Diabetic nephropathy（DN） is a common and severe microvascular complication of diabetes. In recent years， the classical herbal formula Shenqi dihuang decoction has demonstrated unique advantages in the clinical treatment of DN. This article conducts a systematic review of the mechanisms of action and clinical applications of Shenqi dihuang decoction in the treatment of DN. It reveals that the mechanism by which this formula improves DN involves multi-target synergistic regulation. For instance, Shenqi dihuang decoction exerts multiple pharmacological effects by regulating signaling pathways including phosphatidy linostiol 3-kinase/protein kinase B， AMP-activated protein kinase/silent information regulator 1/forkhead box O1， and nuclear factor erythroid 2-related factor 2/heme oxygenase-1 pathways.These effects include regulating glucose and lipid metabolism， inhibiting oxidative stress， reducing inflammation， improving insulin resistance， modulating cell death （apoptosis/autophagy/ferroptosis/pyroptosis）， and preventing renal fibrosis. Existing clinical studies indicate that Shenqi dihuang decoction and its modified formulas， alone or in combination with other therapeutic methods， can significantly improve glucose and lipid metabolism， reduce proteinuria， and delay renal function decline in patients with DN. These effects are superior to those of Western medicines such as irbesartan， valsartan， and empagliflozin， and the treatment demonstrates good safety. Future research should leverage systems biology and artificial intelligence technologies to further elucidate the integrated mechanisms in the treatment of DN by Shenqi dihuang decoction， thereby advancing the precision and standardization of its clinical application.

ObjectiveTo construct B cell conditional knockout Spi1 gene mice and analysis their genotypes to provide an animal model basis for disease pathogenesis and drug target research. MethodsMb1-cre transgenic mice were crossed with Spi1^flox/flox mice. PCR combined with agarose gel electrophoresis was performed for genotyping， and Mb1-cre×Spi1^flox/flox homozygous knockout mice were screened out. Flow cytometry was used to detect the expression of PU.1 protein in various immune cell subsets to verify the specificity of gene knockout. ResultsPCR identification results showed that when using flox primers for detection， only a 220 bp band was amplified， indicating a genotype of Spi1^flox/flox in the mice. When using Mb1-Cre primers for detection， a 383 bp band was amplified， indicating a genotype of Mb1-cre×Spi1^flox/flox in the mice； Flow cytometry results showed that compared to Spi1^flox/flox mice， Mb1-cre×Spi1^flox/flox mice exhibited significantly reduced PU.1 expression levels in bone marrow-derived mononuclear cells， peripheral blood mononuclear cells， and spleen-derived B cells. Moreover， PU.1 exhibited normal expression in other immune cells， such as CD4⁺ T cells and macrophages. ConclusionBy utilizing the Cre/LoxP system and CRISPR/Cas9 technology， this study successfully generates mice with B cell-specific conditional knockout of the Spi1 gene， providing a reliable animal model for in-depth exploration of the specific role of PU.1 in B cell-related diseases.

Objective To study the plasma metabolomics of mice poisoned by different dosage of the combination of diazepam and ethanol,and to reveal the toxicological mechanisms of combined poisoning of diazepam and ethanol.Methods Female Kunming mice were randomly divided into blank group,single and combined poisoning group(n=6),Based on the LD50 of diazepam co-administered with graded ethanol doses,mice in the single-drug and combined groups received oral gavage at 1/2,1,and 2 × LD50.Retro-orbital blood samples(～500 μL)were collected within 24 hours post-administration and analyzed by UPLC-QE-MS technology.Principal component analysis and orthogonal partial least squares discriminant analysis were used to identify differential metabolites and associated metabolic pathways.Results A total of 387 differential metabolites were identified in the combined poisoning group of diazepam and ethanol implicating the key pathways including tryptophan metabolism,phenylalanine metabolism,arginine and proline metabolism,Glycerophospholipid metabolism,phenylalanine,tyrosine and tryptophan biosynthesis.Conclusion Combined diazepam and ethanol poisoning exerts significant systemic effects by disrupting neurotransmitters conduction,exacerbating oxidative stress response and dysregulating energy metabolism.

