ObjectiveTo investigate the therapeutic effects and mechanism of Shenqi Dihuang decoction （SQDHD） on diabetic kidney disease （DKD）， with a focus on its impact on arachidonic acid-related ferroptosis. MethodsSixty C57BL/6 mice were allocated into a normal group （n=10） and a modeling group （n=50）， with 43 mice successfully modeled. The successfully modeled mice were further allocated into model， low-， medium-， and high-dose （4.68， 9.36， and 18.72 g·kg^-1， respectively） SQDHD， and dapagliflozin （0.13 mg·kg^-1） groups. The drug treatment groups were administrated with corresponding agents by gavage， and the normal and model groups were administrated with equal volumes of normal saline by gavage. An electronic balance and a glucometer were used to monitor the body weight and fasting blood glucose level from the tail tip， respectively. Serum creatinine （Scr） and blood urea nitrogen （BUN） levels were measured by enzyme-linked immunosorbent assay （ELISA）. Histopathological changes in the renal tissue were assessed by hematoxylin-eosin staining， Masson staining， and periodic acid-Schiff （PAS） staining. The fluorescence intensity of reactive oxygen species （ROS） in frozen sections was observed by an inverted fluorescence microscope to evaluate the levels of ferrous ions （Fe²⁺） and lipid peroxidation in the renal tissue. Immunofluorescence staining of glutathione peroxidase 4 （GPX4） and acyl-CoA synthetase long-chain family member 4 （ACSL4） in the renal tissue was performed to detect their localization and expression. Western blot was employed to assess the expression levels of key ferroptosis proteins such as GPX4 and cystine/glutamate antiporter （xCT）， as well as the arachidonic acid metabolic pathway-related proteins， including ACSL4， lysophosphatidylcholine acyltransferase 3 （LPCAT3）， and arachidonate 15-lipoxygenase （ALOX15）. Real-time PCR was employed to measure the mRNA levels of key ferroptosis proteins， including solute carrier family 7 member 11 （SLC7A11） and GPX4， as well as arachidonic acid metabolism-related factors （ACSL4， LPCAT3， and ALOX15） in the renal tissue. ResultsCompared with the normal group， DKD model mice exhibited a decrease in body weight （P<0.01）， increases in levels of blood glucose （P<0.01）， 24-hour urinary protein， Scr， and BUN （P<0.01）， along with severe pathological changes， such as mesangial cell proliferation， basement membrane thickening， tubular atrophy， and interstitial inflammatory cell infiltration. In addition， the modeling elevated the levels of Fe²⁺， MDA， LPO， and ROS （P<0.01）， lowered the GPX4 and xCT levels （P<0.01）， raised the ACSL4， LPCAT3， and ALOX15 levels （P<0.01）， down-regulated the mRNA levels of GPX4 and SLC7A11 （P<0.01）， and up-regulated the mRNA levels of ACSL4， LPCAT3， and ALOX15 （P<0.01） in the renal tissue. Compared with the model group， low-， medium-， and high-dose SQDHD groups and the dapagliflozin group showed an increase in body weight （P<0.01）， decreases in levels of blood glucose （P<0.01）， 24-hour urinary protein， and Scr （P<0.01）， alleviated pathological changes in glomeruli and tubules， and reduced degree of glomerular and tubular fibrosis. The high-dose SQDHD group and the dapagliflozin group showed reductions in Fe²⁺， MDA， LPO， and ROS levels （P<0.01）. The medium- and high-dose SQDHD groups and the dapagliflozin group exhibited increased levels of GPX4 and xCT （P<0.01）， decreased levels of ACSL4， LPCAT3， and ALOX15 （P<0.05， P<0.01）， and down-regulated mRNA levels of ACSL4， LPCAT3， and ALOX15 （P<0.01）. ConclusionSQDHD ameliorates DKD by inhibiting ferroptosis potentially by reducing iron ion levels， inhibiting lipid peroxidation， up-regulating GPX4 expression， and down-regulating ACSL4 expression. This study provides new insights and a theoretical basis for the treatment of DKD with traditional Chinese medicine and identifies potential targets for developing novel therapeutics for DKD.

