ObjectiveTo investigate the effects of Chaihu Jia Longgu Mulitang（CJLM） on depression-like behaviors and neuroinflammation in rats subjected to social isolation combined with chronic unpredictable mild stress（CUMS）， and to explore the potential underlying mechanisms. MethodsSixty male SD rats were randomly divided into a normal group， a model group， and low-， medium-， and high-dose CJLM groups（2.89， 5.78， 11.56 g·kg^-1）， as well as a fluoxetine group（10 mg·kg^-1）. Except for the normal group， all other groups were subjected to social isolation combined with CUMS for 63 d. During the first 35 d， depression models were established only， and from day 36 onward， modeling and drug administration were conducted simultaneously for a total intervention period of 28 d. Depression-like behaviors were evaluated using the sucrose preference test， open-field test， and forced swimming test. Hematoxylin-eosin（HE） staining was performed to observe hippocampal histomorphology. Immunohistochemistry（IHC） was used to detect the expression levels of ionized calcium-binding adapter molecule 1（Iba1） and gasdermin D（GSDMD） proteins in the hippocampus. Western blot analysis was employed to determine the protein expression levels of adenosine 5′-monophosphate（AMP）-activated protein kinase（AMPK） and phosphorylated（p）-AMPK， silent information regulator 1（SIRT1）， nuclear factor-κB（NF-κB） and p-NF-κB， NOD-like receptor protein 3（NLRP3）， and Caspase-1 in the hippocampus. Real-time quantitative polymerase chain reaction（Real-time PCR） was used to detect the mRNA expression levels of tumor necrosis factor-α（TNF-α）， interleukin（IL）-6， and IL-1β in the hippocampus. ResultsCompared with the normal group， the model group showed a decreased sucrose preference rate（P<0.01）， reduced total movement distance（P<0.01）， prolonged immobility time（P<0.01）， and decreased central zone residence time（P<0.01） in the open-field test， and increased immobility time in the forced swimming test（P<0.01）. Hippocampal neuronal structure was damaged. The contents of Iba1 and GSDMD in the hippocampus were significantly increased（P<0.01）. The protein expression levels of p-AMPK and SIRT1 in the hippocampus were significantly decreased（P<0.01）， whereas the protein expression levels of p-NF-κB， NLRP3， and Caspase-1 were significantly increased（P<0.01）. The mRNA expression levels of IL-1β， IL-6， and TNF-α in the hippocampus were significantly upregulated（P<0.01）. Compared with the model group， the low-， medium-， and high-dose CJLM groups and the fluoxetine group all were able to reverse depression-like behavioral changes， as evidenced by increased sucrose preference rate， increased total movement distance with shortened immobility time in the open-field test， prolonged central zone residence time， and reduced immobility time in the forced swimming test（P<0.05， P<0.01）. Meanwhile， hippocampal neuronal structural damage was alleviated. In the hippocampus， the expression levels of Iba1 and GSDMD were downregulated， the expression levels of p-AMPK and SIRT1 were upregulated， and the abnormal elevations of p-NF-κB， NLRP3， Caspase-1， as well as IL-1β， IL-6， and TNF-α mRNA were suppressed（P<0.05， P<0.01）. ConclusionCJLM can ameliorate depression-like behaviors in rats subjected to social isolation combined with CUMS and attenuate hippocampal neuroinflammation and pyroptosis， suggesting that its effects may be associated with the regulation of AMPK/SIRT1/NF-κB/NLRP3 signaling pathway.

