搜索结果：European journal of histochemistry : EJH

Colorectal cancer (CRC) is prone to metastasis, leading to a poor prognosis. miR-411-3p exhibits a tumor-suppressive function in CRC, but its exact mechanism is unclear. The malignant biological properties of CRC cells were detected by Carboxyfluorescein diacetate succinimidyl ester (CFSE) staining, scratch-wound and transwell assay. Levels of markers associated with macrophage polarization were evaluated by flow cytometry and ELISA kits. Bioinformatics analysis to screen whether the downstream target mRNA of miR-411-3p is matrix metalloproteinase 7 (MMP7), and Dual-Luciferase reporter assay verified the targeting relationship between the two. qRT-PCR tested miR-411-3p and MMP7 levels. MMP7 level was quantified by Western blot. Additionally, a nude mouse subcutaneous graft tumor model was constructed, Ki-67 expression was detected by immunohistochemistry, and the impact of miR-411-3p/MMP7 on the polarization of M2 macrophages was explored. miR-411-3p expression is downregulated in CRC. Knockdown of miR-411-3p elevated the amount of CFSE-positive, migrating, and invading cells, decreased apoptosis, and elevated the levels of M2 macrophage polarization markers. After overexpression of miR-411-3p, all of the above metrics were reversed in CRC cells. miR-411-3p targeted negative regulation of MMP7 expression, and MMP7 overexpression further enhanced the promotional effect of knockdown of miR-411-3p on the malignant progression of CRC and M2 macrophage polarization. Furthermore, knockdown of miR-411-3p upregulated the MMP7 level, elevated Ki-67-positive cells count, and induced M2 macrophage polarization in vivo. Knockdown of miR-411-3p upregulates MMP7 and induces M2 macrophage polarization, which in turn promotes malignant biological progression of CRC.

This study aimed to investigate the crosstalk mechanism between pancreatic cancer (PAC) cells and M2 tumor-associated macrophages induced by tumor-derived exosomal miR-34a. MicroRNA and mRNA expression levels were detected using RT-qPCR. Cell Counting Kit-8, wound-healing, transwell assays and flow cytometry were respectively employed to assess cell proliferation, migration, invasion and apoptosis. Enzyme-linked immunosorbent assay was utilized to determine cytokine secretion. Transmission electron microscopy and nanoparticle tracking analyses were performed to detect the exosome morphology and particle size. Phagocytosis of exosomes by macrophages was verified by PKH26 labeling. The effects of exosome-treated macrophages on the epithelial-mesenchymal transition, invasion, and migration of PANC-1 cells were investigated using coculture experiments. The identification of miR-34a's potential targets were determined with TargetScan and validated by a dual-luciferase reporter assay. miR-34a was expressed at low levels in PAC tissues, cells, and exosomes. The overexpression of miR-34a restrains the malignant progression of PANC-1 cells. After miR-34a-overexpressed PANC-1-derived exosomes were phagocytosed by macrophages, the process of M2 polarization in macrophages was obstructed, leading to the suppression of epithelial-mesenchymal transition, migration, and invasion of the cocultured PANC-1 cells. Suppressor of cytokine signaling 3 is a direct target of miR-34a. MiR-34a negatively modulates the suppressor of cytokine signaling 3 to prevent the M2 polarization of macrophages by engaging the Janus kinase/signal transducers and activators of the transcription pathway and influencing the malignancy of PAC cells.  miR-34a in cancer cell-derived exosomes inhibits the malignant progression of pancreatic cancer cells by restraining M2 polarization of macrophages.

