Proteomic Analysis of Surgery-induced Stress Post-Tracheal Transplantation Highlights Changes in Matrisome.

OBJECTIVE: Investigate the impact of Surgery-induced stress (SIS) on the normal airway repair process after airway reconstruction using a mouse microsurgery model, mass spectrometry (MS), and bioinformatic analysis. METHODS: Tracheal tissue from non-surgical (N = 3) and syngeneic tracheal grafts at 3 months post-replacement (N = 3) were assessed using mass spectrometry. Statistical analysis was done using MASCOT via Proteome Discoverer™. Proteins were categorized into total, dysregulated, suppressed, and evoked proteins in response to SIS. Dysregulated proteins were identified using cut-off values of -1 1 and t-test (p value <0.05). Enriched pathways were determined using STRING and Metascape. RESULTS: At the three-month post-operation mark, we noted a significant increase in submucosal cellular infiltration (14343 ± 1286 cells/mm2, p = 0.0003), despite reduced overall thickness (30 ± 3 µm, p = 0.01), compared to Native (4578 ± 723 cells/mm2; 42 ± 6 µm). Matrisome composition remained preserved, with proteomic analysis identifying 193 commonly abundant proteins, encompassing 7.2% collagens, 34.2% Extracellular matrix (ECM) glycoproteins, 6.2% proteoglycans, 33.2% ECM regulators, 14.5% Extracellular matrix-affiliated, and 4.7% secreted factors. Additionally, our analysis unveiled a unique proteomic signature of 217 "Surgery-evoked proteins" associated with SIS, revealing intricate connections among neutrophils, ECM remodeling, and vascularization through matrix metalloproteinase-9 interaction. CONCLUSIONS: Our study demonstrated the impact of SIS on the extracellular matrix, particularly MMP9, after airway reconstruction. The novel identification of MMP9 prompts further investigation into its potential role in repair. LEVEL OF EVIDENCE: NA Laryngoscope, 2024 Laryngoscope, 2024.

Dynamic flow for efficient partial decellularization of tracheal grafts: A preliminary rabbit study.

Objective: Bioengineered tracheal grafts are a potential solution for the repair of long-segment tracheal defects. A recent advancement is partially decellularized tracheal grafts (PDTGs) which enable regeneration of host epithelium and retain viable donor chondrocytes for hypothesized benefits to mechanical properties. We propose a novel and tunable 3D-printed bioreactor for creating large animal PDTG that brings this technology closer to the bedside. Methods: Conventional agitated immersion with surfactant and enzymatic activity was used to partially decellularize New Zealand white rabbit (Oryctolagus cuniculus) tracheal segments (n = 3). In parallel, tracheal segments (n = 3) were decellularized in the bioreactor with continuous extraluminal flow of medium and alternating intraluminal flow of surfactant and medium. Unprocessed tracheal segments (n = 3) were also collected as a control. The grafts were assessed using the H&E stain, tissue DNA content, live/dead assay, Masson's trichrome stain, and mechanical testing. Results: Conventional processing required 10 h to achieve decellularization of the epithelium and submucosa with poor chondrocyte viability and mechanical strength. Using the bioreactor reduced processing time by 6 h and resulted in chondrocyte viability and mechanical strength similar to that of native trachea. Conclusion: Large animal PDTG created using our novel 3D printed bioreactor is a promising approach to efficiently produce tracheal grafts. The bioreactor offers flexibility and adjustability favorable to creating PDTG for clinical research and use. Future research includes optimizing flow conditions and transplantation to assess post-implant regeneration and mechanical properties. Level of Evidence: NA.

Protection of Proanthocyanidins Against HSP Serum-Induced Inflammation and Oxidative Stress on Human Umbilical Vein Endothelial Cells.

