Transforming Growth Factor-β Research Articles

The Protective Effects of Acteoside and Total Glycosides of the Leaves of Rehmannia on Three Types of Glomerular Structural Cells Under High Glucose and Inflammatory Stimulation

ABSTRACT Background: The challenge of chronic glomerulonephritis necessitates innovative strategies for preventing renal function deterioration. Acteoside (ACT), the primary bioactive compound of total glycosides of the leaves of Rehmannia (DHYZG), has been demonstrated availability and safety in reducing proteinuria, showing promising prospects in the treatment of kidney disease. This study aimed to elucidate the impact of ACT and DHYZG on glomerular structural cells and key proteins of rat glomeruli under several injury states. Methods: Employing 3-(4,5)-dimethylthiazolyl(-Z-Y1)-3,5-diphenyltetrazolium bromide (MTT) and Western blot methodologies, the investigation assessed varying concentrations of ACT and clinical concentration of DHYZG on cell viability and intracellular biomarkers. Specifically, the study explored the influence of ACT on connective tissue growth factor (CTGF), transforming growth factor-β (TGF-β), matrix metalloproteinase-2 (MMP-2), and MPP in rat mesangial cells, endothelial cells, and podocytes stimulated by lipopolysaccharide (LPS), high glucose, and interleukin-1β (IL-1β). Additionally, the impact of high glucose on angiotensin II (Ang II) expression in endothelial cells was investigated. Results: ACT and DHYZG demonstrated a protective effect on all cell types, and ACT administration shows a significant dose-dependent response. These compounds attenuated the expression of CTGF and TGF-β in LPS-stimulated mesangial cells and reduced the expression of MMP-2 and MMP-9 in all three cell types following IL-1β stimulation. Moreover, the high glucose-induced expression of Ang II in endothelial cells was mitigated. Conclusion: ACT and DHYZG exhibit a pronounced protective effect on glomerular cells, with ACT being the primary contributor to the therapeutic efficacy of DHYZG. Notably, ACT demonstrates a significant superiority over DHYZG in terms of both proliferation and cytokine expression in glomerular endothelial cells.

Targeted delivery of factors and cells for improving cardiac tissue regeneration and heart function following myocardial infarction

Following myocardial infarction (MI), cardiac tissue undergoes irreversible cellular alterations, with cardiomyocytes being replaced by fibrotic tissue. In order to improve tissue regeneration, a previously characterized chitosan-based cryogel, which was designed by our group, was used. The treatment regimen involved the sequential delivery of the cryogel loaded with specific cytokines and growth factors, followed by a separate injection of pre-differentiated cells. Initially, the cryogel loaded with interleukin-10 and transforming growth factor-β was injected into infarcted tissue immediately after MI induction, targeting the acute inflammatory response. On day four post-MI, a second injection was administered, this time utilizing cryogel loaded with vascular endothelial growth factor and fibroblast growth factor-2, aimed at promoting tissue regeneration and angiogenesis. Subsequently, on day six post-MI, the experimental group received cardiomyocyte-like cells, smooth muscle cells, and endothelial cells. The purpose of these cells, in synergy with cytokines and growth factors, was to repopulate the lost cellular populations, thereby enhancing myocardial repair. The treatment improved myocardial tissue regeneration, increased cardiac output, ejection fraction, and reduced fibrotic regions. Thus, the chitosan-based cryogel, enriched with anti-inflammatory and proangiogenic factors and supplemented with pre-differentiated cells, offers a promising platform for controlled release of therapeutics, promoting substantial tissue repair and regeneration following MI.

PRG4 represses the genesis and metastasis of osteosarcoma by inhibiting PDL1 expression

BackgroundOsteosarcoma is originated from skeletal system. Recombinant human proteoglycan 4 (rhPRG4) can inhibit cell proliferation and migration in multiple cancers. This research is designed to dig out the role and mechanism of PRG4 in osteosarcoma. MethodsHuman osteosarcoma cell lines, MG63 and 143B, were transfected with programmed death 1 (PD-L1) overexpression vectors and/or treated with 20, 50, and 100μg/mL rhPRG4, followed by the determination of cell viability, colony formation, sphere formation, invasion, migration, apoptosis, and the expressions of matrix metalloproteinases (MMPs), PD-L1 and apoptosis-related proteins. Tumor-bearing mouse models were constructed by injection of 143B cells and treatment of anti-PD-L1 antibody and/or adenovirus PRG4 (AdPRG4). Tumor volume was monitored, and immunohistochemical location of Ki67 was performed. Expressions of MMPs, transforming growth factor-β (TGF-β), PD-L1, and epithelial mesenchymal transition (EMT)-related proteins were measured in tumors. ResultsRhPRG4 (20, 50, and 100μg/mL) inhibited the viability, colony formation, sphere formation, invasion, migration, and the expressions of MMP2, MMP9 and Bcl2 in osteosarcoma cells, while promoting cell apoptosis as well as Bax and c-caspase3 expressions, at a dose-dependent manner; by contrast, PD-L1 overexpression reversed the above effects of 100μg/mL rhPRG4. AdPRG4 or anti-PD-L1 antibody decreased tumor volume, number of pulmonary metastasis nodule, Ki67 location, and expressions of TGF-β, PD-L1, MMP2, MMP9, Vimentin, and Snail, but increased E-cadherin expression in tumor cells. Moreover, anti-PD-L1 antibody and AdPRG4 together functioned more effectively than them alone in reducing tumor burden. ConclusionPRG4 represses the genesis and metastasis of osteosarcoma via inhibiting PD-L1 expression, and AdPRG4 enhances the effectiveness of anti-PD-L1 therapy.