Objective:To evaluate the role of inositol 1, 4, 5 triphosphate receptor 1 (IP3R1)-regulated changes in mitochondria-associated endoplasmic reticulum membrane (MAM) structure in the long-term cognitive impairment induced by multiple exposures to sevoflurane anesthesia in neonatal mice.Methods:Sixty SPF-grade healthy neonatal C57BL/6J mice of either sex, aged 6 days, weighing 6-10 g, were divided into 3 groups ( n=20 each) using a random number table method: control group (group C), multiple sevoflurane anesthesia group (group S), and IP3R antagonist 2-APB+ multiple sevoflurane anesthesia group (group I+ S). Group S and group I+ S inhaled 3% sevoflurane anesthesia for 2 h starting from 6, 8 and 10 days after birth. In group I+ S, 2-APB 3 mg/kg was intraperitoneally injected before each sevoflurane anesthesia. The open field test was performed at day 31 after birth to assess the spontaneous mobility. The Morris water maze test was performed at days 31-36 after birth to assess the cognitive function. Mice were sacrificed at the end of the water maze test, hippocampal CA1 region was isolated and hippocampal tissues were obtained for determination of the intracellular calcium ion concentration ([Ca 2+ ] i) and rate of necroptosis (using Flow cytometry) and expression of IP3R1, G protein-coupled receptor 75 (GRP75), receptor-interacting protein kinase 1 (RIPK1), RIPK3, and phosphorylated human mixed-series protein kinase-like structural domains (p-MLKL) (by Western blot). Transmission electron microscopy was performed to observe and record the partial length of MAMs, endoplasmic reticulum circumference and mitochondrial circumference. Results:There were no statistically significant differences in the speed, distance, and time of staying at the center in open field tests among the three groups ( P>0.05). Compared with group C, the escape latency was significantly prolonged on postnatal days 33-35, the number of crossing the original platform was reduced, the necroptosis rate in the hippocampal CA1 region and [Ca 2+ ] i were increased, the expression of IP3R, GRP75, RIPK1, RIPK3 and p-MLKL was up-regulated, and the ratio of MAMs partial length/endoplasmic reticulum perimeter and ratio of MAMs partial length/mitochondria perimeter in hippocampal neurons were elevated in group S ( P<0.05). Compared with group S, the escape latency was significantly shortened on postnatal days 32-35, the number of crossing the original platform was increased, the necroptosis rate in the hippocampal CA1 region and [Ca 2+ ] i were decreased, the expression of IP3R, GRP75, RIPK1, RIPK3 and p-MLKL was down-regulated, and the ratio of MAMs partial length/endoplasmic reticulum perimeter and ratio of MAMs partial length/mitochondria perimeter in hippocampal neurons were decreased in group I+ S ( P<0.05). Conclusions:Structural changes in MAMs in the hippocampal CA1 region mediated by the up-regulation of IP3R1 expression are involved in the process of long-term cognitive impairment induced by multiple exposures to sevoflurane anesthesia in neonatal mice.

Objective:To evaluate the relationship between sevoflurane preconditioning-induced reduction of cognitive impairment and hippocampal necroptosis after cardiopulmonary bypass (CPB) in rats.Methods:Sixty SPF healthy male Sprague-Dawley rats, aged 6 months, weighing 400-450 g, were divided into 4 groups ( n=15 each) using the random number table method: control group (group C), sevoflurane group (Sev group), CPB group and CPB+ sevoflurane preconditioning group (CPB+ Sev group). The rats were exposed to 0.4% sevoflurane for 2 h in CPB+ Sev group and Sev group. The CPB model was established at 30 min after the end of sevoflurane preconditioning in CPB+ Sev group. The open field test was performed to assess the autonomic movement ability on the 2nd day after CPB. The Morris water maze test was used to assess the cognitive function on the 3rd day after CPB. The hippocampal tissues were removed after the end of the Morris water maze test for determination of the necroptosis rate and cytosolic calcium concentration of hippocampal neuron ([Ca 2+ ] i) (by flow cytometry) and the expression of phosphorylated receptor-interacting protein kinase 1 (p-RIPK1), phosphorylated RIPK3 and phosphorylated mixed-lineage kinase-like domain (p-MLKL) (by Western blot) and for microscopic examination of the ultrastructure of hippocampal neurons (by transmission electron microscopy). Results:There was no statistically significant difference in the parameters of the open field test among the four groups ( P>0.05). Compared with group C, the escape latency was significantly prolonged, the number of crossing the original platform was decreased, the time of staying at the original platform quadrant was shortened, the hippocampal necroptosis rate and [Ca 2+ ] i were increased, the expression of p-RIPK1, p-RIPK3 and p-MLKL was up-regulated ( P<0.05), the organelles of hippocampal neurons swelled, lysosomes broke, and some chromatin in nuclei dissoluted in CPB group. Compared with CPB group, the escape latency was significantly shortened, the number of crossing the original platform was increased, the time of staying at the original platform quadrant was prolonged, the hippocampal necroptosis rate and [Ca 2+ ] i were decreased, the expression of p-RIPK1, p-RIPK3 and p-MLKL was down-regulated ( P<0.05), and the damage to the ultrastructure of hippocampal neurons was sinificantly reduced in CPB+ Sev group ( P<0.05). Conclusions:The mechanism by which sevoflurane preconditioning attenuates cognitive impairment may be related to the inhibition of calcium overload-mediated hippocampal necroptosis in a rat model of CPB.