E-cadherin (E-cad) is a crucial regulatory factor in rescue Epithelial-mesenchymal transition and is involved in the occurrence of various malignant tumor. However, the mechanisms by which E-cadherin regulates tumor metastasis in CRC remain unclear. We established sh-E-cad (silenced by short hairpin RNA) and rescue-E-cad (overexpressed by E-cad plasmid transfection) CRC cell lines to investigate the role of E-cad in CRC in vitro. Immunohistochemistry, clonogenic assays, scratch wound healing assays, CCK-8 assays, flow cytometry, Transwell assay, real time-PCR and Western blot were employed to investigate the underlying mechanisms by which E-cad involve the progression of CRC. In CRC tissues, E-cad expression was significantly reduced, while YAP expression was markedly elevated. Silencing E-cad induced a significant increase of clonogenic ability in CRC cells, which was reduced upon rescue of E-cad expression. Transwell assays indicate that low expression of E-cad enhances the cell migration, a finding corroborated by scratch wound healing experiments. CCK-8 results demonstrate that silencing E-cad promotes the proliferation of CRC cells. Importantly, we found that E-cad influences apoptosis rather than the cell cycle. Analysis of Hippo signaling pathway-related factors revealed that silencing E-cad resulted in significantly decreased expression of MST1/2 and LATS1/2, as well as reduced phosphorylation levels of YAP, while YAP expression was significantly increased. Additionally, immunofluorescence confirmed the nuclear translocation of YAP. Our study indicates that E-cad regulates the malignant progression of CRC via the Hippo signaling pathway, offering a potential new strategy for CRC treatment.

Hepatocellular cancer (HCC) is the sixth most common type of cancer worldwide. Guanosine monophosphate synthase (GMPS) participates in the regulation of chromatin and genes in various organisms, and is highly expressed in a number of human malignant tumors. However, the role of GMPS in HCC has not yet been fully studied and clarified. In this study, the differential fold changes in gene expression levels between HCC cancer tissues and correspondent adjacent normal tissue in The Cancer Genome Atlas Program and GEO datasets were analyzed using R language. GMPS expression levels in HCC cells were knocked down using specific siRNAs. In addition, CCK-8, EdU, TUNEL and immunofluorescence staining were conducted to explore the effects of GMPS siRNAs on HCC cell viability, proliferation, apoptosis and the STAT pathway level, respectively. The results indicated GMPS expression was significantly increased in HCC tumor tissues compared with the corresponding adjacent normal tissues. In addition, high expression of GMPS is negatively associated with the survival rate of patients with HCC. In vitro studies illustrated the knockdown of GMPS notably prevented HCC cell proliferation and induced HCC cell (Hep3B2.1-7 and MHCC97H) apoptosis by regulating the STAT3/c-Myc pathway. The apoptosis-specific marker cleaved caspase was significantly upregulated by GMPS knockdown in HCC cells. The findings of the present study revealed the association between GMPS and the prognosis of HCC. The results suggested that GMPS may serve as a promising marker for the prognosis of HCC, and it may also be a potential therapeutic target for HCC. These findings may lay the theoretical foundation for the clinical application of GMPS.

Newborns' intestinal adhesions have been reported in 4.7% infants who underwent a laparotomy, but adhesions can also appear idiopathically. Etiology and pathogenesis of adhesions is still to be determined, but evidence shows relation to inflammation, formation of fibrin bands, hypoxia and tissue remodelation. Multiple candidate genes have been associated with adhesion development. The aim of this study was to evaluate the appearance of Sonic Hedgehog (SHH), Indian Hedgehog (IHH), Forkhead-box F1 (FOXF1), caudal type homeobox 1 (CDX1), HCLS1-associated protein X-1 (HAX-1), GATA Binding Protein 4 (GATA4) and Granzyme-B (GZMB) proteins in infant adhesions and to describe possible interfactorial correlations. Adhesion affected tissue samples were collected from 14 patients under one year of age that underwent abdominal surgery to treat partial or complete intestinal obstruction. The control group consisted of 6 individuals that had surgical repairment of inguinal hernia. Routine staining and immunohistochemistry were performed. Immunopositive fibroblasts, macrophages, endotheliocytes, smooth muscle myocytes of blood vessel wall and mesotheliocytes were investigated. The relative distribution of all factors was evaluated by the semiquantitative counting method. Statistical analysis was done using non-parametric tests and correlations were calculated based on Spearman's correlation analysis. A statistically significant decrease was observed for SHH, IHH, FOXF1, GATA4 and partially for GZMB in the adhesion group. There were also decreased HAX-1 and CDX1 immunopositive structures in the adhesion group, however, without any statistical significance. SHH, IHH, FOXF1, GATA4 and GZMB might have a role in adhesion development among infant patients which could suggest a dysregulation of cellular events. Abundance of correlations between the gene protein appearances in different structures indicate the affected blood vessels, fibroblasts and macrophages, however, mesothelium seems not to be the key driver in the morphopathogenesis of adhesion development.