Exploring the anti-inflammatory and anti-fibrotic activity of NFκB decoy oligodeoxynucleotide-loaded spermine-functionalized acetalated nanoparticles

Chronic inflammation, oxidative stress, and airway remodelling represent the principal pathophysiological features of chronic respiratory disorders. Inflammation stimuli like lipopolysaccharide (LPS) activate macrophages and dendritic cells, with concomitant M1 polarization and release of pro-inflammatory cytokines. Chronic inflammation and oxidative stress lead to airway remodelling causing irreversible functional and structural alterations of the lungs. Airway remodelling is multifactorial, however, the hormone transforming growth factor-β (TGF-β) is one of the main contributors to fibrotic changes. The signalling pathways mediating inflammation and remodelling rely both on the transcription factor nuclear factor-κB (NFκB), underlying the potential of NFκB inhibition as a therapeutic strategy for chronic respiratory disorders. In this study, we encapsulated an NFκB-inhibiting decoy oligodeoxynucleotide (ODN) in spermine-functionalized acetalated dextran (SpAcDex) nanoparticles and tested the in vitro anti-inflammatory and anti-remodelling activity of this formulation. We show that NF-κB ODN nanoparticles counteract inflammation by reversing LPS-induced expression of the activation marker CD40 in myeloid cells and counteracts remodelling features by reversing the TGF-β-induced expression of collagen I and α-smooth muscle actin in human dermal fibroblast. In summary, our study highlights the great potential of inhibiting NFκB via decoy ODN as a therapeutic strategy tackling multiple pathophysiological features underlying chronic respiratory conditions.

The antiprotozoal drug nitazoxanide improves experimental liver fibrosis in mice

Nitazoxanide is an FDA-approved antiprotozoal drug. Our previous studies find that nitazoxanide and its metabolite tizoxanide affect AMPK, STAT3, and Smad2/3 signals which are involved in the pathogenesis of liver fibrosis, therefore, in the present study, we examined the effect of nitazoxanide on experimental liver fibrosis and elucidated the potential mechanisms. The in vivo experiment results showed that oral nitazoxanide (75, 100 mg·kg−1) significantly improved CCl4- and bile duct ligation-induced liver fibrosis in mice. Oral nitazoxanide activated the inhibited AMPK and inhibited the activated STAT3 in liver tissues from liver fibrosis mice. The in vitro experiment results showed that nitazoxanide and its metabolite tizoxanide activated AMPK and inhibited STAT3 signals in LX-2 cells (human hepatic stellate cells). Nitazoxanide and tizoxanide inhibited cell proliferation and collagen I expression and secretion of LX-2 cells. Nitazoxanide and tizoxanide inhibited transforming growth factor-β1 (TGF-β1)- and IL-6-induced increases of cell proliferation, collagen I expression and secretion, inhibited TGF-β1- and IL-6-induced STAT3 and Smad2/3 activation in LX-2 cells. In mouse primary hepatic stellate cells, nitazoxanide and tizoxanide also activated AMPK, inhibited STAT3 and Smad2/3 activation, inhibited cell proliferation, collagen I expression and secretion. In conclusion, nitazoxanide inhibits liver fibrosis and the underlying mechanisms involve AMPK activation, and STAT3 and Smad2/3 inhibition.