ObjectiveTo investigate the therapeutic effects and mechanism of Shenqi Dihuang decoction （SQDHD） on diabetic kidney disease （DKD）， with a focus on its impact on arachidonic acid-related ferroptosis. MethodsSixty C57BL/6 mice were allocated into a normal group （n=10） and a modeling group （n=50）， with 43 mice successfully modeled. The successfully modeled mice were further allocated into model， low-， medium-， and high-dose （4.68， 9.36， and 18.72 g·kg^-1， respectively） SQDHD， and dapagliflozin （0.13 mg·kg^-1） groups. The drug treatment groups were administrated with corresponding agents by gavage， and the normal and model groups were administrated with equal volumes of normal saline by gavage. An electronic balance and a glucometer were used to monitor the body weight and fasting blood glucose level from the tail tip， respectively. Serum creatinine （Scr） and blood urea nitrogen （BUN） levels were measured by enzyme-linked immunosorbent assay （ELISA）. Histopathological changes in the renal tissue were assessed by hematoxylin-eosin staining， Masson staining， and periodic acid-Schiff （PAS） staining. The fluorescence intensity of reactive oxygen species （ROS） in frozen sections was observed by an inverted fluorescence microscope to evaluate the levels of ferrous ions （Fe²⁺） and lipid peroxidation in the renal tissue. Immunofluorescence staining of glutathione peroxidase 4 （GPX4） and acyl-CoA synthetase long-chain family member 4 （ACSL4） in the renal tissue was performed to detect their localization and expression. Western blot was employed to assess the expression levels of key ferroptosis proteins such as GPX4 and cystine/glutamate antiporter （xCT）， as well as the arachidonic acid metabolic pathway-related proteins， including ACSL4， lysophosphatidylcholine acyltransferase 3 （LPCAT3）， and arachidonate 15-lipoxygenase （ALOX15）. Real-time PCR was employed to measure the mRNA levels of key ferroptosis proteins， including solute carrier family 7 member 11 （SLC7A11） and GPX4， as well as arachidonic acid metabolism-related factors （ACSL4， LPCAT3， and ALOX15） in the renal tissue. ResultsCompared with the normal group， DKD model mice exhibited a decrease in body weight （P<0.01）， increases in levels of blood glucose （P<0.01）， 24-hour urinary protein， Scr， and BUN （P<0.01）， along with severe pathological changes， such as mesangial cell proliferation， basement membrane thickening， tubular atrophy， and interstitial inflammatory cell infiltration. In addition， the modeling elevated the levels of Fe²⁺， MDA， LPO， and ROS （P<0.01）， lowered the GPX4 and xCT levels （P<0.01）， raised the ACSL4， LPCAT3， and ALOX15 levels （P<0.01）， down-regulated the mRNA levels of GPX4 and SLC7A11 （P<0.01）， and up-regulated the mRNA levels of ACSL4， LPCAT3， and ALOX15 （P<0.01） in the renal tissue. Compared with the model group， low-， medium-， and high-dose SQDHD groups and the dapagliflozin group showed an increase in body weight （P<0.01）， decreases in levels of blood glucose （P<0.01）， 24-hour urinary protein， and Scr （P<0.01）， alleviated pathological changes in glomeruli and tubules， and reduced degree of glomerular and tubular fibrosis. The high-dose SQDHD group and the dapagliflozin group showed reductions in Fe²⁺， MDA， LPO， and ROS levels （P<0.01）. The medium- and high-dose SQDHD groups and the dapagliflozin group exhibited increased levels of GPX4 and xCT （P<0.01）， decreased levels of ACSL4， LPCAT3， and ALOX15 （P<0.05， P<0.01）， and down-regulated mRNA levels of ACSL4， LPCAT3， and ALOX15 （P<0.01）. ConclusionSQDHD ameliorates DKD by inhibiting ferroptosis potentially by reducing iron ion levels， inhibiting lipid peroxidation， up-regulating GPX4 expression， and down-regulating ACSL4 expression. This study provides new insights and a theoretical basis for the treatment of DKD with traditional Chinese medicine and identifies potential targets for developing novel therapeutics for DKD.