To elucidate the proteins associated with cochlear development and auditory formation from a histomorphological point of view, this study examined the spatio-temporal expression pattern of nestin, parvalbumin, and otoferlin in the mouse cochlea from embryonic day 17 (E17) to postnatal day 28 (P28) using immunofluorescence. Our findings revealed that nestin was broadly expressed in developing otic mesenchyme cells beneath the basilar membrane, medial to the greater epithelial ridge, and adjacent to the developing stria vascularis during late embryonic stages (E17 and E18.5). From P1 to the onset of hearing (P14), nestin was primarily expressed in fibrocytes derived from otic mesenchyme cells in the spiral ligament and spiral limbus, as well as in tympanic border cells. Dual immunofluorescence staining of nestin with Isolectin B4 (IB4), a specific vascular endothelial marker, showed the location of nestin in the blood vessels within the cochlear lateral wall. Notably, in adults (P28), nestin expression was downregulated in the fibrocytes of the spiral ligament and spiral limbus but persisted in the tympanic border cells. Parvalbumin immunolabeling was consistently observed in spiral ganglion neurons (SGNs) and inner hair cells (IHCs) from E17 through adulthood. By P1, parvalbumin expression extended to all three rows of outer hair cells (OHCs) and persisted into adulthood. Transient parvalbumin expression was also noted in afferent nerve fibers innervating the IHCs during early postnatal stages. Otoferlin labeling was predominantly detected in the cytoplasm of IHCs, with limited temporal expression in OHCs from P6 to P10. Taken together, these results illustrated the dynamic expression of nestin, parvalbumin and otoferlin during cochlear development and suggested their important function in cochlear development.

Epithelial-mesenchymal transition (EMT) is closely linked to liver cancer prognosis, invasiveness, and aggressiveness. One promising treatment for liver cancer is cell therapy, where stem cells are stimulated to develop into functional liver cells. This study aimed to investigate the effect of miR-122-5p on the differentiation of human induced pluripotent stem cells (hiPSCs) into hepatocyte-like cells and its impact on the EMT process in liver cancer cells. MiR-122-5p was overexpressed or silenced in hiPSCs to analyze the expression of liver-specific markers, including AFP, ALB and ASGPR, to confirm hepatocyte-like differentiation. A co-culture system with HepG2 liver cancer cells was also used to evaluate the effect of miR-122-5p-overexpressing hiPSCs or miR-122-5p-silencing hiPSCs on the expression of EMT markers. Results revealed that overexpression of miR-122-5p in hiPSCs induced hepatocyte-like characteristics, as evidenced by increased levels of AFP, ALB, and ASGPR. However, knockdown of miR-122-5p had the opposite effect. In the co-culture system, hiPSCs overexpressing miR-122-5p inhibited the EMT process of HepG2 cells, resulting in increased levels of mesenchymal markers and decreased levels of epithelial markers. Taken together, miR-122-5p promotes the differentiation of hiPSCs into hepatocyte-like cells and inhibits EMT process of liver cancer cells. Targeting miR-122-5p may be a novel approach to prevent liver cancer progression through cell therapy.