Background: Immune-mediated inflammation and oxidative stress play pivotal roles in Henoch-Schonlein purpura (HSP), primarily through the TLR4/MyD88/NF-κB pathway. Proanthocyanidins (PCs) exert anti-inflammatory and antioxidant effects by regulating some signals like TLR4/MyD88/NF-κB. Previous research uncovered that PCs could alleviate purpura-like lesions and pathological changes on rats likely through attenuating inflammation and OS damage. The mechanism of PCs on HSP deserves further investigation. Objective: To clarify the potential mechanism of PCs to HUVECs induced by the serum of HSP patients. Methods: HUVECs were randomly divided into blank, control, model, and low-, medium-, and high-concentration PCs group. Then, 25% HSP serum was assigned to the latter four groups, while 25% serum from healthy subjects to control group and serum-free culture medium to blank one. The last three groups separately received different concentrations of PCs. In addition, TAK-242, a TLR4 inhibitor, was applied to investigate the effect of TLR4-related signals in PCs against HSP serum-induced damage. Finally, inflammatory and OS-related parameters were detected by using cytological/molecular-biological techniques. Results: Treated with HSP serum later, the levels of immuno-inflammatory and oxidative indicators obviously went up (P < 0.05), and those of antioxidants remarkably went down (P < 0.05). PCs, however, reversed above phenomena (P < 0.05). Moreover, TLR4, MyD88 and NF-κB proteins/genes highly expressed in the model group; but significantly fell off in the presence of PCs (P < 0.05). Amazingly, all of above indicators showed no significant difference among the groups of different PCs concentrations (P > 0.05). These alterations likewise occurred after TAK-242 pretreatment with or without PCs, ie a notable drop of TLR4, MyD88 and NF-κB appeared in TAK-242 presence, few differences existing when compared to the PCs groups. Conclusion: PCs effectively protect HUVECs from inflammatory and OS damage provoked by HSP serum via blocking TLR4/MyD88/NF-κB signals.

Guhan Yangsheng Jing mitigates hippocampal neuronal pyroptotic injury and manifies learning and memory capabilities in sleep deprived mice via the NLRP3/Caspase1/GSDMD signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Guhan Yangsheng Jing (GHYSJ) is a traditional Chinese patent medicine, that has the function of nourishing the kidney and replenishing the essence, invigorating the brain and calming the mind. It is often used to treat dizziness, memory loss, sleep disorders, fatigue, and weakness, etc. However, its mechanism for improving sleep has not yet been determined. AIM OF THE STUDY: This study aims to explore the effects of GHYSJ on Sleep Deprivation (SD)-induced hippocampal neuronal pyroptotic injury, learning and cognitive abilities, and sleep quality in mice. METHODS: In this study, a PCPA-induced SD mouse model was established. We assessed the influence of GHYSJ on sleep quality and mood by using the pentobarbital-induced sleep test (PIST) and sucrose preference test (SPT). The pharmacological effects of GHYSJ on learning and memory impairment were evaluated by the Morris Water Maze (MWM) and Open Field Test (OFT). Pathological changes in the hippocampal tissue of the SD rats were observed via HE staining and Nissl staining. The severity of neuronal damage was evaluated by detecting the expression of the neuronal marker Microtubule-associated protein 2 (MAP2), via immunohistochemistry and immunofluorescence. Furthermore, the levels of neurotransmitter 5-hydroxytryptophan (5-HTP), 5-hydroxy tryptamine (5-HT), γ-aminobutyric acid (GABA), and Glutamic acid (Glu) in hippocampal tissues, as well as the expression of inflammatory factors Interleukin-1ß (IL-1ß) and Interleukin-18 (IL-18) in serum, were determined by ELISA. The expressions of mRNA and protein NOD-like receptor thermal protein domain associated protein 3 (NLRP3), Gasdermin D (GSDMD), Cysteinyl aspartate specific proteinase1 (Caspase1), High mobility group box-1 protein (HMGB1) and Apoptosis-associated speck-like protein containing CARD (ASC) related to the cellular ferroptosis pathway were tested and analyzed by RT-PCR and WB respectively. RESULTS: PCPA significantly diminishes the sleep span of experimental animals by expediting the expenditure of 5-HT, consequently establishing an essentially direct SD model. The intervention of GHYSJ displays remarkable efficacy in mitigating insomnia symptoms, encompassing difficulties in initiating sleep and insufficient sleep duration. Likewise, it ameliorates memory function impairments induced by sleep deprivation, along with symptoms such as fatigue and depletion of vitality. GHYSJ exerts a protective influence on hippocampal neurons facilitated by inhibiting the down regulation of MAP2 and maintaining the equilibrium of neurotransmitters (5-HTP, 5-HT, GABA, and Glu). It diminishes the expression of intracellular pyroptosis-associated inflammatory factors (IL-1ß and IL-18) and curbs the activation of the NLRP3/Caspase1/GSDMD pyroptosis-related signaling pathways, thereby alleviating the damage caused by hippocampal neuronal pyroptosis.