The protein phosphatase PP6 promotes RIPK1-dependent PANoptosis

BackgroundThe innate immune system serves as the first line of host defense. Transforming growth factor-β–activated kinase 1 (TAK1) is a key regulator of innate immunity, cell survival, and cellular homeostasis. Because of its importance in immunity, several pathogens have evolved to carry TAK1 inhibitors. In response, hosts have evolved to sense TAK1 inhibition and induce robust lytic cell death, PANoptosis, mediated by the RIPK1-PANoptosome. PANoptosis is a unique innate immune inflammatory lytic cell death pathway initiated by an innate immune sensor and driven by caspases and RIPKs. While PANoptosis can be beneficial to clear pathogens, excess activation is linked to pathology. Therefore, understanding the molecular mechanisms regulating TAK1 inhibitor (TAK1i)-induced PANoptosis is central to our understanding of RIPK1 in health and disease.ResultsIn this study, by analyzing results from a cell death-based CRISPR screen, we identified protein phosphatase 6 (PP6) holoenzyme components as regulators of TAK1i-induced PANoptosis. Loss of the PP6 enzymatic component, PPP6C, significantly reduced TAK1i-induced PANoptosis. Additionally, the PP6 regulatory subunits PPP6R1, PPP6R2, and PPP6R3 had redundant roles in regulating TAK1i-induced PANoptosis, and their combined depletion was required to block TAK1i-induced cell death. Mechanistically, PPP6C and its regulatory subunits promoted the pro-death S166 auto-phosphorylation of RIPK1 and led to a reduction in the pro-survival S321 phosphorylation.ConclusionsOverall, our findings demonstrate a key requirement for the phosphatase PP6 complex in the activation of TAK1i-induced, RIPK1-dependent PANoptosis, suggesting this complex could be therapeutically targeted in inflammatory conditions.

Transforming growth factor-β1 protects mechanically injured cortical murine neurons by reducing trauma-induced autophagy and apoptosis

Transforming growth factor β1 (TGF-β1) has a neuroprotective function in traumatic brain injury (TBI) through its anti-inflammatory and immunomodulatory properties. However, the precise mechanisms underlying the neuroprotective actions of TGF-β1 on the cortex require further investigation. In this study, we were aimed to investigate the regulatory function of TGF-β1 on neuronal autophagy and apoptosis using an in vitro primary cortical neuron trauma-injury model. LDH activity was assayed to measure cell viability, and intracellular [Ca2+] was measured using Fluo-4-AM in an in vitro primary cortical neuron trauma-injury model. RNA-sequencing (RNAseq), immunofluorescent staining, transmission electron microscopy (TEM), western blot and CTSD activity detection were employed. We observed significant enrichment of DEGs related to autophagy, apoptosis, and the lysosome pathway in trauma-injured cortical neurons. TEM confirmed the presence of autophagosomes as well as autophagolysosomes. Western blot revealed upregulation of autophagy-related protein light chain 3 (LC3-II/LC3-I), sequestosome 1 (SQSTM1/p62), along with apoptosis-related protein cleaved-caspase 3 in trauma-injured primary cortical neurons. Furthermore, trauma-injured cortical neurons showed an upregulation of lysosomal marker protein (LAMP1) and lysosomal enzyme mature cathepsin D (mCTSD), but a decrease in the activity of CTSD enzyme. These results indicated that apoptosis was up-regulated in trauma- injured cortical neurons at 24 h, accompanied by lysosomal dysfunction and impaired autophagic flux. Notably, TGF-β1 significantly reversed these changes. Our results suggested that TGF-β1 exerted neuroprotective effects on trauma- injured cortical neurons by reducing lysosomal dysfunction, decreasing the accumulation of autophagosomes and autophagolysosomes, and enhancing autophagic flux.

M6A reader YTHDF1 promotes cardiac fibrosis by enhancing AXL translation.

Cardiac fibrosis caused by ventricular remodeling and dysfunction such as post-myocardial infarction (MI) can lead to heart failure. RNA N6-methyladenosine (m6A) methylation has been shown to play a pivotal role in the occurrence and development of many illnesses. In investigating the biological function of the m6A reader YTHDF1 in cardiac fibrosis, adeno-associated virus 9 was used to knock down or overexpress the YTHDF1 gene in mouse hearts, and MI surgery in vivo and transforming growth factor-β (TGF-β)-activated cardiac fibroblasts in vitro were performed to establish fibrosis models. Our results demonstrated that silencing YTHDF1 in mouse hearts can significantly restore impaired cardiac function and attenuate myocardial fibrosis, whereas YTHDF1 overexpression could further enhance cardiac dysfunction and aggravate the occurrence of ventricular pathological remodeling and fibrotic development. Mechanistically, zinc finger BED-type containing 6 mediated the transcriptional function of the YTHDF1 gene promoter. YTHDF1 augmented AXL translation and activated the TGF-β-Smad2/3 signaling pathway, thereby aggravating the occurrence and development of cardiac dysfunction and myocardial fibrosis. Consistently, our data indicated that YTHDF1 was involved in activation, proliferation, and migration to participate in cardiac fibrosis in vitro. Our results revealed that YTHDF1 could serve as a potential therapeutic target for myocardial fibrosis.

Nuclear factor-κB activation by transforming growth factor-β1 drives tumour microenvironment-mediated drug resistance in neuroblastoma.