Hypoxia is a key driver of glioblastoma (GBM) progression. Serine/arginine-rich splicing factor 3 (SRSF3) is associated with the malignant progression of GBM, but its role in the hypoxic microenvironment of GBM remains unclear. This study aimed to explore the regulatory role and molecular mechanisms of SRSF3 in hypoxia adaptation in GBM. The expression of SRSF3 in normal astrocytes and GBM cells was detected. The effects of knockdown or overexpression of SRSF3 combined with hypoxia treatment on malignant phenotypes and hypoxia stress adaptation in GBM cells were evaluated. Cell viability, colony formation, migration, invasion, and cell death assays were performed to assess phenotypic changes. Mechanisms were investigated using mRFP-GFP-LC3, autophagy, and unfolded protein response (UPR)-related molecular detection. SRSF3 was highly expressed in GBM cells. Knockdown of SRSF3 inhibited cell viability, migration, invasion, and colony formation, whereas overexpression of SRSF3 promoted malignant behaviors. Further studies revealed that hypoxia induction significantly increased the expression levels of GRP78, CHOP, ATF4, LC3-II/I, and p62; upregulated the GFP/mRFP ratio; and increased cleaved-caspase3 expression, promoting cell death. Mechanistic studies revealed that SRSF3 overexpression promoted XBP1s formation, alleviated hypoxia-induced autophagic flux blockage, and reduced cell death. The IRE1 RNase inhibitor 4μ8C weakened the SRSF3-mediated promotion of XBP1s generation. SRSF3 enhances adaptive UPR output by promoting IRE1-dependent XBP1 splicing, thereby maintaining autophagic flux and promoting GBM cell survival under hypoxic conditions.

The bone marrow (BM) niche plays a pivotal role in regulating the fate of hematopoietic stem cells (HSCs), and its integrity changes significantly during aging and in rare hematological disease, as in myelofibrosis (MF). In this study, we investigated how the localization and dynamics of HSCs are influenced under physiological and pathological conditions by a newly identified by HSC-supportive megakaryocytes (MKs) subpopulation. Using huCD34tTA/TetO-H2BGFP reporter mice, we analyzed HSCs distribution within the BM and quantified nuclear green fluorescent protein (GFP) intensity to assess the repopulating potential of aged controls and mutated Gata1low mice for MF. In the control group of aged mice, cells with high levels of GFP are clustered, and adjacent to cells morphologically identifiable as supportive MKs. These clusters displayed homogeneous GFP intensity, indicating that HSCs with similar functional properties tend to co-localize in proximity to supportive MKs. By contrast, in aged huCD34/TET/Gata1low mice, GFP cells were predominantly isolated and showed reduced fluorescence intensity. Although the frequency of MKs with a supportive phenotype was increased in MF mice, analyses of GFP revealed that the ability of these MKs to maintain the HSCs in their niche was significantly impaired. Our results provide new insights on the maladaptive remodeling of the BM niche. They highlight the supportive role of MKs as potential key regulators of HSCs homeostasis. Despite their numerical expansion in MF, these cells are functionally compromised, thereby contributing to altered HSCs localization, mobilization, and to hematopoietic failure.

Ozone (O3) is an oxidizing natural gas widely applied as adjunctive therapeutic treatment for a variety of pathological conditions. Currently, O3-based therapies rely on the low-dose concept i.e., the administration of low O3 concentrations able to induce a mild oxidative stress stimulating antioxidant and anti-inflammatory response without causing cell damage. In addition, low O3 concentrations are thought to activate cellular and molecular mechanisms responsible for analgesic and regenerative effects. Due to these properties, in the last decade interest has arisen in the fields of orthopedics and regenerative medicine on the potential of O3 to counteract joint diseases involving cartilage degeneration. In this pilot study, we have explored the anti-degenerative potential of O3 on knee articular cartilage explanted from a healthy adult rabbit and maintained in vitro. Light and transmission electron microscopy were used to monitor chondrocyte and extracellular matrix features of cartilage samples undergoing O3 treatment every three days for two weeks. Results demonstrated that low O3 concentrations act on chondrocytes and the molecular components of the extracellular matrix of articular cartilage explants, significantly improving their preservation under in vitro conditions, likely by promoting both protective and pro-regenerative pathways. This opens promising perspectives for further investigations on the therapeutic potential of O3 for the treatment of cartilage degeneration not only as painkilling and anti-inflammatory agent but also as a cartilage regenerative agent.