Long-Term Chondrocyte Retention in Partially Decellularized Tracheal Grafts.

OBJECTIVE: Decellularized tracheal grafts possess the biological cues necessary for tissue regeneration. However, conventional decellularization approaches to target the removal of all cell populations including chondrocytes lead to a loss of mechanical support. We have created a partially decellularized tracheal graft (PDTG) that preserves donor chondrocytes and the mechanical properties of the trachea. In this study, we measured PDTG chondrocyte retention with a murine microsurgical model. STUDY DESIGN: Murine in vivo time-point study. SETTING: Research Institute affiliated with Tertiary Pediatric Hospital. METHODS: PDTG was created using a sodium dodecyl sulfate protocol. Partially decellularized and syngeneic grafts were orthotopically implanted into female C57BL/6J mice. Grafts were recovered at 1, 3, and 6 months postimplant. Pre- and postimplant grafts were processed and analyzed via quantitative immunofluorescence. Chondrocytes (SOX9+, DAPI+) present in the host and graft cartilage was evaluated using ImageJ. RESULTS: Partial decellularization resulted in the maintenance of gross tracheal architecture with the removal of epithelial and submucosal structures on histology. All grafts demonstrated SOX9+ chondrocytes throughout the study time points. Chondrocytes in PDTG were lower at 6 months compared to preimplant and syngeneic controls. CONCLUSION: PDTG retained donor graft chondrocytes at all time points. However, PDTG exhibits a reduction in chondrocytes at 6 months. The impact of these histologic changes on cartilage extracellular matrix regeneration and repair remains unclear.

Assessing the Biocompatibility and Regeneration of Electrospun-Nanofiber Composite Tracheal Grafts.

OBJECTIVE: Composite tracheal grafts (CTG) combining decellularized scaffolds with external biomaterial support have been shown to support host-derived neotissue formation. In this study, we examine the biocompatibility, graft epithelialization, vascularization, and patency of three prototype CTG using a mouse microsurgical model. STUDY DESIGN: Tracheal replacement, regenerative medicine, biocompatible airway splints, animal model. METHOD: CTG electrospun splints made by combining partially decellularized tracheal grafts (PDTG) with polyglycolic acid (PGA), poly(lactide-co-ε-caprolactone) (PLCL), or PLCL/PGA were orthotopically implanted in mice (N = 10/group). Tracheas were explanted two weeks post-implantation. Micro-Computed Tomography was conducted to assess for graft patency, and histological analysis was used to assess for epithelialization and neovascularization. RESULT: Most animals (greater than 80%) survived until the planned endpoint and did not exhibit respiratory symptoms. MicroCT confirmed the preservation of graft patency. Grossly, the PDTG component of CTG remained intact. Examining the electrospun component of CTG, PGA degraded significantly, while PLCL+PDTG and PLCL/PGA + PDTG maintained their structure. Microvasculature was observed across the surface of CTG and infiltrating the pores. There were no signs of excessive cellular infiltration or encapsulation. Graft microvasculature and epithelium appear similar in all groups, suggesting that CTG did not hinder endothelialization and epithelialization. CONCLUSION: We found that all electrospun nanofiber CTGs are biocompatible and did not affect graft patency, endothelialization and epithelialization. Future directions will explore methods to accelerate graft regeneration of CTG. LEVEL OF EVIDENCE: N/A Laryngoscope, 134:1155-1162, 2024.

Methyl protodioscin reduces c-Myc to ameliorate diabetes mellitus erectile dysfunction via downregulation of AKAP12.

