Holothurian Wall Hydrolysate Ameliorates Cyclophosphamide-Induced Immunocompromised Mice via Regulating Immune Response and Improving Gut Microbiota

Some biologically active compounds isolated from sea cucumbers stimulate the body’s immune response by activating immune cells. Immune function is closely related to the integrity intestinal barrier and balanced gut microbiota. However, it is unknown whether the daily administration of holothurian wall hydrolysate (HWH) ameliorated intestinal dysbiosis and barrier injury induced by immunodeficiency. This study aimed to investigate the immunomodulatory effect and the underlying mechanism of HWH in cyclophosphamide (CTX)-induced immunocompromised mice. BALB/c mice received CTX (80 mg/kg, intraperitoneally) once a day for 3 days to induce immunodeficiency, and then they received the oral administration of HWH (80 or 240 mg/kg) or levamisole hydrochloride (LH, 40 mg/kg, positive control), respectively, once a day for 7 days. We utilized 16S rRNA sequencing for microbial composition alterations, histopathological analysis for splenic and colonic morphology, Western blotting for expressions of tight junction proteins (TJs), and quantitative real-time (qRT)-PCR for measurements of pro-inflammatory cytokines. HWH attenuated the immune organ damage induced by CTX, increased the secretions of interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α, and promoted the recovery of goblet cells and the production of TJs (claudin-1, occludin, and ZO-1) in the colon of the immunocompromised mice. Moreover, HWH promoted the growth of beneficial microorganisms such as Lactobacillus, Lachnospiraceae, Christensenellaceae, and Bifidobacterium, while it suppressed the populations of Ruminococcus, Staphylococcus, and Streptococcus. These results demonstrate that HWH elicits intestinal mucosal immunity, repairs the damage to intestinal mucosal integrity, and normalizes the imbalanced intestinal microbial profiles in immunocompromised mice. It may be helpful to identify the biological activities of HWH to support its potential use in new prebiotics, immunomodulatory agents, and medical additives for intestinal repair

Improvement of cardiomyocyte function by in\ua0vivo hexarelin treatment in streptozotocin-induced diabetic rats

Diabetic cardiomyopathy is characterized by diastolic and systolic cardiac dysfunction, yet no therapeutic drug to specifically treat it. Hexarelin has been demonstrated to improve heart function in various types of cardiomyopathy via its receptor GHS-R. This experiment aims to test the effect of hexarelin on cardiomyocytes under experimental diabetes. Streptozotocin (STZ, 65\ua0mg/kg)-induced diabetic rat model was employed with vehicle injection group as control. Daily hexarelin (100\ua0μg/kg) treatment was performed for 2\ua0weeks after 4-week STZ-induced diabetes. Cardiomyocytes were isolated by enzyme treatment under O-saturated perfusion for single-cell shortening, [Ca] transient, and electrophysiology recordings. GHS-R expression and apoptosis-related signaling proteins Bax, Bcl-2, caspase-3 and 9, were assessed by western blot. Experimental data demonstrated a reduced cell contraction and relaxation in parallel with depressed rise and fall of [Ca] transients in diabetic cardiomyocytes. Hexarelin reversed the changes in both contraction and [Ca]. Action potential duration and transient outward potassium current (I) density were dramatically increased in diabetic cardiomyocytes and hexarelin treatment reverse such changes. Upregulated GHS receptor (GHS-R) expression was observed in both control and diabetic groups after hexarelin treatment, which also caused antiapoptotic changes of Bax, Bcl-2, caspase-3 and 9 expression. In STZ-induced diabetic rats, hexarelin is able to improve cardiomyocyte function through recovery of I K currents, intracellular Ca homeostasis and antiapoptotic signaling pathways

Glomerular endothelial cell injury and damage precedes that of podocytes in adriamycin-induced nephropathy

The role of podocytes in the development and progression of glomerular disease has been extensively investigated in the past decade. However, the importance of glomerular endothelial cells in the pathogenesis of proteinuria and glomerulosclerosis has been largely ignored. Recent studies have demonstrated that endothelial nitric oxide synthatase (eNOS) deficiency exacerbates renal injury in anti-GBM and remnant kidney models and accelerates diabetic kidney damage. Increasing evidence also demonstrates the importance of the glomerular endothelium in preventing proteinuria. We hypothesize that endothelial dysfunction can initiate and promote the development and progression of glomerulopathy. Administration of adriamycin (ADR) to C57BL/6 mice, normally an ADR resistant strain, with an eNOS deficiency induced overt proteinuria, severe glomerulosclerosis, interstitial fibrosis and inflammation. We also examined glomerular endothelial cell and podocyte injury in ADR-induced nephropathy in Balb/c mice, an ADR susceptible strain, by immunostaining, TUNEL and Western blotting. Interestingly, down-regulation of eNOS and the appearance of apoptotic glomerular endothelial cells occurred as early as 24 hours after ADR injection, whilst synaptopodin, a functional podocyte marker, was reduced 7 days after ADR injection and coincided with a significant increase in the number of apoptotic podocytes. Furthermore, conditioned media from mouse microvascular endothelial cells over-expressing GFP-eNOS protected podocytes from TNF-α-induced loss of synaptopodin. In conclusion, our study demonstrated that endothelial dysfunction and damage precedes podocyte injury in ADR-induced nephropathy. Glomerular endothelial cells may protect podocytes from inflammatory insult. Understanding the role of glomerular endothelial dysfunction in the development of kidney disease will facilitate in the design of novel strategies to treat kidney disease

Endothelial dysfunction exacerbates renal interstitial fibrosis through enhancing fibroblast Smad3 linker phosphorylation in the mouse obstructed kidney.