Morphological analysis of neuronal processes and their networks is a key aspect of neuroscience, with relevance from basic research to clinical practice due to the central role of neuronal development and plasticity in many neurological disorders. More than a century after its introduction, Golgi staining, a technique based on the random precipitation of metallic deposits in different neuronal subtypes, remains a highly valuable method for investigating the cellular morphology of neurons in the nervous system. Despite the wide range of protocols developed over the years, several limitations of the technique remain a matter of discussion. Among these is the need to extend sample preservation during the interval between staining and sectioning procedures without compromising the quality of the histochemical labeling. By adopting a specific processing method, the present study demonstrates that it is possible to embed murine nervous tissue following Golgi staining and to preserve the samples for extended periods prior to sectioning, while maintaining well-preserved and clearly detectable histochemical labeling across different regions and neurons of the mouse central nervous system.

Inflammatory bowel disease (IBD) including ulcerative colitis (UC) and Crohn's disease has become a global disease in the 21st century, with increasing incidence rates in almost every industrialized country. Previous studies have suggested that the traditional Chinese medicine herb, cryptotanshinone (CTN), a major liposoluble extract of Salvia miltiorrhiza, alleviates the symptoms of experimental colitis in vitro and in vivo. However, the mechanisms underlying the protective effects of CTN against IBD remain exclusive. The present study found that CTN reversed lipopolysaccharide-induced inflammation in human colon epithelial cells (HIEC-6) by inhibiting the NF-κB pathway. In addition, CTN alleviated dextran sulfate sodium (DSS)-induced inflammatory bowel disease in mice by regulating the balance of TH17/Treg cells. CTN also exerted its role by inhibiting the polarization of M1 macrophages in mice with DSS-induced colitis. Of note, the effects of CTN on these immune cells may be mediated via changes in the levels of TNF-α and IL-6 directly in mice. Taken together, these findings may provide new insight regarding the therapeutic potential of CTN for UC.

Cold shock domain (CSD) proteins, such as YB-1, play a crucial role in the regulation of transcription, mRNA stability, and translation. Consequently, YB-1 is implicated in processes such as cell differentiation, oncogenesis and oxidative stress response. The development of the eye is a complex process that involves the differentiation of numerous highly specialized cell types. We hypothesized that YB-1 is involved in both eye development and stress defense mechanisms. As an initial step, we investigated the expression of YB-1 during the embryology of the mouse eye. YB-1 mRNA could be detected by RT-PCR and sequencing the PCR product in retinal tissue of adult mice. To elucidate the expression pattern of YB-1 protein during mouse eye development, we analyzed its expression in the developing mouse eye at embryonic day 13 (E13), E15, E18 and postnatal day 14 (P14) using immunohistochemistry. Expression of the YB-1 protein was detected in all retinal cells, as well as in the corneal and lens epithelial cells, throughout all stages of eye development examined. These findings suggest that YB-1 could have a significant role in the eye, potentially related to development and differentiation.

The bone morphogenetic protein (BMP) pathway, which plays a crucial role in the control of intestinal epithelial cell homeostasis, has been studied in mice and humans, leading to an understanding of its involvement in several intestinal pathologies. However, the expression and localization of the various actors (ligands, antagonists, receptors) of this pathway remain unknown in the rat intestine, although this species is widely used in pathophysiology studies. Here, we aimed to determine the expression and localization of the various players in the BMP pathway in the jejunum and colon of the rat using RT-qPCR and immunohistochemistry. BMP2, mainly localized in epithelial cells, was the most expressed ligand in the jejunum and colon in comparison with BMP4, BMP6 and BMP7. We showed for the first time that BMP7 was highly expressed in epithelial cells in both tissues. BMP2, BMP6 and BMP7 ligands were also present in the enteric nervous plexuses, as the BMP receptors and antagonists Noggin and Chordin-like 1. The expression of BMP antagonists and ligands in enterocytes and mature colonocytes could suggest a paracrine or autocrine feedback modulation at the cellular level. Finally, all the studied BMP actors were present in colonic vessel walls including GREM1, a BMP antagonist described as pro-angiogenic and also being a ligand for VEGFR receptors. These data provided a good correlation between the observations in rats compared to those in humans and highlighted the importance of the BMP pathway not only in the intestinal epithelium, but also in both the enteric nervous system and vascular system. Our work lays the foundations for further studies on the involvement of the BMP pathway in rat models of intestinal pathophysiology.

