ObjectiveTo explore the role of cucurbitacin B （CuB） in inducing ferroptosis in 4T1 cells and its mechanism. MethodsThe effects of CuB（0.2， 0.4， 0.8 μmol·L^-1）on the proliferation ability of 4T1 cells in vitro were detected using the methyl thiazolyl tetrazolium （MTT） assay. The clonogenic ability of 4T1 cells was detected by the plate cloning assay， and the levels of lactate dehydrogenase （LDH） in 4T1 cells were detected by the use of a kit. The mitochondrial membrane potential and reactive oxygen species （ROS） levels in 4T1 cells were detected by flow cytometry， and the mitochondrial ultrastructure of 4T1 cells was observed by transmission electron microscopy. The western blot was used to detect the expression of ferroptosis-related protein p53 in 4T1 cells， solute carrier family 7 member 11 （SCL7A11）， glutathione peroxidase 4 （GPX4）， long-chain acyl-CoA synthetase 4 （ACSL4）， transferrin receptor protein 1 （TFR1）， and ferritin heavy chain 1 （FTH1）. ResultsCompared with that in the blank group， the survival rate of 4T1 cells in CuB groups was significantly decreased （P<0.05）， and the number of cell clones in CuB groups was significantly reduced （P<0.01）. In addition， compared with that in the blank group， the leakage of LDH in cells in CuB groups was significantly increased （P<0.01）， and the mitochondrial membrane potential of cells in CuB groups decreased significantly （P<0.01）. Cellular ROS levels were significantly elevated in CuB groups （P<0.01）. The mitochondria of cells in CuB groups were obviously wrinkled， and the mitochondrial cristae were reduced or even disappeared. Compared with that in the blank group， the protein expression of p53， ACSL4， and TFR1 were significantly up-regulated in CuB groups （P<0.05）， and that of SLC7A11， GPX4， and FTH1 were significantly down-regulated （P<0.05）. ConclusionCuB may inhibit SLC7A11 and GPX4 expression by up-regulating the expression of p53， which in turn regulates the p53/SLC7A11/GPX4 signaling pathway axis and accelerates the generation of lipid peroxidation substrate by up-regulating the expression of ACSL4. It up-regulates TFR1 expression to promote cellular uptake of Fe³⁺ and down-regulates the expression of FTH1 to reduce the ability of iron storage， resulting in an elevated free Fe²⁺ level. It catalyzes the Fenton reaction， generates excess ROS， imbalances the antioxidant system and iron metabolism， and then induces ferroptosis in 4T1 cells.

ObjectiveTo explore the role of cucurbitacin B （CuB） in inducing ferroptosis in 4T1 cells and its mechanism. MethodsThe effects of CuB（0.2， 0.4， 0.8 μmol·L^-1）on the proliferation ability of 4T1 cells in vitro were detected using the methyl thiazolyl tetrazolium （MTT） assay. The clonogenic ability of 4T1 cells was detected by the plate cloning assay， and the levels of lactate dehydrogenase （LDH） in 4T1 cells were detected by the use of a kit. The mitochondrial membrane potential and reactive oxygen species （ROS） levels in 4T1 cells were detected by flow cytometry， and the mitochondrial ultrastructure of 4T1 cells was observed by transmission electron microscopy. The western blot was used to detect the expression of ferroptosis-related protein p53 in 4T1 cells， solute carrier family 7 member 11 （SCL7A11）， glutathione peroxidase 4 （GPX4）， long-chain acyl-CoA synthetase 4 （ACSL4）， transferrin receptor protein 1 （TFR1）， and ferritin heavy chain 1 （FTH1）. ResultsCompared with that in the blank group， the survival rate of 4T1 cells in CuB groups was significantly decreased （P<0.05）， and the number of cell clones in CuB groups was significantly reduced （P<0.01）. In addition， compared with that in the blank group， the leakage of LDH in cells in CuB groups was significantly increased （P<0.01）， and the mitochondrial membrane potential of cells in CuB groups decreased significantly （P<0.01）. Cellular ROS levels were significantly elevated in CuB groups （P<0.01）. The mitochondria of cells in CuB groups were obviously wrinkled， and the mitochondrial cristae were reduced or even disappeared. Compared with that in the blank group， the protein expression of p53， ACSL4， and TFR1 were significantly up-regulated in CuB groups （P<0.05）， and that of SLC7A11， GPX4， and FTH1 were significantly down-regulated （P<0.05）. ConclusionCuB may inhibit SLC7A11 and GPX4 expression by up-regulating the expression of p53， which in turn regulates the p53/SLC7A11/GPX4 signaling pathway axis and accelerates the generation of lipid peroxidation substrate by up-regulating the expression of ACSL4. It up-regulates TFR1 expression to promote cellular uptake of Fe³⁺ and down-regulates the expression of FTH1 to reduce the ability of iron storage， resulting in an elevated free Fe²⁺ level. It catalyzes the Fenton reaction， generates excess ROS， imbalances the antioxidant system and iron metabolism， and then induces ferroptosis in 4T1 cells.