Objective To investigate the expression characteristics of the transcription factor E2F2 in clear cell renal cell carcinoma (ccRCC), its impact on patient prognosis, and its potential role in the tumor immune microenvironment, with validation using clinical samples and functional assays. Methods RNA sequencing data and clinical information of ccRCC patients were obtained from the TCGA database; GSE53757 and GSE66272 datasets were downloaded from the GEO database as external validation cohorts. The expression difference of E2F2 between ccRCC tissue and normal tissue was compared, and the relationship between E2F2 expression and clinical pathological characteristics was analyzed. Kaplan-Meier (KM) survival analysis with log-rank test was performed to evaluate the effects of E2F2 on overall survival (OS), progression-free survival (PFS), and disease-specific survival (DSS). The prognostic efficacy of E2F2 in predicting 1-, 3- and 5-year survival was evaluated using receiver operating characteristic (ROC) curve. GSEA was performed to identify E2F2-related signaling pathways, and the ESTIMATE and CIBERSORT algorithms were used to assess the relationship between E2F2 expression level and tumor immune cell infiltration and immune microenvironment. Twenty pairs of surgically resected and pathologically confirmed ccRCC tissues and matched adjacent non-tumor tissues were collected from our hospital, and immunohistochemistry (IHC) was performed to detect the protein expression of E2F2. Human ccRCC cell line 786-O was used for functional assays;shRNA was used to knock down E2F2 expression (constructing stable shE2F2#1 and shE2F2#2 cell lines), and qRT-PCR and Western blot were performed to verify knockdown efficiency, followed by MTT, colony formation, and Transwell migration assays to evaluate the effects of E2F2 on the biological behaviors of ccRCC cells. Results E2F2 was significantly upregulated in ccRCC tissues (the same results in the verification set). Higher E2F2 expression was associated with advanced histological grade, clinical stage and higher TNM classification (P＜0.05). KM analysis showed that patients in the E2F2 high-expression group had worse OS, PFS, and DSS (P=0.019, 0.036, ＜0.001); the ROC curves showed area under the curve (AUC) of E2F2 of predicting 1-, 3- and 5- year survival of ccRCC patients were 0.844, 0.851 and 0.815, respectively. GSEA revealed that the E2F2 low-expression group was enriched in multiple metabolism-related pathways, whereas the E2F2 high-expression group was associated with immune-related pathways. Immune analysis demonstrated that high E2F2 expression was associated with increased immune scores, increased proportion of immune cells, higher tumor mutation burden, and potentially stronger immunotherapy response. IHC results showed that the E2F2 immunoreactivity score in ccRCC tissues was significantly higher than that in adjacent non-tumor tissues (P＜0.05). Functional assays indicated that E2F2 knockdown significantly inhibited the proliferation, colony formation, and migration of ccRCC cells. Conclusion E2F2 is highly expressed in ccRCC and may be involved in tumorigenesis and progression through modulation of the immune microenvironment. Its expression level is closely associated with patient prognosis, and E2F2 has the potential to serve as a prognostic biomarker and immunotherapeutic target in ccRCC.

Chronic visceral pain is a persistent and debilitating condition arising from dysfunction or sensitization of the visceral organs and their associated nervous pathways. Increasing evidence suggests that imbalances in central nervous system function play an essential role in the progression of visceral pain, but the exact mechanisms underlying the neural circuitry and molecular targets remain largely unexplored. In the present study, the ventral tegmental area (VTA) was shown to mediate visceral pain in mice. Visceral pain stimulation increased c-Fos expression and Ca²⁺ activity of glutamatergic VTA neurons, and optogenetic modulation of glutamatergic VTA neurons altered visceral pain. In particular, the upregulation of NMDA receptor 2A (NR2A) subunits within the VTA resulted in visceral pain in mice. Administration of a selective NR2A inhibitor decreased the number of visceral pain-induced c-Fos positive neurons and attenuated visceral pain. Pharmacology combined with chemogenetics further demonstrated that glutamatergic VTA neurons regulated visceral pain behaviors based on NR2A. In summary, our findings demonstrated that the upregulation of NR2A in glutamatergic VTA neurons plays a critical role in visceral pain. These insights provide a foundation for further comprehension of the neural circuits and molecular targets involved in chronic visceral pain and may pave the way for targeted therapies in chronic visceral pain.