Muscle spindles are skeletal muscle sensory receptors composed of intrafusal fibres, partially encapsulated by connective tissue capsule. This capsule encloses the central A and B regions while leaving the distal C region extracapsular. Several past studies in rat have shown that muscle spindles typically contain a single bag1 fibre, a single bag2 fibre, and two smaller chain fibres. Intrafusal fibres co-express multiple myosin heavy chain (MyHC) isoforms: -slow or -1, -slow-tonic, -α, -2a, -2b, -embryonic, and -neonatal. While MyHC-2x was previously thought absent, the recently discovered MyHC-15 isoform has been identified in the C region of rat bag fibres. Using antibodies specific for nine MyHC isoforms and analyzing four different rat skeletal muscles-soleus, extensor digitorum longus, and the lateral and medial heads of gastrocnemius-we aimed to further characterize the co-expression pattern of MyHC-15 with other known isoforms and to determine whether MyHC-2x is expressed in rat intrafusal fibres. While rodents are widely used as animal models in skeletal muscle research, notable species-specific differences in MyHC isoform expression exist. Our findings revealed that MyHC-15 expression in rat intrafusal fibres is less abundant than in human fibres. MyHC-15 was primarily observed in bag fibres but was not detected in the C region, contrary to previous reports in both rat and human. We confirmed the absence of MyHC-2x in rat intrafusal fibres. Similarly, MyHC-embryonic and -neonatal were not detected in the analyzed spindles, suggesting that previously used antibodies may have cross-reacted with MyHC-2a and -2b. While our results partially corroborate previous extensive studies, discrepancies suggest that MyHC expression in intrafusal fibres varies not only along the fibre length but also across muscles.

Myelofibrosis (MF) is a rare chronic hematological disorder, within the family of myeloproliferative neoplasms. The MF patients present clinical abnormalities such as anemia, and thrombosis, as well as alterations in the bone marrow (BM) microenvironment, an increased number of megakaryocytes (MKs), most of which are found in emperipolesis with neutrophils. In MF, the MKs emperipolesis is induced by an altered MK secretome, containing increased levels of pro-inflammatory cytokines, proteins, and growth factors such as interleukin-8 (IL-8) and P-selectin (P-sel). These, allow the altered cell-to-cell interactions and cause the transforming growth factor-β (TGF-β) to be released into the BM microenvironment. This fibrogenic cytokine contributes to BM fibrosis and disease progression. Emperipolesis has already been identified as a pathobiological event that contributes to MF and it is widely recognized in the most advanced stages of the disease. In this study, we evaluated the role of P-sel in BM alterations associated with emperipolesis in the Gata1low mouse model of MF. Our data show that emperipolesis is driven by P-sel. Genetic ablation of P-sel rescued the BM microenvironment, by decreasing fibrosis, suggesting that pharmacological targeting of P-sel could contribute to reduce the BM dysfunction and disease progression.

Astragaloside IV (AS IV) inhibits the malignant phenotype of nasopharyngeal carcinoma (NPC), but whether its mechanism involves the regulation of immune checkpoint programmed cell death-ligand 1 (PD-L1) is not clear. Human NPC cells were treated with AS IV. The effects of AS IV on PD-L1 expression were assessed using RT-qPCR and Western blot. SATB2/Wnt/β-catenin signaling axis regulation was analyzed by siRNA interference, plasmid overexpression and Wnt pathway inhibitor DKK-1. T cell killing activity and tumor malignant phenotype were evaluated by LDH release, ELISA, flow cytometry and Transwell experiments. huHSC-NCG tumor-bearing mice were established to detect tumor growth, immune cell infiltration and related protein expression. AS IV dose-dependently inhibited PD-L1 expressions within NPC cells, and enhanced the activation and killing function of CD8+ T cells. Mechanism studies have shown that AS IV significantly lowered the expression of SATB2, thereby inhibiting Wnt/β-catenin axis and c-MYC and Axin2 expressions, and ultimately reducing PD-L1 levels. Overexpression of SATB2 reversed AS IV's suppression of this signaling and PD-L1. Animal experiments confirmed that AS IV effectively inhibited tumor growth, enhanced CD8+ T cell infiltration and activity within tumor tissues, and down-regulated the SATB2/β-catenin/PD-L1 signal axis. AS IV inhibited PD-L1 expression in NPC via targeting the SATB2/Wnt/β-catenin axis, thereby activating CD8+ T cells, amplifying immunological responses, and ultimately inhibiting NPC growth. Graphical Abstract.