BACKGROUND: Diabetes mellitus erectile dysfunction (DMED) is one of common complications of diabetes. We aimed to investigate the potential efficacy of methyl protodioscin (MPD) in DMED and explored the underlying mechanism. METHODS: Diabetic mice were induced by streptozotocin, while vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs) were stimulated with high glucose. MPD was administrated in vitro and in vivo to verify its efficacy on DMED. The interaction of c-Myc and AKAP12 was determined by luciferase reporter assay and chromatin immunoprecipitation assay. RESULTS: c-Myc and AKAP12 were upregulated in penile tissues in DMED mice. In high glucose-stimulated VSMCs or VECs, MPD intervention enhanced cell viability, inhibited apoptosis, decreased c-Myc and AKAP12, as well as elevated p-eNOS Ser1177. MPD-induced apoptosis inhibition, AKAP12 reduction and p-eNOSSer1177 elevation were reversed by AKAP12 overexpression. c-Myc functioned as a positive regulator of AKAP12. Overexpression of c-Myc reversed the effects induced by MPD in vitro, which was neutralized by AKAP12 silencing. MPD ameliorated erectile function in diabetic mice via inhibiting AKAP12. CONCLUSIONS: MPD improved erectile dysfunction in streptozotocin-caused diabetic mice by regulating c-Myc/AKAP12 pathway, indicating that MPD could be developed as a promising natural agent for the treatment of DMED.

Partial decellularization eliminates immunogenicity in tracheal allografts.

There is currently no suitable autologous tissue to bridge large tracheal defects. As a result, no standard of care exists for long-segment tracheal reconstruction. Tissue engineering has the potential to create a scaffold from allografts or xenografts that can support neotissue regeneration identical to the native trachea. Recent advances in tissue engineering have led to the idea of partial decellularization that allows for the creation of tracheal scaffolds that supports tracheal epithelial formation while preserving mechanical properties. However, the ability of partial decellularization to eliminate graft immunogenicity remains unknown, and understanding the immunogenic properties of partially decellularized tracheal grafts (PDTG) is a critical step toward clinical translation. Here, we determined that tracheal allograft immunogenicity results in epithelial cell sloughing and replacement with dysplastic columnar epithelium and that partial decellularization creates grafts that are able to support an epithelium without histologic signs of rejection. Moreover, allograft implantation elicits CD8+ T-cell infiltration, a mediator of rejection, while PDTG did not. Hence, we establish that partial decellularization eliminates allograft immunogenicity while creating a scaffold for implantation that can support spatially appropriate airway regeneration.

A newly improved method of primary cell culture: Tissue block with continuous adhesion subculture in skin fibroblast.

BACKGROUND: Fibroblasts (FBs) have been widely used as a typical in vitro cell model for investigating the biological processes and cell pathophysiological mechanisms. However, FBs are prone to senescence in cell culture process after several passages. Thus, a new approach to cell culture is quite required to enhance the viability of cells. OBJECTIVE: To explore a novel method of cell culture based on skin FBs. METHODS: Dermal tissue blocks were obtained from BALB/c neonatal mice and randomly divided into experimental group and control group. The experimental group received the newly improved culture method, namely, continuous adherence subculture of tissue block (CASTB) method; while the traditional subculture method was applied in the control group. Cells at 1st, 5th and 10th passages were collected and identified by using histological/immunohistochemical and western blot analysis. Cellular viability, proliferation, senescence and apoptosis were analyzed through application of cell growth curve, CCK-8 assay, Ki67 assay, PCNA protein analysis, ß-galactosidase staining, flow cytometry and western blot analysis. RESULTS: Cells under two culture patterns exhibited spindle/irregular shape and vimentin positive expression. With the increase of passage times, the cellular growth rate in the control group gradually decreased, but no alterations emerged from the experimental group. CASTB method remarkably promoted cell growth and proliferation. Moreover, a greatly lower apoptosis and senescence tendency appeared in the experimental group than the control group with passages increasing. CONCLUSION: The method of CASTB is superior to traditional subculture, offering a large number of primary FBs with higher efficiency and success rate and being worth of further popularization and application.

Guijiajiao (Colla Carapacis et Plastri, CCP) prevents male infertility via gut microbiota modulation.