Intrinsic and extrinsic factors in the tumour microenvironment (TME) contribute to therapeutic resistance. Here we demonstrate that transforming growth factor (TGF)-β1 produced in the TME increased drug resistance of neuroblastoma (NB) cells. Human NB cell lines were tested in vitro for their sensitivity to Doxorubicin (DOX) and Etoposide (ETOP) in the presence of tumour-associated macrophages (TAM) and mesenchymal stromal cells/cancer-associated fibroblasts (MSC/CAF). These experiments were validated in xenotransplanted and primary tumour samples. Drug resistance was associated with an increased expression of efflux transporter and anti-apoptotic proteins. Upregulation was dependent on activation of nuclear factor (NF)-κB by TGF-β-activated kinase (TAK1) and SMAD2. Resistance was reversed upon pharmacologic and genetic inhibitions of NF-κB, and TAK1/SMAD2. Interleukin-6, leukaemia inhibitory factor and oncostatin M were upregulated by this TGF-β/TAK1/NF-κB/SMAD2 signalling pathway contributing to drug resistance via an autocrine loop activating STAT3. An analysis of xenotransplanted NB tumours revealed an increased presence of phospho (p)-NF-κB in tumours co-injected with MSC/CAF and TAM, and these tumours failed to respond to Etoposide but responded if treated with a TGF-βR1/ALK5 inhibitor. Nuclear p-NF-κB was increased in patient-derived tumours rich in TME cells. The data provides a novel insight into a targetable mechanism of environment-mediated drug resistance.

Effect of high plant protein diet supplemented with Lysophospholipids-butyrate on the growth performance, liver health and intestinal morphology of Largemouth bass (Micropterus salmoides)

This study was conducted to evaluate the effects of dietary supplementation with lysophospholipids-butyrate (LPB) in a high plant protein diet on the growth performance, liver health, and intestinal morphology of Largemouth bass (Micropterus salmoides). Three experimental diets were formulated: a positive control diet (PC) containing 15.00 % soybean meal (SBM) and 13 % chicken meal；a high-plant-protein diet (HPP) with 28.00 % SBM; a diet (LPB) supplemented LPB at 0.1 % in HPP. Largemouth bass with an average initial body weight of 9.3 g were fed for 60 days, with 4 replicates per group and 20 fish per replicate. The results demonstrated that the HPP group exhibited significantly lower final body weight (FBW), weight gain rate (WGR) and specific growth rate (SGR) compared to the PC group. Moreover, the HPP diet induced a notable increase in liver collagen fiber and liver lipid content, which was alleviated by LPB supplementation. In comparison to the HPP group, the LPB group showed significant increases in serum albumin and albumin/globulin, along with a significant decrease in alanine aminotransferase activity (P < 0.05). Compared with the PC group, the mRNA levels of acetyl-CoA carboxylase-1 (ACC-1), diacylglycerol acyltransferase (DGAT), carnitine palmitoyl transferase-1 (CPT-1) and tumor necrosis factor-α (TNF-α) in HPP group were increased significantly (P < 0.05), while adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL) as well as apoptotic gene B-cell lymphoma-2 (Bcl-2) were significantly down-regulated. The mRNA levels of fatty acid synthase (FAS), ACC-1, peroxisome proliferator activated receptor-α (PPAR-α), ATGL, interleukin-1β (IL-1β), transforming growth factor-β (TGF-β) and Bcl-2 were up-regulated (P < 0.05), while DGAT and TNF-α were down-regulated in the LPB supplemented groups than that in the HPP group (P < 0.05). In conclusion, the supplementation of 0.1 % LPB can alleviate liver lipid accumulation and fibrosis caused by high level of soybean meal.

Molecular mechanisms of extracellular-ATP-mediated colorectal cancer progression: Implication of purinergic receptors-mediated nucleocytoplasmic shuttling of HuR.

One of the leading causes of cancer-related deaths worldwide is colorectal cancer (CRC). Extracellular ATP (e-ATP) and purinergic receptors (P2R) play a central role in CRC proliferation and progression. Human antigen R (HuR) is becoming more and more understood to be essential for the expression of genes linked to cancer. The current study demonstrates that ATP can mediate CRC (Caco-2 cells) progression via induction of HuR nucleocytoplasmic shuttling and subsequent expression of cancer-related genes, a consequence mostly mediated via the P2R receptor. It was also noted that suppression of HuR activity by using dihydrotanshinone I (DHTS) prevents cancer-related gene expression and subsequent CRC (Caco-2 cells) progression induced by ATP. The expression of cyclin A2/cyclin-dependent kinase 2 (CDK2), Bcl-2, ProT-α, hypoxia-inducible factor1-α (HIF1-α), vascular endothelial growth factor A (VEGF-A), transforming growth factor-β (TGF-β) and matrix metallopeptidase 9 (MMP-9) induced by ATP were highly reduced in the presence of either PPADS (non-selective P2R antagonist) or DHTS. In addition, e-ATP-induced Caco-2 cell proliferation as well as cell survival were highly reduced in the presence of either PPADS or DHTS or selective CDK-2 inhibitor (Roscovitine) or selective Bcl-2 inhibitor (ABT-263). Furthermore, it was found that MMP-9 is critical for Caco-2 cells migration induced by e-ATP as demonstrated by a clear reduction in cells migration in the presence of a selective MMP-9 inhibitor (Marimastat). Collectively, these data demonstrate that ATP through P2R activation can induce HuR nucleocytoplasmic shuttling that could be translated into an increase in cancer-related genes expression and subsequent, cell proliferation and progression.