Animals ; Male ; Visceral Pain/metabolism* ; Up-Regulation/physiology* ; Ventral Tegmental Area/metabolism* ; Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors* ; Neurons/drug effects* ; Mice, Inbred C57BL ; Mice ; Proto-Oncogene Proteins c-fos/metabolism* ; Chronic Pain/metabolism* ; Glutamic Acid/metabolism*

This study aimed to comprehensively examine the association of gallstones, cholecystectomy, and cancer risk. Multivariable logistic regressions were performed to estimate the observational associations of gallstones and cholecystectomy with cancer risk, using data from a nationwide cohort involving 239 799 participants. General and gender-specific two-sample Mendelian randomization (MR) analysis was further conducted to assess the causalities of the observed associations. Observationally, a history of gallstones without cholecystectomy was associated with a high risk of stomach cancer (adjusted odds ratio (aOR)=2.54, 95% confidence interval (CI) 1.50-4.28), liver and bile duct cancer (aOR=2.46, 95% CI 1.17-5.16), kidney cancer (aOR=2.04, 95% CI 1.05-3.94), and bladder cancer (aOR=2.23, 95% CI 1.01-5.13) in the general population, as well as cervical cancer (aOR=1.69, 95% CI 1.12-2.56) in women. Moreover, cholecystectomy was associated with high odds of stomach cancer (aOR=2.41, 95% CI 1.29-4.49), colorectal cancer (aOR=1.83, 95% CI 1.18-2.85), and cancer of liver and bile duct (aOR=2.58, 95% CI 1.11-6.02). MR analysis only supported the causal effect of gallstones on stomach, liver and bile duct, kidney, and bladder cancer. This study added evidence to the causal effect of gallstones on stomach, liver and bile duct, kidney, and bladder cancer, highlighting the importance of cancer screening in individuals with gallstones.
Humans ; Mendelian Randomization Analysis ; Gallstones/complications* ; Female ; Male ; Cholecystectomy/statistics & numerical data* ; Middle Aged ; Risk Factors ; Aged ; Adult ; Neoplasms/etiology* ; Stomach Neoplasms/epidemiology*

Combined with elastic network model (ENM), the perturbation response scanning (PRS) has emerged as a robust technique for pinpointing allosteric interactions within proteins. Here, we proposed the PRS analysis of drug-target networks (DTNs), which could provide a promising avenue in network medicine. We demonstrated the utility of the method by introducing a deep learning and network perturbation-based framework, for drug repurposing of multiple sclerosis (MS). First, the MS comorbidity network was constructed by performing a random walk with restart algorithm based on shared genes between MS and other diseases as seed nodes. Then, based on topological analysis and functional annotation, the neurotransmission module was identified as the "therapeutic module" of MS. Further, perturbation scores of drugs on the module were calculated by constructing the DTN and introducing the PRS analysis, giving a list of repurposable drugs for MS. Mechanism of action analysis both at pathway and structural levels screened dihydroergocristine as a candidate drug of MS by targeting a serotonin receptor of serotonin 2B receptor (HTR2B). Finally, we established a cuprizone-induced chronic mouse model to evaluate the alteration of HTR2B in mouse brain regions and observed that HTR2B was significantly reduced in the cuprizone-induced mouse cortex. These findings proved that the network perturbation modeling is a promising avenue for drug repurposing of MS. As a useful systematic method, our approach can also be used to discover the new molecular mechanism and provide effective candidate drugs for other complex diseases.

Hepatocellular carcinoma (HCC) expresses abundant glycolytic enzymes and displays comprehensive glucose metabolism reprogramming. Aldolase A (ALDOA) plays a prominent role in glycolysis; however, little is known about its role in HCC development. In the present study, we aim to explore how ALDOA is involved in HCC proliferation. HCC proliferation was markedly suppressed both in vitro and in vivo following ALDOA knockout, which is consistent with ALDOA overexpression encouraging HCC proliferation. Mechanistically, ALDOA knockout partially limits the glycolytic flux in HCC cells. Meanwhile, ALDOA translocated to nuclei and directly interacted with c-Jun to facilitate its Thr93 phosphorylation by P21-activated protein kinase; ALDOA knockout markedly diminished c-Jun Thr93 phosphorylation and then dampened c-Jun transcription function. A crucial site Y364 mutation in ALDOA disrupted its interaction with c-Jun, and Y364S ALDOA expression failed to rescue cell proliferation in ALDOA deletion cells. In HCC patients, the expression level of ALDOA was correlated with the phosphorylation level of c-Jun (Thr93) and poor prognosis. Remarkably, hepatic ALDOA was significantly upregulated in the promotion and progression stages of diethylnitrosamine-induced HCC models, and the knockdown of A ldoa strikingly decreased HCC development in vivo. Our study demonstrated that ALDOA is a vital driver for HCC development by activating c-Jun-mediated oncogene transcription, opening additional avenues for anti-cancer therapies.