Chronic kidney disease (CKD) is a progressive disorder characterized by renal fibrosis, inflammation, and dysregulated autophagy and apoptosis. High-mobility group box 1 (HMGB1) plays a crucial role in regulating autophagy in CKD. Hirudin, a potent thrombin inhibitor, has demonstrated antifibrotic and anti-inflammatory properties, but its effects on autophagy and apoptosis in CKD remain unclear. In this study, a rat model of renal interstitial fibrosis (RIF) and an HK-2 cell culture model were established to assess the effects of varying doses of hirudin and HMGB1 interference. Molecular and histological analyses, including RTqPCR, Western blot, TUNEL staining, hematoxylin-eosin (H&E) staining, immunofluorescence, and immunohistochemistry (IHC), were performed to assess renal injury, fibrosis, apoptosis, and autophagy-related markers. Hirudin treatment significantly reduced the expression of LC3, ATG12, ATG5, &#x3b1;-SMA, COL1A1, caspase-3, and caspase-9 while increasing P62 levels (p<0.05). It also lowered the renal coefficient (p<0.001) and apoptosis levels. The optimal effective concentration of hirudin in vitro was determined to be 4.8 ATU/mL (p<0.001). HMGB1 interference suppressed autophagy and apoptosis, as indicated by decreased LC3-II/LC3-I, ATG12, ATG5, caspase-3, and caspase-9 levels, increased P62 expression (p<0.001), and reduced apoptosis. However, simultaneous HMGB1 interference in hirudin-treated cells weakened the therapeutic effects of hirudin, leading to increased autophagy and apoptosis markers, decreased P62 levels, and a higher renal coefficient. These findings indicate that hirudin exerts protective effects in CKD by modulating autophagy and apoptosis, potentially through HMGB1 regulation. These findings highlight the therapeutic potential of targeting these mechanisms in renal dysfunction and underscore the necessity for further research to support clinical applications.

Sepsis remains a major cause of morbidity and mortality worldwide, yet its prolonged pathophysiological consequences are poorly understood. Here, we employed a murine cecal ligation and puncture (CLP) model to investigate the prolonged impact of sepsis on survival, systemic inflammation, and organ pathology. Adult male C57BL/6 mice underwent CLP or sham surgery and were monitored for 28 days. Survival was recorded daily, while serial assessments of hematology, serum biochemistry, bacterial load, and cytokine levels were performed. Tissue immunofluorescence was used to characterize myeloid-derived suppressor cells (MDSCs), which are potent immunosuppressive cells that inhibit both adaptive and innate immune responses in sepsis, contributing to sepsis-induced immunosuppression. Histopathological analyses were conducted to evaluate structural changes in major organs. CLP mice displayed markedly reduced long-term survival compared with sham controls. Hematological profiling revealed persistent leukocytosis and an inflammatory response, while serum analyses showed sustained elevations in in bilirubin, creatinine, and blood urea nitrogen, reflecting hepatic and renal injury. Bacterial cultures confirmed systemic microbial persistence, and cytokine measurements indicated ongoing inflammatory activity. Tissue immunofluorescence demonstrated the infiltration of MDSCs across multiple organs, consistent with post-sepsis immunosuppression. Histopathological examination revealed widespread, chronic injury in the lungs, liver, kidneys, and spleen, including inflammatory infiltration, tissue degeneration, and architectural disruption. In conclusion, sepsis induces not only acute systemic inflammation but also enduring immune dysregulation and progressive organ damage. These findings highlight the CLP model as a robust platform for studying post-sepsis sequelae and underscore the need for therapeutic strategies that target long-term organ protection and immune restoration.