Background: In acute and chronic renal inflammatory diseases, the activation of inflammatory cells is involved in the defect of erythropoietin (EPO) production. Ras guanine nucleotide-releasing protein-4 (RasGRP4) promotes renal inflammatory injury in type 2 diabetes mellitus (T2DM). Our study aimed to investigate the role and mechanism of RasGRP4 in the production of renal EPO in diabetes. Methods: The degree of tissue injury was observed by pathological staining. Inflammatory cell infiltration was analyzed by immunohistochemical staining. Serum EPO levels were detected by enzyme-linked immunosorbent assay, and EPO production and renal interstitial fibrosis were analyzed by immunofluorescence. Quantitative real-time polymerase chain reaction and Western blotting were used to detect the expression of key inflammatory factors and the activation of signaling pathways. In vitro, the interaction between peripheral blood mononuclear cells (PBMCs) and C3H10T1/2 cells was investigated via cell coculture experiments. Results: RasGRP4 decreased the expression of hypoxia-inducible factor 2-alpha (HIF2A) via the ubiquitination–proteasome degradation pathway and promoted myofibroblastic transformation by activating critical inflammatory pathways, consequently reducing the production of EPO in T2DM mice. Conclusion RasGRP4 participates in the production of renal EPO in diabetic mice by affecting the secretion of proinflammatory cytokines in PBMCs, degrading HIF2A, and promoting the myofibroblastic transformation of C3H10T1/2 cells.

Phakic intraocular lens implantation has become one of the important means of correcting refractive errors today. Among them,the implantable collamer lens(ICL)is favored for its wide range of correction, excellent optical quality, and high safety, but the risks of postoperative complications such as glaucoma and anterior subcapsular opacification still exist. Vault is an important indicator for evaluating the safety after ICL implantation, and its ideal state is crucial for preventing complications. Studies have shown that iris morphology has a significant impact on vault. In order to further optimize surgical outcomes and improve surgical safety, this review comprehensively reviews the research progress of iris-related parameters in ICL implantation and discusses the importance of various parameters in preoperative evaluation and postoperative follow-up.

Background: In acute and chronic renal inflammatory diseases, the activation of inflammatory cells is involved in the defect of erythropoietin (EPO) production. Ras guanine nucleotide-releasing protein-4 (RasGRP4) promotes renal inflammatory injury in type 2 diabetes mellitus (T2DM). Our study aimed to investigate the role and mechanism of RasGRP4 in the production of renal EPO in diabetes. Methods: The degree of tissue injury was observed by pathological staining. Inflammatory cell infiltration was analyzed by immunohistochemical staining. Serum EPO levels were detected by enzyme-linked immunosorbent assay, and EPO production and renal interstitial fibrosis were analyzed by immunofluorescence. Quantitative real-time polymerase chain reaction and Western blotting were used to detect the expression of key inflammatory factors and the activation of signaling pathways. In vitro, the interaction between peripheral blood mononuclear cells (PBMCs) and C3H10T1/2 cells was investigated via cell coculture experiments. Results: RasGRP4 decreased the expression of hypoxia-inducible factor 2-alpha (HIF2A) via the ubiquitination–proteasome degradation pathway and promoted myofibroblastic transformation by activating critical inflammatory pathways, consequently reducing the production of EPO in T2DM mice. Conclusion RasGRP4 participates in the production of renal EPO in diabetic mice by affecting the secretion of proinflammatory cytokines in PBMCs, degrading HIF2A, and promoting the myofibroblastic transformation of C3H10T1/2 cells.