Male infertility is a significant cause of psychosocial and marital distress in approximately 50% of couples who are unable to conceive, with male factors being the underlying cause. Guijiajiao (Colla Carapacis et Plastri, CCP) is a Traditional Chinese Medicine commonly used to treat male infertility. The present study aimed to investigate the potential mechanisms underlying the preventive effects of CCP on male infertility. An infertile male rat model was established using cyclophosphamide (CTX), and CCP was administered for both treatment and prevention. Fecal microbiota transplantation (FMT) was also performed to explore the role of gut microbiota in the CCP-mediated prevention of male infertility in rats. Sperm motility and concentration were determined using a semi-automatic sperm classification analyzer. Subsequently, histopathological analysis using HE staining was performed to examine the changes in the small intestine and testis. Moreover, the serum levels of lipopolysaccharide (LPS) and testosterone were measured by ELISA. In addition, immunohistochemistry was conducted to detect CD3 expression in the small intestine, while RT-qPCR was employed to assess the expressions of interleukin-1 beta (IL-1ß), cluster of differentiation 3 (CD3), Monocyte chemoattractant protein-1 (MCP-1), and C-X-C motif chemokine ligand 10 (CXCL-10) in the small intestine and epididymis. Finally, gut microbiota was analyzed by 16S rRNA sequencing. CCP improved sperm motility, number, and concentration in CTX-induced infertile male rats. CCP increased the serum testosterone level, inhibited the immune cell infiltration of the intestinal lamina propria, and promoted the aggregation of CD3+ T cells in CTX-induced male infertility rats. CCP also inhibited the expressions of MCP-1, CXCL-10, and IL-1ß in the epididymis of male infertility rats. At the genus level, CTX led to a reduction in the abundance of Lactobacillus, Clostridia_UCG.014, and Romboutsia in the intestinal tract of rats. In contrast, CCP decreased the abundance of Ruminococcus and increased the abundance of Romboutsia in infertile male rats. Additionally, FMT experiments proved that the gut microbiota of CCP-treated rats facilitated testicular tissue recovery and spermatogenesis while also reducing the serum LPS level in infertile male rats. CCP improves the spermatogenic ability of infertile male rats by restoring gut microbiota diversity and inhibiting epididymal inflammation.

Regeneration of tracheal neotissue in partially decellularized scaffolds.

Extensive tracheal injury or disease can be life-threatening but there is currently no standard of care. Regenerative medicine offers a potential solution to long-segment tracheal defects through the creation of scaffolds that support the generation of healthy neotissue. We developed decellularized tracheal grafts (PDTG) by removing the cells of the epithelium and lamina propria while preserving donor cartilage. We previously demonstrated that PDTG support regeneration of host-derived neotissue. Here, we use a combination of microsurgical, immunofluorescent, and transcriptomic approaches to compare PDTG neotissue with the native airway and surgical controls. We report that PDTG neotissue is composed of native tracheal cell types and that the neoepithelium and microvasculature persisted for at least 6 months. Vascular perfusion of PDTG was established within 2 weeks and the graft recruited multipotential airway stem cells that exhibit normal proliferation and differentiation. Hence, PDTG neotissue recapitulates the structure and function of the host trachea and has the potential to regenerate.

Seven lower toxicity celastrol derivatives by biotransformation of Pestalotiopsis sp. LGT-1.

Biotransformation of toxic components by plant endophytes has become an effective method to reduce the toxicity of target compounds and discover lead compounds. In this context, an endophytic fungus, Pestalotiopsis sp. LGT-1, from Tripterygium wilfordii Hook F. (TwHF), was used to reduce the toxicity of celastrol which is also produced by TwHF and is considered an attractive molecule with a variety of biological activities. Seven celastrol derivatives (1-7) were isolated from the coculture fermentation broth of LGT-1 and celastrol. Their structures were elucidated by spectroscopic data analysis including 1D and 2D NMR, as well as HRESIMS. Their absolute configurations were determined by analysis of NOESY, ECD data and NMR calculations. In cell proliferation experiments, the toxicity of seven compounds was 10.11- to 1.24-fold lower in normal cells than the prototype compound celastrol. These derivatives serve as potential candidates for future pharmaceutical applications.

Optimization of Chondrocyte Viability in Partially Decellularized Tracheal Grafts.