OBJECTIVE: To elucidate the molecular mechanisms underlying sepsis-induced injury in mouse intestinal organoids and investigate the possible mechanisms or potential drug targets of myeloid differentiation factor 88 inhibitor [TJ-M2010-5 (TJ5)] on this condition. METHODS: Small intestinal organoids from C57BL/6 mice aged 6-8 weeks were established and characterized using immunofluorescence for cell growth and proliferation marker nuclear antigen Ki-67, goblet cell marker mucin-2 (MUC-2), epithelial cell marker E-cadherin, and Paneth cell marker lysozyme (Lyz). Small intestinal organoids after 3 days of passaging were divided into different groups: a normal control group treated with culture medium containing 0.2% dimethyl sulfoxide (DMSO) for 10 hours, a lipopolysaccharide (LPS) group treated with culture medium containing 200 mg/L LPS and 0.2% DMSO for 10 hours, and a TJ5 group pre-treated with 10 mmol/L TJ5 for 2 hours followed by treatment with culture medium containing 200 mg/L LPS for 10 hours. Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to measure the expression levels of interleukin-6 (IL-6) and zonula occludens-1 (ZO-1) in the small intestinal organoids. RNA transcriptome sequencing was performed on the small intestinal organoids from each group to analyze differentially expressed genes between groups, and significant enrichment was analyzed using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). RESULTS: By the 7th day of primary culture, mature organoids had formed, and their growth rate increased after passaging. Immunofluorescence identification showed expressions of Ki-67, MUC-2, E-cadherin, and Lyz, indicating that the mouse small intestinal organoids maintained their cellular composition and functional characteristics under in vitro culture conditions. RT-qPCR results showed that compared with the normal control group, the mRNA expression of IL-6 in the small intestinal organoids of the LPS group was significantly increased (2^-ΔΔCT: 1.83±0.16 vs. 1.02±0.28, P < 0.05), while the mRNA expression of ZO-1 was significantly decreased (2^-ΔΔCT: 0.53±0.11 vs. 1.01±0.18, P < 0.05). In contrast, the mRNA expression trends of both IL-6 and ZO-1 were reversed in the TJ5 group, showing statistically significant differences as compared with the LPS group (2^-ΔΔCT: IL-6 mRNA was 1.24±0.01 vs. 1.83±0.16, ZO-1 mRNA was 1.97±0.29 vs. 0.53±0.11, both P < 0.05). RNA transcriptome sequencing showed 49 differentially expressed genes in the LPS group compared to the normal control group, with 42 upregulated and 7 downregulated. Compared to the LPS group, the TJ5 group showed 84 differentially expressed genes, with 47 upregulated and 37 downregulated. GO enrichment analysis of these differentially expressed genes showed that the significantly enriched biological processes of the differentially expressed genes between the normal control group and the LPS group included responses to LPS, responses to molecule of bacterial origin and responses to bacterium. The significantly enriched biological processes of the differentially expressed genes between the LPS group and the TJ5 group included glutathione metabolic processes, responses to stress cellular and responses to chemical stimulus. In molecular function groups, glutathione binding and oligopeptide binding were significantly enriched by the differentially expressed genes. In cellular component classifications, the enrichment of the differentially expressed genes was mainly observed in the cytoplasm, endoplasmic reticulum, and microsomes. KEGG pathway enrichment analysis indicated that the differentially expressed genes between the normal control group and LPS group were enriched in IL-17 signaling pathways, tumor necrosis factor (TNF) signaling pathways, viral protein interactions with cytokines and cytokine receptors signaling pathways, and cytokine-cytokine receptor interaction signaling pathways. In contrast, the differentially expressed genes between the LPS and TJ5 groups were mainly enriched in atherosclerosis signaling pathways, ferroptosis signaling pathways, glutathione metabolism signaling pathways, and cytochrome P450-mediated drug metabolism signaling pathways. CONCLUSIONS Mouse small intestinal organoids were successfully extracted and cultured. TJ5 may exert its protective effects by regulating gene expression and related signaling pathways (fluid shear stress and atherosclerosis, ferroptosis, glutathione metabolism, cytochrome P450 drug metabolism, etc.) in sepsis-injured mouse small intestinal organoids. These genes and signaling pathways may be key targets for treating sepsis-induced intestinal injury.