The regulatory effect of electroacupuncture on endometrial M1-type macrophages in rats with intrauterine adhesions.

To observe the effect of electroacupuncture(EA) on endometrial fibrosis and M1-type macrophages in rats with intrauterine adhesions(IUA), so as to explore the possible mechanism of EA in the treatment of IUA. Fifteen female SD rats were randomly divided into blank group, model group and EA group, with 5 rats in each group. The IUA rat model was established by double damage method using mechanical scraping combined with lipopolysaccharide infection. Rats in the EA group were treated with acupuncture at "Guanyuan"(CV4), and EA at bilateral "Zusanli"(ST36) and "Sanyinjiao"(SP6)for 20 minutes each time, once a day, for 3 consecutive cycles of estrus. Five rats in each group were sampled during the estrous period, and the endometrial morphology, endometrial thickness and the number of blood vessels and glands were observed after HE staining. The fibrotic area of the uterus was observed after Masson staining. The positive expressions of Runt-related transcription factor(RUNX1), transforming growth factor-β1(TGF-β1), connective tissue growth factor(CTGF), α-smooth muscle actin(α-SMA), collagen type I(Col-Ⅰ), cluster of differentiation 86(CD86), interleukin-1β(IL-1β), and tumor necrosis factor-α(TNF-α) in endometrial tissue were detected by immunohistochemistry. Western blot was used to detect relative protein expressions of RUNX1, TGF-β1, α-SMA, CD86, and TNF receptor 2 (TNFR2), and real-time fluorescence quantitative PCR was used to detect mRNA expressions of RUNX1, TGF-β1, α-SMA, CD86, and TNF-α in the endometrium. During the estrous phase, the endometrial layer in the model group was damaged, with reduced folds, disordered arrangement of epithelial cells, loose fibrous connective tissue, significant narrowing and adhesions in the uterine cavity, interstitial congestion, edema, and a significant infiltration of inflammatory cells with sparse glands. While uterine tissue structure of the EA group was basically intact, resembling a normal uterus, with more newly formed glands and a small amount of inflammatory cell infiltration. In comparison with the blank group, the endometrial thickness, the number of blood vessels, and the number of glands were significantly decreased(P<0.001) in the model group, while the ratio of uterine fibrosis area, the positive expressions of RUNX1, TGF-β1, CTGF, α-SMA, Col-Ⅰ, CD86, IL-1β, and TNF-α, the protein relative expressions of RUNX1, TGF-β1, α-SMA, CD86 and TNFR2, and the mRNA relative expression levels of RUNX1, TGF-β1, α-SMA, CD86 and TNF-α in the endometrium were significantly increased (P<0.001, P<0.01). Compared to the model group, the endometrial thickness, the number of blood vessels, and the number of glands were significantly increased(P<0.01, P<0.05) in the EA group, while the ratio of uterine fibrosis area, the positive expressions of RUNX1, TGF-β1, CTGF, α-SMA, Col-Ⅰ, CD86, IL-1β and TNF-α in the endometrial tissue, the protein expressions of RUNX1, TGF-β1, α-SMA, CD86 and TNFR2, and the mRNA relative expressions of RUNX1, TGF-β1, α-SMA, CD86 and TNF-α in the endometrium were significantly decreased (P<0.001, P<0.01, P<0.05). EA can improve endometrial fibrosis in IUA rats, which may be related to its function in decreasing the level of endometrial M1-type macrophages and the secretion of related inflammatory factors.

MicroRNA-19a-3p augments TGF-β1-induced cardiac fibroblast activation via targeting BAMBI.