OBJECTIVE: Advancements in tissue-engineered tracheal replacement (TETR) show promise for the use of partially decellularized tracheal grafts (PDTG) to address critical gaps in airway management and reconstruction. In this study, aiming to leverage the immunoprivileged nature of cartilage to preserve tracheal biomechanics, we optimize PDTG for retention of native chondrocytes. STUDY DESIGN: Comparison in vivo murine study. SETTING: Research Institute affiliated with Tertiary Pediatric Hospital. METHODS: PDTG were created per a shortened decellularization protocol using sodium dodecyl sulfate, then biobanked via cryopreservation technique. Decellularization efficiency was characterized by DNA assay and histology. Viability and apoptosis of chondrocytes in preimplanted PDTG and biobanked native trachea (control) was assessed with live/dead and apoptosis assays. PDTG (N = 5) and native trachea (N = 6) were orthotopically implanted in syngeneic recipients for 1-month. At the endpoint, microcomputed tomography (micro-CT) was employed to interrogate graft patency and radiodensity in vivo. Vascularization and epithelialization were qualitatively analyzed using histology images following explant. RESULTS: PDTG exhibited complete decellularization of all extra-cartilaginous cells and reduced DNA content compared to control. Chondrocyte viability and nonapoptotic cell populations were improved utilizing biobanking and shorter decellularization time. All grafts remained patent. Evaluation of graft radiodensity at 1 month revealed elevation of Hounsfield units in both PDTG and native compared to host, with PDTG showing higher radiodensity than native. PDTG supported complete epithelialization and functional reendothelialization 1-month postimplantation. CONCLUSION: Optimizing PDTG chondrocyte viability is a key component to successful tracheal replacement. Ongoing research seeks to evaluate the acute and chronic immunogenicity of PDTG.

Successful Early Neovascularization in Composite Tracheal Grafts.

OBJECTIVE: Long-segment tracheal defects require tissue replacement for successful reconstruction. Rapid revascularization is imperative to maintain graft function. We previously showed that partially decellularized tracheal grafts (PDTG) and composite tracheal grafts (CTG; PDTG supported by a 3-dimensionally printed external splint) regenerate respiratory epithelium and may support the regeneration of endothelial cells (CD31+). However, the capability of graft endothelial cells to organize or contribute to tracheal revascularization remains unclear. In this study, we quantified endothelial cells (CD31+) and neovessel formation in PDTG and CTG. We hypothesize that PDTG and CTG support tracheal neovascularization to a similar extent as surgical (syngeneic tracheal graft [STG]) and native trachea (NT) controls. STUDY DESIGN: The animal study, a randomized control trial. SETTING: Center for Regenerative Medicine, Nationwide Children's Hospital. METHODS: PDTG was created via an established decellularization protocol. Segmental tracheal reconstruction was performed with STG, PDTG, or CTG using a mouse microsurgical model. NT was used as a nonsurgical control. At 1 month, mice were euthanized, grafts harvested, sectioned, and stained with CD31 and hematoxylin and eosin. Neovessel formation was quantified by the number of formed blood vessels in the lamina propria and vessel size (vessel/graft area, mm2 ). RESULTS: Decellularization eliminated all endothelial cells and there were no perfused vessels at implantation. At 1 month, PDTG and CTG supported neovessel formation with tubular vessels lined with endothelial cells. There was no difference in the number or size of vessels compared to controls. CONCLUSION: PDTG and CTG support tracheal endothelial cell regeneration and neovessel formation. Future directions to assess the function, kinetics, and distribution of graft neovessels are needed.

De novo Assembly and Comparative Analyses of Mitochondrial Genomes in Piperales.

The mitochondrial genome of Liriodendron tulipifera exhibits many ancestral angiosperm features and a remarkably slow evolutionary rate, while mitochondrial genomes of other magnoliids remain yet to be characterized. We assembled nine new mitochondrial genomes, representing all genera of perianth-bearing Piperales, as well as for a member of the sister clade: three complete or nearly complete mitochondrial genomes from Aristolochiaceae and six additional draft assemblies including Thottea, Asaraceae, Lactoridaceae, and Hydnoraceae. For comparative purpose, a complete mitochondrial genome was assembled for Saururus, a member of the perianth-less Piperales. The average number of short repeats (50-99 bp) was much larger in genus Aristolochia than in other angiosperm mitochondrial genomes, and approximately 30% of repeats (<350 bp) were found to have the capacity to mediate recombination. We found mitochondrial genomes in perianth-bearing Piperales comprising conserved repertories of protein-coding genes and rRNAs but variable copy numbers of tRNA genes. We identified several shifts from cis- to trans-splicing of the Group II introns of nad1i728, cox2i373, and nad7i209. Two short regions of the cox1 and atp8 genes were likely derived from independent horizontal gene transfer events in perianth-bearing Piperales. We found biased enrichment of specific substitution types in different lineages of magnoliids and the Aristolochiaceae family showed the highest ratio of A:T > T:A substitutions of all other investigated angiosperm groups. Our study reports the first mitochondrial genomes for Piperales and uses this new information for a better understanding of the evolutionary patterns of magnoliids and angiosperms in general.