OBJECTIVE: To assess the independent and combined effects of air pollutants, meteorological factors, and greenspace exposure on new tuberculosis (TB) cases. METHODS: TB case data from Shanghai (2013-2018) were obtained from the Shanghai Center for Disease Control and Prevention. Environmental data on air pollutants, meteorological variables, and greenspace exposure were obtained from the National Tibetan Plateau Data Center. We employed a distributed-lag nonlinear model to assess the effects of these environmental factors on TB cases. RESULTS: Increased TB risk was linked to PM _2.5, PM ₁₀, and rainfall, whereas NO ₂, SO ₂, and air pressure were associated with a reduced risk. Specifically, the strongest cumulative effects occurred at various lags: PM _2.5 ( RR = 1.166, 95% CI: 1.026-1.325) at 0-19 weeks; PM ₁₀ ( RR = 1.167, 95% CI: 1.028-1.324) at 0-18 weeks; NO ₂ ( RR = 0.968, 95% CI: 0.938-0.999) at 0-1 weeks; SO ₂ ( RR = 0.945, 95% CI: 0.894-0.999) at 0-2 weeks; air pressure ( RR = 0.604, 95% CI: 0.447-0.816) at 0-8 weeks; and rainfall ( RR = 1.404, 95% CI: 1.076-1.833) at 0-22 weeks. Green space exposure did not significantly impact TB cases. Additionally, low temperatures amplified the effect of PM _2.5 on TB. CONCLUSION Exposure to PM _2.5, PM ₁₀, and rainfall increased the risk of TB, highlighting the need to address air pollutants for the prevention of TB in Shanghai.
China/epidemiology* ; Humans ; Air Pollutants/analysis* ; Tuberculosis/epidemiology* ; Incidence ; Meteorological Concepts ; Particulate Matter/adverse effects* ; Environmental Exposure ; Male ; Female ; Adult ; Air Pollution ; Middle Aged

Objective To explore the effect of rehmanniae radix extract(RRE)on chondrocyte apoptosis in the rabbit model of knee osteoarthritis(KOA)by regulating the miR-485-5p/heat shock protein 90 beta family member 1(Hsp90b1)axis.Methods New Zealand rabbits were randomly assigned into control,KOA,low-dose RRE,medium-dose RRE,high-dose RRE,celecoxib,high-dose RRE+antagonist control,and high-dose RRE+miR-485-5p antagonist groups,with 12 rabbits in each group.Rabbits in other groups except the control group were modeled for KOA with the improved Hulth method.After modeling for 8 weeks,the rabbits were administrated with corresponding agents for 4 weeks.The changes in the activity rating of rabbits were recorded.ELISA was employed to measure the levels of tumor necrosis factor-α(TNF-α)and interleukin(IL)-6 in the serum.Safranine O-fast green staining was conducted to reveal the pathological changes in the cartilage tissue and Mankin scoring was performed.TUNEL was employed to detect chondrocyte apoptosis.Real-time fluorescence quantitative PCR was performed to determine the expression of miR-485-5p in the cartilage tissue.Western blot was employed to determine the protein levels of Hsp90b1,cleaved cysteinyl aspartate-specific proteinase-3(Caspase-3),and Bcl2-associated-X(Bax)in the cartilage tissue.The dual-luciferase reporter assay was employed to examine the relationship between miR-485-5p and Hsp90b1.Results Compared with the control group,the KOA group showed down-regulated expression of miR-485-5p,elevated levels of TNF-α and IL-6 in the serum,cartilage erosion and losses,and increases in activity rating,Mankin score,chondrocyte apoptosis rate,and protein levels of Hsp90b1,cleaved Caspase-3,and Bax(all P<0.001).Compared with the KOA group,RRE at low,medium,and high doses,and celecoxib up-regulated the expression of miR-485-5p,lowered the levels of TNF-α and IL-6 in the serum,alleviated the pathological damage to the cartilage tissue,and decreased the activity rating,Mankin score,chondrocyte apoptosis rate,and protein levels of Hsp90b1,cleaved Caspase-3,and Bax(all P<0.05).Compared with the high-dose RRE group and the high-dose RRE+antagonist control group,high-dose RRE+miR-485-5p antagonist down-regulated the expression of miR-485-5p,elevated the levels of TNF-α and IL-6 in the serum,exacerbated the pathological damage to the cartilage tissue,and increased the activity rating,Mankin score,chondrocyte apoptosis rate,and protein levels of Hsp90b1,cleaved Caspase-3,and Bax(all P<0.05).The results indicated that there was a targeted regulatory relationship between miR-485-5p and Hsp90b1.Conclusion RRE may inhibit the expression of Hsp90b1 by up-regulating miR-485-5p,thereby inhibiting chondrocyte apoptosis in the rabbit model of KOA.