Background: In acute and chronic renal inflammatory diseases, the activation of inflammatory cells is involved in the defect of erythropoietin (EPO) production. Ras guanine nucleotide-releasing protein-4 (RasGRP4) promotes renal inflammatory injury in type 2 diabetes mellitus (T2DM). Our study aimed to investigate the role and mechanism of RasGRP4 in the production of renal EPO in diabetes. Methods: The degree of tissue injury was observed by pathological staining. Inflammatory cell infiltration was analyzed by immunohistochemical staining. Serum EPO levels were detected by enzyme-linked immunosorbent assay, and EPO production and renal interstitial fibrosis were analyzed by immunofluorescence. Quantitative real-time polymerase chain reaction and Western blotting were used to detect the expression of key inflammatory factors and the activation of signaling pathways. In vitro, the interaction between peripheral blood mononuclear cells (PBMCs) and C3H10T1/2 cells was investigated via cell coculture experiments. Results: RasGRP4 decreased the expression of hypoxia-inducible factor 2-alpha (HIF2A) via the ubiquitination–proteasome degradation pathway and promoted myofibroblastic transformation by activating critical inflammatory pathways, consequently reducing the production of EPO in T2DM mice. Conclusion RasGRP4 participates in the production of renal EPO in diabetic mice by affecting the secretion of proinflammatory cytokines in PBMCs, degrading HIF2A, and promoting the myofibroblastic transformation of C3H10T1/2 cells.

Background: In acute and chronic renal inflammatory diseases, the activation of inflammatory cells is involved in the defect of erythropoietin (EPO) production. Ras guanine nucleotide-releasing protein-4 (RasGRP4) promotes renal inflammatory injury in type 2 diabetes mellitus (T2DM). Our study aimed to investigate the role and mechanism of RasGRP4 in the production of renal EPO in diabetes. Methods: The degree of tissue injury was observed by pathological staining. Inflammatory cell infiltration was analyzed by immunohistochemical staining. Serum EPO levels were detected by enzyme-linked immunosorbent assay, and EPO production and renal interstitial fibrosis were analyzed by immunofluorescence. Quantitative real-time polymerase chain reaction and Western blotting were used to detect the expression of key inflammatory factors and the activation of signaling pathways. In vitro, the interaction between peripheral blood mononuclear cells (PBMCs) and C3H10T1/2 cells was investigated via cell coculture experiments. Results: RasGRP4 decreased the expression of hypoxia-inducible factor 2-alpha (HIF2A) via the ubiquitination–proteasome degradation pathway and promoted myofibroblastic transformation by activating critical inflammatory pathways, consequently reducing the production of EPO in T2DM mice. Conclusion RasGRP4 participates in the production of renal EPO in diabetic mice by affecting the secretion of proinflammatory cytokines in PBMCs, degrading HIF2A, and promoting the myofibroblastic transformation of C3H10T1/2 cells.

OBJECTIVE: To observe the effects of Xingnao Kaiqiao acupuncture technique (for regaining consciousness and opening orifice) on methylation of N6-methyladenosine (m6A), and key methyltransferases and demethylases, so as to clarify the mechanism of acupuncture on cerebral ischemia-reperfusion injury (CIRI). METHODS: Of 68 male Sprague-Dawley rats of SPF grade, 15 rats were randomly selected as a sham-operation group, and the remaining rats were subjected to the model of middle cerebral artery occlusion using the suture ligation. CIRI was induced by ischemia for 2 h followed by reperfusion. Rats that failed to modeling or died were excluded. The rest 45 rats were randomly divided into three groups, i.e. model group, acupuncture group, and non-point acupuncture group, with 15 rats in each group. The rats in the acupuncture group were treated with acupuncture at bilateral "Neiguan" (PC6) and "Shuigou" (GV26). In the non-point acupuncture group, acupuncture was delivered at three non-points, located 3 mm below the bilateral midaxillary line and 3 mm lateral to the tip of the coccyx. One intervention was operated in these two acupuncture groups and the needles were retained for 30 min. Before modeling start and 2 h after ischemia, a laser speckle flowmeter was used to monitor the cerebral blood perfusion. In 2 h of ischemia and 24 h of reperfusion, the neurological behavioral score was evaluated. The volume of rat cerebral infarction was determined by triphenyltetrazolium chloride (TTC) staining, and the level of m6A methylation in ischemic cortical area was detected by Dot blot, and the protein and mRNA expression of the demethylase i.e. fat mass and obesity-associated protein (FTO), AlkB homolog 5 (ALKBH5) and key methyltransferases, i.e. methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), and Wilms' tumor 1-associated protein (WTAP) in ischemic cortical area were detected by Western blot and real-time PCR. RESULTS: Cerebral blood perfusion decreased by>70% after 2 h ischemia. Compared with the sham-operation group, the neurobehavioral score and the percentage of cerebral infarction volume increased in the model group (P<0.01); the level of m6A methylation in the ischemic cortical area increased (P<0.01), the protein and mRNA expression of FTO decreased (P<0.01), and that of ALKBH5, METTL3, and METTL14 increased (P<0.01, P<0.05) in the model group. When compared with the model group and the non-point acupuncture group, the acupuncture group showed a decrease in the neurobehavioral score and the percentage of cerebral infarction volume (P<0.01), the level of m6A methylation in the ischemic cortical area was reduced (P<0.01, P<0.05), and the protein and mRNA expression of FTO was elevated (P<0.01). CONCLUSION Xingnao Kaiqiao acupuncture technique presents its protective effect on the brain in the rats with CIRI, which is related to up-regulating the expression of FTO and modulating m6A methylation.
Animals ; Rats, Sprague-Dawley ; Male ; Acupuncture Therapy ; Reperfusion Injury/genetics* ; Rats ; Brain Ischemia/genetics* ; Humans ; Adenosine/metabolism* ; Methylation ; Acupuncture Points ; Cerebral Cortex/metabolism*