Guilu-Erxian-Glue alleviates Tripterygium wilfordii polyglycoside-induced oligoasthenospermia in rats by resisting ferroptosis via the Keap1/Nrf2/GPX4 signaling pathway.

CONTEXT: Guilu-Erxian-Glue (GLEXG) is a traditional Chinese formula used to improve male reproductive dysfunction. OBJECTIVE: To investigate the ferroptosis resistance of GLEXG in the improvement of semen quality in the oligoasthenospermia (OAS) rat model. MATERIALS AND METHODS: Male Sprague-Dawley (SD) rats were administered Tripterygium wilfordii polyglycoside, a compound extracted from Tripterygium wilfordii Hook F. (Celastraceae), at a dose of 40 mg/kg/day, to establish an OAS model. Fifty-four SD rats were randomly divided into six groups: sham, model, low-dose GLEXG (GLEXGL, 0.25 g/kg/day), moderate-dose GLEXG (GLEXGM, 0.50 g/kg/day), high-dose GLEXG (GLEXGH, 1.00 g/kg/day) and vitamin E (0.01 g/kg/day) group. The semen quality, structure and function of sperm mitochondria, histopathology, levels of oxidative stress and iron, and mRNA levels and protein expression in the Keap1/Nrf2/GPX4 pathway, were analyzed. RESULTS: Compared with the model group, GLEXGH significantly improved sperm concentration (35.73 ± 15.42 vs. 17.40 ± 4.12, p < 0.05) and motility (58.59 ± 11.06 vs. 28.59 ± 9.42, p < 0.001), and mitigated testicular histopathology. Moreover, GLEXGH markedly reduced the ROS level (5684.28 ± 1345.47 vs. 15500.44 ± 2307.39, p < 0.001) and increased the GPX4 level (48.53 ± 10.78 vs. 23.14 ± 11.04, p < 0.01), decreased the ferrous iron level (36.31 ± 3.66 vs. 48.64 ± 7.74, p < 0.05), and rescued sperm mitochondrial morphology and potential via activating the Keap1/Nrf2/GPX4 pathway. DISCUSSION AND CONCLUSIONS: Ferroptosis resistance from GLEXG might be driven by activation of the Keap1/Nrf2/GPX4 pathway. Targeting ferroptosis is a novel approach for OAS therapy.

A review focusing on the benefits of plant-derived polysaccharides for osteoarthritis.

Osteoarthritis (OA) is a chronic joint disease characterized by progressive cartilage degeneration, which imposes a heavy physical and financial burden on the middle-aged and elderly population. As the pathogenesis of OA has not been fully elucidated, it is of great importance to develop targeted therapeutic or preventive medications. Traditional therapeutic drugs, such as non-steroidal anti-inflammatory drugs, steroids and opioids, have significant side effects, making the exploration for safe and effective alternative therapeutic drugs urgent. In recent years, many studies have reported the role of plant-derived polysaccharides in anti-inflammation, anti-oxidation, regulation of chondrocyte metabolism and proliferation, and cartilage protection, and have demonstrated their great potential in the treatment of OA. Therefore, by focusing on studies related to the intervention of plant-derived polysaccharides in OA, including in vivo and in vitro experiments, this review aimed to classify and summarize the existing research findings according to different mechanisms of action. In addition, reports on plant-derived polysaccharides as nanoparticles were also explored. Then, candidate monomers and theoretical bases were provided for the further development and application of novel drugs in the treatment of OA.

A Multimodal Approach to Quantify Chondrocyte Viability for Airway Tissue Engineering.

