological findings before and after the intervention, and complications caused by the stents were obtained and analyzed with chi-squared, Mann-Whitney U, independent-samples t-tests, Kaplan-Meier methods, and log-rank test. Results: Ten patients (seven patients in group 1 and three in group 2) were included. No procedure complications occurred in both groups. The mean Karnofsky Performance Score after the procedure significantly improved compared to the pre-procedure (57.14 vs. 77.14, P=0.001) in group 1, while decreased in group 2 (50 vs. 40, P=0.026). The control of pneumonia in group 1 patients is better than that in group 2. There was significant improvement in the degree of dysphagia after the procedure (3.86 vs. 2.43, P=0.002) in group 1, while no improvement was found in group 2 (4.00 vs. 3.33, P=0.423). The mean survival of group 1 was significantly longer group 2 (381.00 vs. 80.33 days, P<0.001, log-rank test). No patient needed stent repositioning due to migration in both groups. Cause of death in the group 1 included disease progression, novel coronavirus pneumonia, massive hemoptysis, and respiratory insufficiency, while group 2 included severe pneumonia and disease progression. No death was directly attributed to the procedure in both groups. Conclusions: Removing the esophageal stent and implanting Y silicone trachea stent through a rigid bronchoscopy is a safe and feasible treatment for malignant esophageal stent ERF. This procedure can effectively seal the fistula, prevent from recurrent aspiration pneumonia, improve the quality of life, and prolong the survival time.

Changes in patterns of multimorbidity and associated with medical costs among Chinese middle-aged and older adults from 2013 to 2023: an analysis of repeated cross-sectional surveys in Xiangyang, China.

Background: Multimorbidity has become a major public health problem among Chinese middle-aged and older adults, and the most costly to the health care system. However, most previous population-based studies of multimorbidity have focused on a limited number of chronic diseases, and diagnosis was based on participants' self-report, which may oversimplify the problem. At the same time, there were few reports on the relationship between multimorbidity patterns and health care costs. This study analyzed the multimorbidity patterns and changes among middle-aged and older people in China over the past decade, and their association with medical costs, based on representative hospital electronic medical record data. Methods: Two cross-sectional surveys based on representative hospital data were used to obtain adults aged 45 years and older in Xiangyang in 2013 (n = 20,218) and 2023 (n = 63,517). Latent Class Analysis was used to analyze changes in the patterns of multimorbidity, gray correlation analysis and ordered logistics model were used to assess the association of multimorbidity patterns with medical expenses. The diagnosis and classification of chronic diseases were based on the International Classification of Diseases, Tenth Revision codes (ICD-10). Results: The detection rate of chronic disease multimorbidity has increased (70.74 vs. 76.63%, p < 0.001), and multimorbidity patterns have increased from 6 to 9 (2013: Malignant tumors pattern, non-specific multimorbidity pattern, ischemic heart disease + hypertension pattern, cerebral infarction + hypertension pattern, kidney disease + hypertension pattern, lens disease + hypertension pattern; new in 2023: Nutritional metabolism disorders + hypertension pattern, chronic lower respiratory diseases + malignant tumors pattern, and gastrointestinal diseases pattern) in China. The medical cost of all multimorbidity patients have been reduced between 2013 and 2023 (RMB: 8216.74 vs. 7247.96, IQR: 5802.28-15,737 vs. 5014.63-15434.06). The top three specific multimorbidity patterns in both surveys were malignancy tumor pattern, ischemic heart disease + hypertension pattern, and cerebral infarction + hypertension pattern. Hypertension and type 2 diabetes are important components of multimorbidity patterns. Compared with patients with a single disease, only lens disorders + hypertension pattern were at risk of higher medical costs in 2013 (aOR:1.23, 95% CI: 1.03, 1.47), whereas all multimorbidity patterns were significantly associated with increased medical costs in 2023, except for lens disorders + hypertension (aOR:0.35, 95% CI: 0.32, 0.39). Moreover, the odds of higher medical costs were not consistent across multimorbidity patterns. Among them, ischemic heart disease + hypertension pattern [adjusted odds ratio (aOR):4.66, 95%CI: 4.31, 5.05] and cerebral infarction + hypertension pattern (aOR: 3.63, 95% CI: 3.35, 3.92) were the two patterns with the highest risk. Meanwhile, men (aOR:1.12, 95CI:1.09, 1.16), no spouse (aOR:1.09, 95CI: 1.03, 1.16) had a positive effect on medical costs, while patients with total self-pay (aOR: 0.45, 95CI: 0.29, 0.70), no surgery (aOR: 0.05, 95CI: 0.05, 0.05), rural residence (aOR: 0.92, 95CI: 0.89, 0.95), hospitalization days 1-5 (aOR: 0.04, 95CI: 0.04, 0.04), and hospitalization days 6-9 (aOR: 0.15, 95CI: 0.15, 0.16) had a negative impact on medical costs. Conclusion: Multimorbidity patterns among middle-aged and older adults in China have diversified over the past decade and are associated with rising health care costs in China. Smart, decisive and comprehensive policy and care interventions are needed to effectively manage NCDS and their risk factors and to reduce the economic burden of multimorbidity on patients and the country.

Ngfr+ cholinergic projection from SI/nBM to mPFC selectively regulates temporal order recognition memory.

Acetylcholine regulates various cognitive functions through broad cholinergic innervation. However, specific cholinergic subpopulations, circuits and molecular mechanisms underlying recognition memory remain largely unknown. Here we show that Ngfr+ cholinergic neurons in the substantia innominate (SI)/nucleus basalis of Meynert (nBM)-medial prefrontal cortex (mPFC) circuit selectively underlies recency judgements. Loss of nerve growth factor receptor (Ngfr-/- mice) reduced the excitability of cholinergic neurons in the SI/nBM-mPFC circuit but not in the medial septum (MS)-hippocampus pathway, and impaired temporal order memory but not novel object and object location recognition. Expression of Ngfr in Ngfr-/- SI/nBM restored defected temporal order memory. Fiber photometry revealed that acetylcholine release in mPFC not only predicted object encounters but also mediated recency judgments of objects, and such acetylcholine release was absent in Ngfr-/- mPFC. Chemogenetic and optogenetic inhibition of SI/nBM projection to mPFC in ChAT-Cre mice diminished mPFC acetylcholine release and deteriorated temporal order recognition. Impaired cholinergic activity led to a depolarizing shift of GABAergic inputs to mPFC pyramidal neurons, due to disturbed KCC2-mediated chloride gradients. Finally, potentiation of acetylcholine signaling upregulated KCC2 levels, restored GABAergic driving force and rescued temporal order recognition deficits in Ngfr-/- mice. Thus, NGFR-dependent SI/nBM-mPFC cholinergic circuit underlies temporal order recognition memory.