Animals ; Mice ; Sepsis/genetics* ; Organoids/drug effects* ; Mice, Inbred C57BL ; Intestine, Small/metabolism* ; Gene Expression Profiling ; Transcriptome ; Lipopolysaccharides

The efficient production of L-glutamate is dependent on the product's rapid efflux, hence researchers have recently concentrated on artificially modifying its transport system and cell membrane wall structure. Considering the unique composition and structure of the cell wall of Corynebacterium glutamicum, we investigated the effects of CmpLs on L-glutamate synthesis and transport in SCgGC7, a constitutive L-glutamate efflux strain. First, the knockout strains of CmpLs were constructed, and it was confirmed that the deletion of CmpL1 and CmpL4 significantly improved the performance of L-glutamate producers. Next, temperature-sensitive L-glutamate fermentation with the CmpL1 and CmpL4 knockout strains were carried out in 5 L bioreactors, where the knockout strains showcased temperature-sensitive characteristics and enhanced capacities for L-glutamate production under high temperatures. Notably, the CmpL1 knockout strain outperformed the control strain in terms of L-glutamate production, showing production and yield increases of 69.2% and 55.3%, respectively. Finally, the intracellular and extracellular metabolites collected at the end of the fermentation process were analyzed. The modification of CmpLs greatly improved the L-glutamate excretion and metabolic flux for both L-glutamate production and transport. Additionally, the CmpL1 knockout strain showed decreased accumulation of downstream metabolites of L-glutamate and intermediate metabolites of tricarboxylic acid (TCA) cycle, which were consistent with its high L-glutamate biosynthesis capacity. In addition to offering an ideal target for improving the stability and performance of the industrial strains for L-glutamate production, the functional complementarity and redundancy of CmpLs provide a novel target and method for improving the transport of other metabolites by modification of the cell membrane and cell wall structures in C. glutamicum.
Corynebacterium glutamicum/genetics* ; Glutamic Acid/biosynthesis* ; Fermentation ; Metabolic Engineering ; Bacterial Proteins/metabolism* ; Bioreactors/microbiology* ; Gene Knockout Techniques

With the increasing life expectancy and lifestyle changes of patients with chronic hepatitis B (CHB), the significance of comorbidities of chronic non-communicable diseases (NCDs) in disease progression and health prognosis of CHB patients is gaining prominence. This study aims to explore the association between CHB and NCDs comorbidities, focusing on the impact of common metabolism-related diseases, such as metabolic syndrome and diabetes, on the health outcomes of CHB patients. We also summarize studies on integrating the management of comorbidities in CHB patients and provide relevant recommendations for effective management. The findings of this study serve as a foundation for understanding the clinical characteristics and prevalence trends, reducing the disease burden of comorbidities among CHB patients, and establishing a comprehensive and coordinated management system for comorbidities.

Objective:To analyze the disease burden in middle-aged and elderly population aged ≥55 in Shenzhen from 2016 to 2030 and provide evidence for the development of healthy aging strategies.Methods:The years of life lost (YLL), years lost due to disability (YLD), and the disability-adjusted life year (DALY) in this population from 2016 to 2022 were calculated. Joinpoint log-linear regression model was used to analyze the time trend. Bayesian age-period-cohort model and grey system model were used to predict YLL, YLD, and DALY in this population in 2030.Results:From 2016 to 2022, the crude DALY rate showed a transient fluctuation in age group 55-74 years, but a pronounced increase in age group ≥85 years. The proportions of YLL and YLD due to non-communicable diseases in all age groups was considerably higher than those due to communicable and nutritional diseases and injuries. In 2022, in all age groups, the YLL due to neoplasms (55-74 years old) and cardiovascular disease (≥75 years old) ranked first, and the YLD due to musculoskeletal disorder ranked first. By 2030, the causes of YLL and YLD ranking first in each age group would be remained, while the ranks of some causes would increase.Conclusions:The age specific characteristics of current and predicted disease burden differed in individuals aged ≥55 years. Therefore, it is necessary to allocate social and medical resources according to the disease burden pattern.