Objective:To evaluate the role of RhoA/ROCK2 pathway in electroacupuncture (EA) preconditioning-induced reduction of the perioperative neurocognitive disorder (PND) in aged rats.Methods:Eighty SPF healthy male Sprague-Dawley rats, aged 20 months, weighing 600-650 g, were divided into 4 groups ( n=20 each) using the random number table method: sham operation group (group S), PND group, EA preconditioning group and EA preconditioning plus RhoA agonist arachidonic acid group (EA+ AA group). The PND model was prepared using exploratory laparotomy performed under 3% sevoflurane anesthesia. In PND, EA and EA+ AA groups, EA preconditioning was initiated 5 days before operation as follows: Bilateral acupoints Zusanli, Hegu and Neiguan were stimulated with sparse-dense waves at 2/15 Hz and an electric current intensity of 1 mA, applied for 30 min a day for 5 consecutive days. Arachidonic acidin 10 mg/kg was intraperitoneally injected at 30 min before surgery in group AA. The open field test was conducted at 3 days postoperatively to measure the autonomous motor function, and the Morris water maze test was conducted at 3-7 days postoperatively to evaluate the cognitive function. After the end of Morris water maze test, the rats were sacrificed, and the hippocampal tissue in CA1 region was obtained for determination of the apoptosis rate of cells and concentrations of cytoplasmic calcium ion ([Ca 2+ ] i) (by flow cytometry) and the expression of phosphorylated RhoA (p-RhoA), ROCK2, and cleaved caspase-3 (by Western blot) and for examination of the ultrastructure of hippocampal neurons (with a transmission electron microscope). Results:There was no statistically significant difference in each parameter of the open field test among the four groups ( P>0.05). Compared with group C, the escape latency was significantly prolonged, the number of crossing the original platform was reduced, the apoptosis rate of hippocampal cells and [Ca 2+ ] i were increased, the expression of p-RhoA, ROCK2 and cleaved-caspase-3 was up-regulated ( P<0.05), and the pathological damage to hippocampal neurons was marked in PND group. Compared with PND group, the escape latency was significantly shortened, the number of crossing the original platform was reduced, the apoptosis rate of hippocampal cells and [Ca 2+ ] i were increased, the expression of p-RhoA, ROCK2 and cleaved-caspase-3 was up-regulated ( P<0.05), and the pathological damage to hippocampal neurons was significantly attenuated in EA group. Compared with EA group, the escape latency was significantly prolonged, the number of crossing the original platform was reduced, the apoptosis rate of hippocampal cells and [Ca 2+ ] i were increased, the expression of p-RhoA, ROCK2 and cleaved-caspase-3 was up-regulated ( P<0.05), and the pathological damage to hippocampal neurons was aggravated in EA+ AA group. Conclusions:The mechanism by which EA preconditioning reduces PND is related to inhibiting the activation of hippocampal RhoA/ROCK2 signaling pathway and reducing calcium overload-mediated apoptosis in cells of aged rats.

Objective To investigate the association between healthy lifestyle factors and cardiovascular biological aging,as well as the relative contributions of different lifestyle factors.Methods Based on the clinical biochemical data and anthropometric data from the baseline survey of the UK Biobank(UKB),the Klemera-Doubal method(KDM)was used to establish cardiovascular biological age(CBA),and CBA acceleration was calculated accordingly.Multiple linear regression models were used to estimate the associations between healthy lifestyle factors and CBA acceleration.Then,the Quantile g-computation(QGC)was applied to evaluate the relative contributions of different lifestyle factors to CBA acceleration,with further analyses conducted separately for male and female populations.Additionally,stratified analyses were performed based on age,sex,body mass index(BMI),racial background,and family history of cardiovascular diseases to examine population heterogeneity.Results A total of 251 478 participants were included in the study.Both the overall healthy lifestyle score and each of the 7 lifestyle factors were negatively associated with CBA acceleration(overall lifestyle score:β=-0.75,95%CI:-0.77 to-0.73).Regarding the relative contributions of different lifestyle factors,alcohol consumption and diet accounted for the highest proportions(25.8%and 25.7%,respectively).However,there were differences by sex—alcohol consumption contributed the most in men(29.5%),followed by diet(23.0%),while in women,diet contributed the most(34.5%)and alcohol consumption accounted for a relatively low proportion(5.5%).Stratified analyses suggested that sex,BMI,and race might be potential effect modifiers.Conclusion Lifestyle factors,as modifiable behaviors,can slow the rate of cardiovascular biological aging.Among these factors,alcohol consumption and diet may represent effective targets for intervention.