Animals ; Rabbits ; Apoptosis/drug effects* ; Chondrocytes/pathology* ; Osteoarthritis, Knee/drug therapy* ; MicroRNAs/metabolism* ; Rehmannia/chemistry* ; Disease Models, Animal ; Tumor Necrosis Factor-alpha/blood* ; Plant Extracts/pharmacology* ; Interleukin-6/blood* ; HSP90 Heat-Shock Proteins/metabolism* ; Male ; Drugs, Chinese Herbal/pharmacology*

ObjectiveTo investigate the mechanisms of mitochondrial dynamics and metabolic reprogramming in the treatment of diabetic nephropathy （DN） by Shenxiao Tongluo prescription via the peroxisome proliferator-activated receptor γ coactivator-1α （PGC-1α）/sirtuin-3 （SIRT3）/hypoxia-inducible factor-1α （HIF-1α） signaling pathway. MethodsSixty-five SD rats were randomized into a sham group （10 rats） and a modeling group （55 rats）， and the modeling rats underwent left nephrectomy and intraperitoneal injection of streptozotocin （35 mg·kg^-1） to prepare a DN model. After successful modeling， the rats were randomized into model， empagliflozin （10 mg·kg^-1）， and low-， medium-， and high-dose （7.656， 15.312， 30.624 g·kg^-1， respectively） Shenxiao Tongluo prescription groups. The urine microalbumin （UmAlb）， blood urea nitrogen （BUN）， and serum creatinine （SCr） levels of rats in each group were assessed after continuous gavage for 8 weeks. The corresponding kits were used to measure the levels of lactate， superoxide dismutase （SOD）， and malondialdehyde （MDA） in the kidney tissue. Hematoxylin-eosin staining， Masson staining， and periodic acid-Schiff staining were performed to observe the pathological changes in the kidney tissue. Transmission electron microscopy was employed to observe mitochondrial morphology. Immunohistochemistry was employed to determine the expression levels of dynamin-related protein 1 （DRP1） and pyruvate kinase M2 （PKM2） in the kidney tissue. Western blot was adopted to assess the protein levels of PGC-1α， SIRT3， HIF-1α， dynamin-related protein 1 （Drp1）， optic atrophy 1 （OPA1）， hexokinase 2 （HK2）， and pyruvate kinase M2 （PKM2） in the kidney tissue. ResultsCompared with the sham group， the model group showed elevated levels of UmAlb， BUN， SCr， lactate， and MDA， decreased SOD level （P<0.05）， glomerular hypertrophy， thickening of the mesangial basement membrane， vacuolar degeneration of renal tubular epithelial cells， and infiltration of renal interstitial inflammatory cells， oval mitochondria with disordered， blurred or disappearing cristae， down-regulated protein levels of PGC-1α， SIRT3， and OPA1， and up-regulated protein levels of HIF-1α， DRP1， HK2， and PKM2 （P<0.05）. Compared with the model group， the treatment in all the groups increased the body weight， lowered the levels of GLU， UmAlb， BUN， and MDA， raised the level of SOD， alleviated the pathological damage in the kidney tissue and mitochondrial damage， up-regulated the expression of PGC-1α， SIRT3， and OPA1， and down-regulated the expression of HIF-1α， DRP1， and PKM2 （P<0.05）. Empagliflozin and Shenxiao Tongluo prescription at medium and high doses lowered the levels of SCr and lactate and down-regulated the expression of HK2 （P<0.05）， which had no statistical significance in the low-dose Shenxiao Tongluo prescription group. ConclusionShenxiao Tongluo prescription may regulate mitochondrial dynamics and metabolic reprogramming by activating the PGC-1α/SIRT3/HIF-1α pathway， thereby alleviating oxidative damage in the kidney tissue and delaying the progression of DN.