Retrograde adeno-associated viruses (AAVs) are capable of infecting the axons of projection neurons and serve as a powerful tool for the anatomical and functional characterization of neural networks. However, few retrograde AAV capsids have been shown to offer access to cortical projection neurons across different species and enable the manipulation of neural function in non-human primates (NHPs). Here, we report the development of a novel retrograde AAV capsid, AAV-DJ8R, which efficiently labeled cortical projection neurons after local administration into the striatum of mice and macaques. In addition, intrastriatally injected AAV-DJ8R mediated opsin expression in the mouse motor cortex and induced robust behavioral alterations. Moreover, AAV-DJ8R markedly increased motor cortical neuron firing upon optogenetic light stimulation after viral delivery into the macaque putamen. These data demonstrate the usefulness of AAV-DJ8R as an efficient retrograde tracer for cortical projection neurons in rodents and NHPs and indicate its suitability for use in conducting functional interrogations.
Animals ; Haplorhini ; Axons ; Motor Neurons ; Interneurons ; Macaca ; Dependovirus/genetics* ; Genetic Vectors

OBJECTIVE To investigate the impact of evodiamine on the proliferation， migration and invasion abilities of trophoblastic cells induced by high glucose and its potential mechanism based on advanced glycation end products （AGE）/receptor for advanced glycation end products （RAGE） signaling pathway. METHODS Human trophoblastic cells HTR-8/SVneo were divided into control group， high glucose group， evodiamine low-dose group （2 μmol/L）， evodiamine high-dose group （4 μmol/L）， pc-NC group （transfected with pc-NC plasmid+4 μmol/L evodiamine）， and pc-RAGE group （transfected with pc-RAGE plasmid+ 4 μmol/L evodiamine）. Cells in all groups except for the control group were cultured in a high sugar （25 mmol/L glucose） environment， and cells in all groups except for the control group and the model group were transfected with the corresponding plasmids and/or received the corresponding drug interventions. The survival rate， apoptotic rate， scratch healing rate， and invasion number， as well as the protein and mRNA expressions of AGE， RAGE， nuclear factor- κB p65 （NF- κB p65）， matrix metalloproteinase-2 （MMP-2）， and MMP-9 were examined in each group. RESULTS Compared with the control group， the cell survival rate， scratch healing rate， invasions number， and the mRNA and protein expressions of MMP-2 and MMP-9 in the high glucose group significantly decreased （P＜0.05）， while the apoptotic rate， the mRNA and protein expressions of AGE and RAGE， the mRNA expression of NF-κB p65， and the phosphorylation level of NF-κB p65 protein significantly increased （P＜0.05）； compared with the high glucose group， the above indexes of cells in evodiamine low-dose and high-dose groups were significantly improved， and the effect of the high-dose group was significantly better than that of the low-dose group （P＜0.05）； overexpression of RAGE attenuated the ameliorative effect of evodiamine onthe above indexes in high glucose-induced trophoblast cells （except for AGE mRNA and protein） （P＜0.05）. CONCLUSIONS Evodiamine can promote the proliferation， migration and invasion of high glucose-induced trophoblast cells and ameliorate their functional impairment， and the above effects are associated with the inhibition of the AGE/RAGE signaling pathway.