The main pathogenic factor leading to cardiac remodeling and heart failure is myocardial fibrosis. Recent research indicates that microRNAs are essential for the progress of cardiac fibrosis. Myocardial fibrosis is considered to be alleviated through the bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI), which does this by blocking the transforming growth factor β1 (TGF-β1) signaling pathway. Here, this study sought to elucidate the post-transcriptional regulation of miR-19a-3p on BAMBI and its role in TGF-β1-induced cardiac fibroblast activation. Transverse aortic constriction (TAC) caused both myocardial interstitial and perivascular collagen deposition. RT-PCR showed that miR-19a-3p was upregulated in the myocardial tissue of cardiac fibrosis, and TGF-β1 induced an increase of miR-19a-3p expression in cardiac fibroblasts. The dual-luciferase reporter test and qRT-PCR confirmed that miR-19a-3p directly combined with BAMBI mRNA 3'UTR, thus reduced BAMBI expression, which diminished the capability of BAMBI to inhibit TGF-β1. Furthermore, miR-19a-3p mimic increased the activation of TGF-β1/SMAD2/3 pathway signaling, which supported cardiac fibroblast activation, which blocked by overexpression of BAMBI. These findings imply that miR-19a-3p enhances the activation of TGF-β1/SMAD2/3 by inhibiting BAMBI, further boosting the activation of cardiac fibroblasts, and may thus offer a novel strategy to tackling myocardial fibrosis.

Bushen Huoxue formula protects against renal fibrosis and pyroptosis in chronic kidney disease by inhibiting ROS/NLRP3-mediated inflammasome activation

Background Renal fibrosis contributes to chronic renal failure and a decline in the quality of life. Bushen Huoxue (BSHX) formula is a Traditional Chinese Medicine used to treat chronic renal failure. However, its mechanisms of action remain unclear. Methods and results In this study, a rat model of renal fibrosis was constructed by 5/6 nephrectomy in vivo, and histopathological changes were analyzed using hematoxylin-eosin and Masson’s trichrome staining. Angiotensin II (Ang II) was used to establish an in vitro renal fibrosis cell model in vitro. Pyroptosis was measured using flow cytometry. Related markers of fibrosis and NOD-like receptor protein 3 (NLRP3) inflammasome activation were measured using western blotting and enzyme-linked immunosorbent assay. Treatment with BSHX (0.25, 0.5, and 1 g/kg) significantly inhibited renal fibrosis and damage in 5/6 nephrectomized rats and simultaneously reduced oxidative stress and NLRP3 inflammasome activation. Similarly, BSHX treatment reduced the levels of hydroxyproline, transforming growth factor-β, matrix metalloproteinase 2, and matrix metalloproteinase 9 and inactivated the Smad2/3 signaling pathway in Ang II-treated HK-2 cells. Our data also showed that treatment with BSHX reduced NLRP3 inflammasome activation and pyroptosis in Ang II-treated HK-2 cells. Moreover, fibrosis and pyroptosis in HK-2 cells induced by NLRP3 overexpression were reduced by treatment with BSHX. Conclusions BSHX significantly reduced renal fibrosis and pyroptosis, and its mechanism was mainly associated with the inhibition of reactive oxygen species (ROS)/NLRP3-mediated inflammasome activation.

Heart failure promotes multimorbidity through innate immune memory.

Patients with heart failure (HF) often experience repeated acute decompensation and develop comorbidities such as chronic kidney disease and frailty syndrome. Although this suggests pathological interaction among comorbidities, the mechanisms linking them are poorly understood. Here, we identified alterations in hematopoietic stem cells (HSCs) as a critical driver of recurrent HF and associated comorbidities. Bone marrow transplantation from HF-experienced mice resulted in spontaneous cardiac dysfunction and fibrosis in recipient mice, as well as increased vulnerability to kidney and skeletal muscle insults. HF enhanced the capacity of HSCs to generate proinflammatory macrophages. In HF mice, global chromatin accessibility analysis and single-cell RNA-seq showed that transforming growth factor-β (TGF-β) signaling was suppressed in HSCs, which corresponded with repressed sympathetic nervous activity in bone marrow. Transplantation of bone marrow from mice in which TGF-β signaling was inhibited similarly exacerbated cardiac dysfunction. Collectively, these results suggest that cardiac stress modulates the epigenome of HSCs, which in turn alters their capacity to generate cardiac macrophage subpopulations. This change in HSCs may be a common driver of repeated HF events and comorbidity by serving as a key carrier of "stress memory."

Linagliptin Mitigates TGF-β1 Mediated Epithelial-Mesenchymal Transition in Tacrolimus-Induced Renal Interstitial Fibrosis via Smad/ERK/P38 and HIF-1α/LOXL2 Signaling Pathways.