ObjectiveTo investigate the mechanisms of mitochondrial dynamics and metabolic reprogramming in the treatment of diabetic nephropathy （DN） by Shenxiao Tongluo prescription via the peroxisome proliferator-activated receptor γ coactivator-1α （PGC-1α）/sirtuin-3 （SIRT3）/hypoxia-inducible factor-1α （HIF-1α） signaling pathway. MethodsSixty-five SD rats were randomized into a sham group （10 rats） and a modeling group （55 rats）， and the modeling rats underwent left nephrectomy and intraperitoneal injection of streptozotocin （35 mg·kg^-1） to prepare a DN model. After successful modeling， the rats were randomized into model， empagliflozin （10 mg·kg^-1）， and low-， medium-， and high-dose （7.656， 15.312， 30.624 g·kg^-1， respectively） Shenxiao Tongluo prescription groups. The urine microalbumin （UmAlb）， blood urea nitrogen （BUN）， and serum creatinine （SCr） levels of rats in each group were assessed after continuous gavage for 8 weeks. The corresponding kits were used to measure the levels of lactate， superoxide dismutase （SOD）， and malondialdehyde （MDA） in the kidney tissue. Hematoxylin-eosin staining， Masson staining， and periodic acid-Schiff staining were performed to observe the pathological changes in the kidney tissue. Transmission electron microscopy was employed to observe mitochondrial morphology. Immunohistochemistry was employed to determine the expression levels of dynamin-related protein 1 （DRP1） and pyruvate kinase M2 （PKM2） in the kidney tissue. Western blot was adopted to assess the protein levels of PGC-1α， SIRT3， HIF-1α， dynamin-related protein 1 （Drp1）， optic atrophy 1 （OPA1）， hexokinase 2 （HK2）， and pyruvate kinase M2 （PKM2） in the kidney tissue. ResultsCompared with the sham group， the model group showed elevated levels of UmAlb， BUN， SCr， lactate， and MDA， decreased SOD level （P<0.05）， glomerular hypertrophy， thickening of the mesangial basement membrane， vacuolar degeneration of renal tubular epithelial cells， and infiltration of renal interstitial inflammatory cells， oval mitochondria with disordered， blurred or disappearing cristae， down-regulated protein levels of PGC-1α， SIRT3， and OPA1， and up-regulated protein levels of HIF-1α， DRP1， HK2， and PKM2 （P<0.05）. Compared with the model group， the treatment in all the groups increased the body weight， lowered the levels of GLU， UmAlb， BUN， and MDA， raised the level of SOD， alleviated the pathological damage in the kidney tissue and mitochondrial damage， up-regulated the expression of PGC-1α， SIRT3， and OPA1， and down-regulated the expression of HIF-1α， DRP1， and PKM2 （P<0.05）. Empagliflozin and Shenxiao Tongluo prescription at medium and high doses lowered the levels of SCr and lactate and down-regulated the expression of HK2 （P<0.05）， which had no statistical significance in the low-dose Shenxiao Tongluo prescription group. ConclusionShenxiao Tongluo prescription may regulate mitochondrial dynamics and metabolic reprogramming by activating the PGC-1α/SIRT3/HIF-1α pathway， thereby alleviating oxidative damage in the kidney tissue and delaying the progression of DN.