Objective To analyze the relationship between plasma mitochondrial DNA(mtDNA),macrophage inflammatory protein-1α(MIP1α)and monocyte chemotactic protein 1(MCP-1)in vastus lateralis tissues and postoperative muscle atrophy,recovery of hip function in patients with hip fractures.Methods A total of 86 patients with hip fractures and 43 patients with coxitis in Jinan Eighth People's Hospital were enrolled as hip fracture group and coxitis group between October 2020 and October 2022,respectively.The lateral muscle tissues were collected as samples during surgery.The level of plasma mtDNA was detected by real-time fluorescence quantitative polymerase chain reaction.Before surgery,levels of serum interleukin-6(IL-6)and tumor necrosis factor α(TNF-α)were detected by enzyme-linked immunosorbent assay.Before surgery,cross-sectional areas of types Ⅰ and Ⅱ vastus lateralis fibers were detected by immunofluorescence method.Before surgery,expression levels of MIP1α and MCP-1 proteins in lateral muscle tissues were detected by Western blot.All patients with hip fracture were effectively followed up for 6 months after surgery.At 3 and 6 months after surgery,total lean mass(TLM)and unaffected limb lean mass(ULLM)were detected by DXA.Results The level of plasma mtDNA in hip fracture group was higher than that in coxitis group before surgery[(4.12±0.53)vs(2.37±0.36),P＜0.05],levels of serum IL-6 and TNF-α were higher than those in coxitis group[(34.68±6.14)pg/ml,(21.54±4.12)pg/ml vs(12.74±3.06)pg/ml,(10.81±2.71)pg/ml,P＜0.05],cross-sectional areas of types Ⅰ and Ⅱ vastus lateralis fibers were smaller than those in coxitis group[(4321.45±441.36)μm2,(2384.38±247.11)μm2 vs(5417.63±553.27)μm2,(3569.24±368.22)μm2,P＜0.05],and expression levels of MIP1α and MCP-1 proteins were higher than those in coxitis group[(2.34±0.25),(2.47±0.28)vs(1.18±0.15),(1.95±0.23),P＜0.05].In patients with hip fracture after 6 months of follow-up,there were 53 cases with good prognosis and 33 cases with poor prognosis.The level of plasma mtDNA in poor prognosis group was higher than that in good prognosis group before surgery[(4.53±0.52)vs(3.87±0.44),P＜0.05],levels of serum IL-6 and TNF-α were higher than those in good prognosis group[(35.97±5.32)pg/ml,(20.74±4.27)pg/ml vs(33.51±5.16)pg/ml,(22.83±4.33)pg/ml,P＜0.05],cross-sectional areas of types Ⅰ and Ⅱ vastus lateralis fibers were smaller than those in good prognosis group[(4174.26±434.60)μm2,(2309.56±246.18)μm2 vs(4394.42±450.12)μm2,(2430.97±250.72)μm2,P＜0.05],and expression levels of MIP1α and MCP-1 proteins were higher than those in good prognosis group[(2.47±0.28),(1.95±0.23)vs(2.26±0.24),(1.82±0.21),P＜0.05].TLM and ULLM at 6 months after surgery were lower than those at 3 months after surgery in good prognosis group and poor prognosis group(P＜0.05).At 3 and 6 months after surgery,there was no significant different in TLM or ULLM between good prognosis group and poor prognosis group(P＞0.05).Conclusion Traumatic stress injury will increase level of plasma mtDNA in patients with hip fracture,which will induce the increase of systemic inflammatory indexes(serum IL-6,TNF-α)and inflammatory factors(MCP-1,MIP1α)levels,aggravate muscle atrophy and cause postoperative decline of hip function.

At present,some studies have identified gain-of-function(GOF)mutations in phosphoinositide 3-kinase(PI3K)genes PIK3CD(which encodes p110δ)lead to activated PI3Kδ syndrome(APDS),which can cause immune deficiency,immune dis-order and even tumor by multiple mechanisms of internal immune system defects,such as reduction/senescence/depletion of T cells,impaired development of B cells,and reduced toxicity of NK cells.Here,we review clinical characteristics of APDS induced by PI3Kδ GOF and molecular mechanism of immune deficiency,focusing on molecular mechanism of lymphocyte development,differentiation and functional defects caused by GOF mutation.