The dipeptidyl peptidase-4 (DPP-4) inhibitors, a novel anti-diabetic medication family, are renoprotective in diabetes, but a comparable benefit in chronic non-diabetic kidney diseases is still under investigation. This study aimed to elucidate the molecular mechanisms of linagliptin's (Lina) protective role in a rat model of chronic kidney injury caused by tacrolimus (TAC) independent of blood glucose levels. Thirty-two adult male Sprague Dawley rats were equally randomized into four groups and treated daily for 28 d as follows: The control group; received olive oil (1 mL/kg/d, subcutaneously), group 2; received Lina (5 mg/kg/d, orally), group 3; received TAC (1.5 mg/kg/d, subcutaneously), group 4; received TAC plus Lina concomitantly in doses as the same previous groups. Blood and urine samples were collected to investigate renal function indices and tubular injury markers. Additionally, signaling molecules, epithelial-mesenchymal transition (EMT), and fibrotic-related proteins in kidney tissue were assessed by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis, immunohistochemical and histological examinations. Tacrolimus markedly induced renal injury and fibrosis as indicated by renal dysfunction, histological damage, and deposition of extracellular matrix (ECM) proteins. It also increased transforming growth factor β1 (TGF-β1), Smad4, p-extracellular signal-regulated kinase (ERK)1/2/ERK1/2, and p-P38/P38 mitogen-activated protein kinase (MAPK) protein levels. These alterations were markedly attenuated by the Lina administration. Moreover, Lina significantly inhibited EMT, evidenced by inhibiting Vimentin and α-smooth muscle actin (α-SMA) and elevating E-cadherin. Furthermore, Lina diminished hypoxia-related protein levels with a subsequent reduction in Snail and Twist expressions. We concluded that Lina may protect against TAC-induced interstitial fibrosis by modulating TGF-β1 mediated EMT via Smad-dependent and independent signaling pathways.

#207 Early single session of hyperbaric oxygen therapy mitigates renal apoptosis in lipopolysaccharides-induced endotoxemia in rats

Abstract Background and Aims Sepsis-associated acute kidney injury (SA-AKI) is a prominent sepsis complication, often having adverse clinical outcomes. Hyperbaric oxygen therapy (HBOT), known for its anti-inflammatory characteristics, antioxidant effects, and ability to deliver high oxygen tension to hypo-perfused tissues, may offer potential benefits for SA-AKI. This study investigated whether HBOT improved renal injury in sepsis and elucidated its underlying mechanisms. Method An lipopolysaccharides (LPS)-induced endotoxemia model was established using eight-week-old C57BL/6 mice. Thirty minutes post-LPS administration, a subset of mice underwent HBOT at a 2.5 atmospheric pressure absolute with 100% oxygen for 60 minutes. After 24 hours, all mice were euthanized for measurements. Results Our results demonstrated that HBOT effectively mitigated renal tubular cell apoptosis, as evidenced by decreased apoptosis-associated proteins, including Bax/Bcl-2 and cleaved caspase 3/caspase 3 ratios. HBOT significantly reduced phosphorylated p53 proteins and cytochrome C levels, suggesting that HBOT may attenuate renal apoptosis through decreased p53 activation and cytochrome C release. Notably, HBOT preserved manganese-dependent levels of superoxide dismutase, an antioxidant enzyme, compared to the LPS group. Furthermore, transforming growth factor-β (TGF- β)/Smad4 and alpha-smooth muscle actin expressions were significantly reduced in the LPS+HBOT group. Conclusion An early single session of HBOT exhibited renoprotective effects in LPS-induced endotoxemia mice models by mitigating apoptosis by suppressing p53 activation and cytochrome C levels. The observed TGF-β decrease, downstream Smad expression reduction, and antioxidant capacity preservation following HBOT may contribute to these effects.

Inhibiting Effect and Mechanism of Aconitum tanguticum (Maxim.) Stapf on Intestinal Fibrosis of CCD-18Co Cells

Abstract Objective: The objective of the study was to explore the potential signaling mechanism of Aconitum tanguticum (Maxim.) Stapf (ATS) and its impact on intestinal fibrosis in vitro. Methods: Network pharmacology was used to screen the active components of ATS and predict their potential targets in intestinal fibrosis. The protein–protein interaction network graph was constructed using drug–disease intersection targets retrieved from the Search Tool for Retrieval of Interacting Genes/Proteins database. The network diagram was analyzed using Cytoscape 3.6.1’stopology function. The gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were conducted using the database for annotation, visualization, and integrated discovery platform. Intestinal fibroblast model in vitro was constructed using transforming growth factor-β1 (TGF-β1)-induced CCD-18Co cells. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to verify the network pharmacology-predicted antifibrotic signaling pathway of ATS and the traditional antifibrotic signaling pathway. Results: Network pharmacology revealed that there were 19 active components in ATS, suggesting that ATS could be involved in the regulation of mitogen-activated protein kinase 1 (MAPK1), PIK3CA, MAPK3, and other important targets that are present in cancer pathways, including proteoglycans, influenza A, and phosphatidylinositol-3-kinase (PI3K)-protein kinase B (Akt) signaling pathways. The results of quantitative reverse transcription-polymerase chain reaction (qRT-PCR) demonstrated that 0.1–1 μg/mL ATS may suppress tissue inhibitor of metalloproteinase 1, collagen I, and alpha-smooth muscle actin expression levels. The expression of the TGF-β/suppressor of the mother against decapentaplegic (Smad) and PI3K/Akt signaling pathways was controlled by ATS. In addition, ATS inhibited the PI3K/Akt pathway by reducing the expression of MAPK1, HSP90AA1, and PIK3CA. Conclusions: It has been confirmed that ATS is a multipathway and multitarget treatment for intestinal fibrosis. This study suggested that ATS might alleviate intestinal fibrosis by blocking both the TGF-β/Smad and the PI3K/Akt pathway.