Objective:To evaluate the role of inositol 1, 4, 5 triphosphate receptor 1 (IP3R1)-regulated changes in mitochondria-associated endoplasmic reticulum membrane (MAM) structure in the long-term cognitive impairment induced by multiple exposures to sevoflurane anesthesia in neonatal mice.Methods:Sixty SPF-grade healthy neonatal C57BL/6J mice of either sex, aged 6 days, weighing 6-10 g, were divided into 3 groups ( n=20 each) using a random number table method: control group (group C), multiple sevoflurane anesthesia group (group S), and IP3R antagonist 2-APB+ multiple sevoflurane anesthesia group (group I+ S). Group S and group I+ S inhaled 3% sevoflurane anesthesia for 2 h starting from 6, 8 and 10 days after birth. In group I+ S, 2-APB 3 mg/kg was intraperitoneally injected before each sevoflurane anesthesia. The open field test was performed at day 31 after birth to assess the spontaneous mobility. The Morris water maze test was performed at days 31-36 after birth to assess the cognitive function. Mice were sacrificed at the end of the water maze test, hippocampal CA1 region was isolated and hippocampal tissues were obtained for determination of the intracellular calcium ion concentration ([Ca 2+ ] i) and rate of necroptosis (using Flow cytometry) and expression of IP3R1, G protein-coupled receptor 75 (GRP75), receptor-interacting protein kinase 1 (RIPK1), RIPK3, and phosphorylated human mixed-series protein kinase-like structural domains (p-MLKL) (by Western blot). Transmission electron microscopy was performed to observe and record the partial length of MAMs, endoplasmic reticulum circumference and mitochondrial circumference. Results:There were no statistically significant differences in the speed, distance, and time of staying at the center in open field tests among the three groups ( P>0.05). Compared with group C, the escape latency was significantly prolonged on postnatal days 33-35, the number of crossing the original platform was reduced, the necroptosis rate in the hippocampal CA1 region and [Ca 2+ ] i were increased, the expression of IP3R, GRP75, RIPK1, RIPK3 and p-MLKL was up-regulated, and the ratio of MAMs partial length/endoplasmic reticulum perimeter and ratio of MAMs partial length/mitochondria perimeter in hippocampal neurons were elevated in group S ( P<0.05). Compared with group S, the escape latency was significantly shortened on postnatal days 32-35, the number of crossing the original platform was increased, the necroptosis rate in the hippocampal CA1 region and [Ca 2+ ] i were decreased, the expression of IP3R, GRP75, RIPK1, RIPK3 and p-MLKL was down-regulated, and the ratio of MAMs partial length/endoplasmic reticulum perimeter and ratio of MAMs partial length/mitochondria perimeter in hippocampal neurons were decreased in group I+ S ( P<0.05). Conclusions:Structural changes in MAMs in the hippocampal CA1 region mediated by the up-regulation of IP3R1 expression are involved in the process of long-term cognitive impairment induced by multiple exposures to sevoflurane anesthesia in neonatal mice.