Endothelial dysfunction and enhanced transforming growth factor-β (TGF-β)/Smad3 signalling are common features of progressive renal fibrosis. This study investigated a potential link between these mechanisms. In unilateral ureteric obstruction (UUO) we observed an acute (6 hr) down-regulation of nitric oxide synthase 3 (NOS3/eNOS) levels and increased phosphorylation of the linker region of Smad3 at T179 and S208 in Smad3/JNK complexes. These events preceded Smad3 C-terminal domain phosphorylation and the induction of myofibroblast proliferation at 48 hrs. Mice deficient in NOS3 showed enhanced myofibroblast proliferation and collagen accumulation compared to wild type mice in a 7 day UUO model. This was associated with enhanced phosphorylation of Smad3 T179 and S208 by 92% and 88%, respectively, whereas Smad3-C-terminal phosphorylation was not affected. Resolvin D1 (RvD1) can suppress renal fibrosis in the UUO model, and further analysis herein showed that RvD1 protected against endothelial dysfunction and suppressed Smad3/JNK complex formation with a consequent reduction in phosphorylation of Smad3 T179 and S208 by 78% and 65%, respectively, while Smad3 C-terminal phosphorylation was unaltered. In vitro, conditioned media from mouse microvascular endothelial cells (MMEC) treated with a general inhibitor of nitric oxide synthase (L-NAME) augmented the proliferation and collagen production of renal fibroblasts (NRK49F cells) compared to control MMEC media and this was associated with increased phosphorylation of JNK and Smad3 T179 and S208, whereas Smad3-C-terminal domain phosphorylation was unaffected. The addition of RvD1 to L-NAME treated MMEC abrogated these effects of the conditioned media on renal fibroblasts. Finally, Smad3 T179/V and S208/A mutations significantly inhibit TGF-β1 induced up-regulation collagen I promoter. In conclusion, these data suggest that endothelial dysfunction can exacerbate renal interstitial fibrosis through increased fibroblast proliferation and collagen production via enhanced Smad3 linker phosphorylation

Scanning Probe Microscopy Bone Marrow Determination of Steogenic Differentiation of Mesenchymal Stem Cells

To address the question of determining the osteogenic differentiation of mesenchymal stem cells, the bone marrow studies were performed using probe microscopy. All adherent bone marrow was used to isolate the bone marrow mesenchymal stem cells and expanded and purified in vitro. Its morphology under an inverted microscope was observed. We used Zuogui Pills to differentiate the separation methods. Alcian blue staining, modified calcium cobalt alkaline phosphatase staining, and neuron-specific enolase immunohistochemical staining were performed. The experimental results are shown below. The morphology of the isolated and purified cells was analyzed with an inverted microscope, and the isolated and purified cells were analyzed with Zuogui Pill. Alcian blue staining, modified calcium cobalt alkaline phosphatase staining, and neuron-specific enolase immunohistochemical staining confirmed that the cells differentiated into cartilage and osteoblasts, and the cell structure and morphology were similar to those of the bone marrow mesenchymal stem cells. The results showed that the adherent mode of cells obtained from the whole bone marrow was the rat bone marrow mesenchymal stem cells, and the Zuogui Pills could induce multidirectional differences in the bone marrow mesenchymal stem cells

Compensatory growth and recovery of cartilage cytoarchitecture after transient cell death in fetal mouse limbs.

Acknowledgements: We thank Jonathan Gleadle, Vincent Wong and Darling Rojas-Canales for inspiring discussions about mTORC1 and the balance between proliferation and cell size in compensatory responses. We also acknowledge the Monash Bioinformatics Platform (especially Kirill Tsyganov), and Trevor Wilson, at the Medical Genomics Facility, Monash Health Translation Precinct, for their excellent technical help. This study is funded by HFSP CDA00021/2019-C (to A.R-D.) and NHMRC Ideas grant 2002084 (to C.H.H. and A.R-D.). The Australian Regenerative Medicine Institute is supported by grants from the State Government of Victoria and the Australian Government.A major question in developmental and regenerative biology is how organ size and architecture are controlled by progenitor cells. While limb bones exhibit catch-up growth (recovery of a normal growth trajectory after transient developmental perturbation), it is unclear how this emerges from the behaviour of chondroprogenitors, the cells sustaining the cartilage anlagen that are progressively replaced by bone. Here we show that transient sparse cell death in the mouse fetal cartilage is repaired postnatally, via a two-step process. During injury, progression of chondroprogenitors towards more differentiated states is delayed, leading to altered cartilage cytoarchitecture and impaired bone growth. Then, once cell death is over, chondroprogenitor differentiation is accelerated and cartilage structure recovered, including partial rescue of bone growth. At the molecular level, ectopic activation of mTORC1 correlates with, and is necessary for, part of the recovery, revealing a specific candidate to be explored during normal growth and in future therapies