#2494 Pharmacological enhancement of circadian clock ameliorated fibrosis in adenine induced chronic kidney disease

Abstract Background and Aims Circadian clock regulates diverse biological processes. Recent studies demonstrating the important role of dysregulated molecular clock system in several metabolic diseases as well as in chronic kidney disease (CKD) suggest that modulation of clock machinery might be an attractive therapeutic target. Method We optimized a substance (compound X) that enhances the circadian molecular rhythm and validated its effect in HK-2 cells as well as in mouse model of adenine induced CKD. Results By using transcriptome analysis of whole kidneys, we identified that 19% of CKD associated genes showed rhythmic expressions in circadian manner. Gene enrichment analysis revealed that the rhythmic genes involved in extracellular matrix/collagen/myofibroblasts showed upregulation while those involved in transport/brush border/proximal tubule showed downregulation in CKD. Treatment with compound X in HK-2 cells resulted in marked attenuation of epithelial mesenchymal transition upon transforming growth factor-b (TGF- b) stimulation. Along with these in vitro data, we also observed the preserved kidney function and less interstitial fibrosis in compound X treated CKD mice. The protective effect was associated with suppression of inflammation, cell cycle arrest/cellular senescence as well as restoration of several transporters in proximal tubular cells. Conclusion This is the first study that showed that circadian rhythm might be a novel target for CKD progression by suppressing inflammation/fibrosis and enhancing tubular functions. Unlike other strategies targeting single pathway, targeting circadian rhythm seems to be more promising due to its multi-target effects. A better understanding of the complex role of circadian clock in kidney diseases may provide new perspectives for the prevention and treatment of CKD.

Study of metal organic framework with siRNA for overcoming matrix barrierin breast cancer

Objective: This study aimed to develop a new delivery strategy that utilized metal organic framework (MOF) loaded with small-interfering RNA (siRNA) targeting ITGAV to overcome tumor matrix barrier, and thus enhance drug penetration and immune accessibility in breast cancer. Methods: MOF@siITGAV particles were constructed and characterized. The uptake of MOF@siITGAV in breast cancer cell line 4T1 was observed by the cellular uptake assay. The toxicity of MOF@siITGAV was detected by cell counting kit 8 (CCK-8). The blank control group, naked siITGAV group and MOF@siITGAV group were set. Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) and Western blot were used to detect the expressions of ITGAV. The level of transforming growth factor β1 (TGF-β1) in the cell culture medium was detected by enzyme-linked immunosorbent assay (ELISA). The penetration of MOF@siITGAV in 4T1 cells was tested by constructing 3D spheroids. Mouse models of triple negative breast cancer were established. The effect of MOF@siITGAV on the growth of transplanted tumors and main organs was verified. Imminohistochemical (IHC) was used to test the expression of collagen and CD8. Results: MOF@siITGAV particles were constructed with sizes of (198.0±3.3) nm and zeta potential of -(20.2±0.4) mV. MOF@siITGAV could be engulfed by 4T1 cells and triggered to release siRNA. Compared to the blank control group, the expression of ITGAV in the MOF@siITGAV group [(46.5±11.3)%] and the naked siITGAV group [(109.9±19.0)%] was lower. TGF-β1 in the cell culture medium of the blank control group, naked siITGAV group, and MOF@siITGAV group was (474.5±34.4) pg/ml, (437.2±16.5) pg/ml, and (388.4±14.4) pg/ml, respectively. MOF@siITGAV could better penetrate into 4T1 spheroids and exhibit no obvious toxicity. The cell viability was (99.7±3.5)%, (98.2±5.2)%, (97.3±6.6)%, (92.1±8.1)%, and (92.4±4.1)%, respectively, after MOF@siITGAV treatment with the concentration of 0, 10, 20, 40, 80, and 160 μg/ml, respectively, for 24 h. The tumor growth in the MOF@siITGAV group was suppressed significantly. After 15-day treatment, the tumor volume of the MOF@siITGAV group was (135.3±41.9) mm3, smaller than that of the blank control group [(691.1±193.0) mm3] (P=0.025). The expression of collagen and the number of CD8 positive cells of the MOF@siITGAV group were lower than those of the other two groups. No significant abnormalities were observed in the main organs of mice. Conclusions: Targeting the integrinαv on the surface of cancer cells could destroy extracellular matrix, improve drug delivery, and increase immune infiltration.