OBJECTIVE: To explore the pain-location interaction between pressure-sensitive points on the body surface and traditional acupoints in patients with chronic non-specific low back pain (CNLBP) under different disease courses, using Euclidean distance and multivariate statistical analysis. METHODS: A pressure-sensitive point detection was performed on 30 CNLBP patients with varying disease courses. A constant pressure was applied using an FDK20 algometer within a designated lumbar area, a total of 50 points were tested, and the tested points were numbered; the visual analogue scale (VAS) pain score was recorded simultaneously. MatlabR2022a9.12. software was used to extract the positions of pressure-sensitive points, and preprocessing and normalization of point location and VAS scores data were conducted. Under constraint conditions (VAS≥8.0 ∩ Euclidean distance to acupoint≤0.5), the proportion of pressure-sensitive points within the Euclidean distance threshold to each acupoint (PVD_acupoint) was calculated, followed by multivariate statistical analysis. RESULTS: ①Constrained analysis of PVD_acupoint showed that PVD_{Qihaishu (BL24)} and PVD_{Dachangshu (BL25)} were positively correlated with disease course (r=0.55, P<0.01). ②Factor analysis and silhouette analysis revealed that PVD_{Shenshu (BL23)} and PVD_{Dachangshu (BL25)} exhibited trends consistent with disease course progression (P>0.05), with different degree (P<0.01). CONCLUSION The PVD_acupoint value based on Euclidean distance can characterize the pressure sensitivity features of traditional acupoints associated with disease. Multivariate statistical analysis of PVD_acupoint confirms that selecting the acupoint combination of Shenshu (BL23) and Dachangshu (BL25) for CNLBP is associated with the distribution of surrounding pressure-sensitive points and the pathological characteristics of the condition.
Humans ; Acupuncture Points ; Low Back Pain/physiopathology* ; Male ; Female ; Middle Aged ; Adult ; Aged ; Acupuncture Therapy ; Young Adult ; Pressure

The tumor microenvironment is a crucial factor in tumor occurrence and progression. The hypoxic microenvironment is widely present in tumor tissue and is a key endogenous factor accelerating tumor deterioration. The "blood stasis toxin" theory, as an emerging perspective in tumor research, is regarded as the unique "soil" in tumor progression from the perspective of traditional Chinese medicine(TCM) due to its dynamic evolution mechanism, which closely resembles the formation of the hypoxic microenvironment. Scientifically integrating TCM theories with the biological characteristics of tumors and exploring precise syndrome differentiation and treatment strategies are key to achieving comprehensive tumor prevention and control. This article focused on the hypoxic microenvironment of the tumor, elucidating its formation mechanisms and evolutionary processes and carefully analyzing the internal relationship between the "blood stasis toxin" theory and the hypoxic microenvironment. Additionally, it explored the interaction among blood stasis, toxic pathogens, and hypoxic environment and proposed micro-level prevention and treatment strategies targeting the hypoxic microenvironment based on the "blood stasis toxin" theory, aiming to provide TCM-based theoretical support and therapeutic approaches for precise regulation of the hypoxic microenvironment.
Humans ; Tumor Microenvironment/drug effects* ; Neoplasms/therapy* ; Animals ; Medicine, Chinese Traditional ; Disease Progression ; Drugs, Chinese Herbal