Objective:To evaluate the relationship between sevoflurane preconditioning-induced reduction of cognitive impairment and hippocampal necroptosis after cardiopulmonary bypass (CPB) in rats.Methods:Sixty SPF healthy male Sprague-Dawley rats, aged 6 months, weighing 400-450 g, were divided into 4 groups ( n=15 each) using the random number table method: control group (group C), sevoflurane group (Sev group), CPB group and CPB+ sevoflurane preconditioning group (CPB+ Sev group). The rats were exposed to 0.4% sevoflurane for 2 h in CPB+ Sev group and Sev group. The CPB model was established at 30 min after the end of sevoflurane preconditioning in CPB+ Sev group. The open field test was performed to assess the autonomic movement ability on the 2nd day after CPB. The Morris water maze test was used to assess the cognitive function on the 3rd day after CPB. The hippocampal tissues were removed after the end of the Morris water maze test for determination of the necroptosis rate and cytosolic calcium concentration of hippocampal neuron ([Ca 2+ ] i) (by flow cytometry) and the expression of phosphorylated receptor-interacting protein kinase 1 (p-RIPK1), phosphorylated RIPK3 and phosphorylated mixed-lineage kinase-like domain (p-MLKL) (by Western blot) and for microscopic examination of the ultrastructure of hippocampal neurons (by transmission electron microscopy). Results:There was no statistically significant difference in the parameters of the open field test among the four groups ( P>0.05). Compared with group C, the escape latency was significantly prolonged, the number of crossing the original platform was decreased, the time of staying at the original platform quadrant was shortened, the hippocampal necroptosis rate and [Ca 2+ ] i were increased, the expression of p-RIPK1, p-RIPK3 and p-MLKL was up-regulated ( P<0.05), the organelles of hippocampal neurons swelled, lysosomes broke, and some chromatin in nuclei dissoluted in CPB group. Compared with CPB group, the escape latency was significantly shortened, the number of crossing the original platform was increased, the time of staying at the original platform quadrant was prolonged, the hippocampal necroptosis rate and [Ca 2+ ] i were decreased, the expression of p-RIPK1, p-RIPK3 and p-MLKL was down-regulated ( P<0.05), and the damage to the ultrastructure of hippocampal neurons was sinificantly reduced in CPB+ Sev group ( P<0.05). Conclusions:The mechanism by which sevoflurane preconditioning attenuates cognitive impairment may be related to the inhibition of calcium overload-mediated hippocampal necroptosis in a rat model of CPB.

Objective:To evaluate the role of RhoA/ROCK2 pathway in electroacupuncture (EA) preconditioning-induced reduction of the perioperative neurocognitive disorder (PND) in aged rats.Methods:Eighty SPF healthy male Sprague-Dawley rats, aged 20 months, weighing 600-650 g, were divided into 4 groups ( n=20 each) using the random number table method: sham operation group (group S), PND group, EA preconditioning group and EA preconditioning plus RhoA agonist arachidonic acid group (EA+ AA group). The PND model was prepared using exploratory laparotomy performed under 3% sevoflurane anesthesia. In PND, EA and EA+ AA groups, EA preconditioning was initiated 5 days before operation as follows: Bilateral acupoints Zusanli, Hegu and Neiguan were stimulated with sparse-dense waves at 2/15 Hz and an electric current intensity of 1 mA, applied for 30 min a day for 5 consecutive days. Arachidonic acidin 10 mg/kg was intraperitoneally injected at 30 min before surgery in group AA. The open field test was conducted at 3 days postoperatively to measure the autonomous motor function, and the Morris water maze test was conducted at 3-7 days postoperatively to evaluate the cognitive function. After the end of Morris water maze test, the rats were sacrificed, and the hippocampal tissue in CA1 region was obtained for determination of the apoptosis rate of cells and concentrations of cytoplasmic calcium ion ([Ca 2+ ] i) (by flow cytometry) and the expression of phosphorylated RhoA (p-RhoA), ROCK2, and cleaved caspase-3 (by Western blot) and for examination of the ultrastructure of hippocampal neurons (with a transmission electron microscope). Results:There was no statistically significant difference in each parameter of the open field test among the four groups ( P>0.05). Compared with group C, the escape latency was significantly prolonged, the number of crossing the original platform was reduced, the apoptosis rate of hippocampal cells and [Ca 2+ ] i were increased, the expression of p-RhoA, ROCK2 and cleaved-caspase-3 was up-regulated ( P<0.05), and the pathological damage to hippocampal neurons was marked in PND group. Compared with PND group, the escape latency was significantly shortened, the number of crossing the original platform was reduced, the apoptosis rate of hippocampal cells and [Ca 2+ ] i were increased, the expression of p-RhoA, ROCK2 and cleaved-caspase-3 was up-regulated ( P<0.05), and the pathological damage to hippocampal neurons was significantly attenuated in EA group. Compared with EA group, the escape latency was significantly prolonged, the number of crossing the original platform was reduced, the apoptosis rate of hippocampal cells and [Ca 2+ ] i were increased, the expression of p-RhoA, ROCK2 and cleaved-caspase-3 was up-regulated ( P<0.05), and the pathological damage to hippocampal neurons was aggravated in EA+ AA group. Conclusions:The mechanism by which EA preconditioning reduces PND is related to inhibiting the activation of hippocampal RhoA/ROCK2 signaling pathway and reducing calcium overload-mediated apoptosis in cells of aged rats.