OBJECTIVES/HYPOTHESIS: Partially decellularized tracheal scaffolds have emerged as a potential solution for long-segment tracheal defects. These grafts have exhibited regenerative capacity and the preservation of native mechanical properties resulting from the elimination of all highly immunogenic cell types while sparing weakly immunogenic cartilage. With partial decellularization, new considerations must be made about the viability of preserved chondrocytes. In this study, we propose a multimodal approach for quantifying chondrocyte viability for airway tissue engineering. METHODS: Tracheal segments (5 mm) were harvested from C57BL/6 mice, and immediately stored in phosphate-buffered saline at -20°C (PBS-20) or biobanked via cryopreservation. Stored and control (fresh) tracheal grafts were implanted as syngeneic tracheal grafts (STG) for 3 months. STG was scanned with micro-computed tomography (µCT) in vivo. STG subjected to different conditions (fresh, PBS-20, or biobanked) were characterized with live/dead assay, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and von Kossa staining. RESULTS: Live/dead assay detected higher chondrocyte viability in biobanked conditions compared to PBS-20. TUNEL staining indicated that storage conditions did not alter the proportion of apoptotic cells. Biobanking exhibited a lower calcification area than PBS-20 in 3-month post-implanted grafts. Higher radiographic density (Hounsfield units) measured by µCT correlated with more calcification within the tracheal cartilage. CONCLUSIONS: We propose a strategy to assess chondrocyte viability that integrates with vivo imaging and histologic techniques, leveraging their respective strengths and weaknesses. These techniques will support the rational design of partially decellularized tracheal scaffolds. LEVEL OF EVIDENCE: N/A Laryngoscope, 133:512-520, 2023.

[Effect of Polygonati Rhizoma in improving pyroptosis injury of diabetic macroangiopathy via NLRP3/caspase-1/GSDMD pathway].

This study aims to explore the influence of Polygonati Rhizoma on the pyroptosis in the rat model of diabetic macroangiopathy via the NOD-like receptor thermal protein domain associated protein 3(NLRP3)/cysteinyl aspartate specific proteinase-1(caspase-1)/gasdermin D(GSDMD) pathway. The rat model of diabetes was established by intraperitoneal injection of streptozotocin(STZ) combined with a high-fat, high-sugar diet. The blood glucose meter, fully automated biochemical analyzer, hematoxylin-eosin(HE) staining, enzyme-linked immunosorbent assay, immunofluorescence, immunohistochemistry, and Western blot were employed to measure blood glucose levels, lipid levels, vascular thickness, inflammatory cytokine levels, and expression levels of pyroptosis-related proteins. The mechanism of pharmacological interventions against the injury in the context of diabetes was thus explored. The results demonstrated the successful establishment of the model of diabetes. Compared with the control group, the model group showed elevated levels of fasting blood glucose, total cholesterol(TC), triglycerides(TG) and low-density lipoprotein cholesterol(LDL-c), lowered level of high-density lipoprotein cholesterol(HDL-c), thickened vascular intima, and elevated serum and aorta levels of tumor necrosis factor-α(TNF-α), interleukin-1ß(IL-1ß) and interleukin-18(IL-18). Moreover, the model group showed increased NLRP3 inflammasomes and up-regulated levels of caspase-1 and GSDMD in aortic vascular cells. Polygonati Rhizoma intervention reduced blood glucose and lipid levels, inhibited vascular thickening, lowered the levels of TNF-α, IL-1ß, IL-18 in the serum and aorta, attenuated NLRP3 inflammasome expression, and down-regulated the expression levels of caspase-1 and GSDMD, compared with the model group. In summary, Polygonati Rhizoma can slow down the progression of diabetic macroangiopathy by inhibiting pyroptosis and alleviating local vascular inflammation.

Chemical Structures and Pharmacological Properties of Typical Bioflavonoids in Polygonati Rhizoma (PGR).

Most medicines are coming with toxic and detrimental side effects. In addition, microbials are resisting the medicine. Therefore, alternative drugs with low toxic and side effects and low microbial resistance are needed. Plants offer good potential candidates due to a broad range of chemicals they contain. These chemicals have been studied, and research is still going on to probe chemical properties of plant chemicals. In China, traditional Chinese medicine is practised, whereby plant extracts are obtained, and then sold in packages for reasons like memory enhancement, cancer treatment, boosting immune system, and so on. Among the herbs cultivated in China is Polygonati rhizoma (PGR). This plant contains various bioflavonoids such as diosgenin, kaempferol, catechin, daidzein, and 3'-methoxydaidzein. In this review, we discussed the pharmacological effects of these chemicals, including luteolin antimicrobial activity in a manner that it circumvents antibiotic resistance; rutin antivenom property; kaempferol as an agent that mitigates neuropathic pain; genistein anticancer property; isorhamnetin's ability to alleviate chronic obstructive pulmonary diseases (COPD); proanthocyanidins' ability to deal with diabetic neuropathy and analgesic property of catechin.