Trigonelline mitigates bleomycin-induced pulmonary inflammation and fibrosis: Insight into NLRP3 inflammasome and SPHK1/S1P/Hippo signaling modulation.

AIMS: Pulmonary fibrosis (PF) is a chronic interstitial lung disease with an increasing incidence following the COVID-19 outbreak. Pirfenidone (Pirf), an FDA-approved pulmonary anti-fibrotic drug, is poorly tolerated and exhibits limited efficacy. Trigonelline (Trig) is a natural plant alkaloid with diverse pharmacological actions. We investigated the underlying prophylactic and therapeutic mechanisms of Trig in ameliorating bleomycin (BLM)-induced PF and the possible synergistic antifibrotic activity of Pirf via its combination with Trig. MATERIALS AND METHODS: A single dose of BLM was administered intratracheally to male Sprague-Dawley rats for PF induction. In the prophylactic study, Trig was given orally 3 days before BLM and then for 28 days. In the therapeutic study, Trig and/or Pirf were given orally from day 8 after BLM until the 28th day. Biochemical assay, histopathology, qRT-PCR, ELISA, and immunohistochemistry were performed on lung tissues. KEY FINDINGS: Trig prophylactically and therapeutically mitigated the inflammatory process via targeting NF-κB/NLRP3/IL-1ß signaling. Trig activated the autophagy process which in turn attenuated alveolar epithelial cells apoptosis and senescence. Remarkably, Trig attenuated lung SPHK1/S1P axis and its downstream Hippo targets, YAP-1, and TAZ, with a parallel decrease in YAP/TAZ profibrotic genes. Interestingly, Trig upregulated lung miR-375 and miR-27a expression. Consequently, epithelial-mesenchymal transition in lung tissues was reversed upon Trig administration. These results were simultaneously associated with profound improvement in lung histological alterations. SIGNIFICANCE: The current study verifies Trig's prophylactic and antifibrotic effects against BLM-induced PF via targeting multiple signaling. Trig and Pirf combination may be a promising approach to synergize Pirf antifibrotic effect.

Promising renoprotective effect of gold nanoparticles and dapagliflozin in diabetic nephropathy via targeting miR-192 and miR-21.

Diabetic nephropathy (DN) is a worldwide issue that eventually leads to end-stage renal failure, with limited therapeutic options. Prior research has revealed that gold nanoparticles (AuNPs) have a substantial antidiabetic impact. In addition, sodium-glucose cotransporter2 (SGLT2) inhibitors, including dapagliflozin (DAPA), had renoprotective impact on DN. Therefore, this research attempted to determine the potential AuNPs and DAPA impacts in ameliorating experimentally DN induction and the underlying mechanisms focusing on miR-192 and miR-21, correlating them with autophagy, apoptosis, fibrosis, and oxidative stress. Diabetes induction was through a single intraperitoneal streptozotocin (55 mg/kg) injection, and rats with diabetes received AuNPs (2.5 mg/kg/day) as well as DAPA (2 mg/kg/day) for 7 weeks as a treatment. AuNPs and DAPA treatment for 7 weeks substantially alleviated DN. AuNPs and DAPA significantly increased catalase (CAT) activity as well as serum total antioxidant capacity (TAC), along with a substantial decline in malondialdehyde (MDA). AuNPs and DAPA treatment alleviated renal fibrosis as they decreased transforming growth factorß1(TGF-ß1) as well as matrix metalloproteinase-2 (MMP-2) renal expression, decreased apoptosis through alleviating the proapoptotic gene (caspase-3) renal expression and increased the antiapoptotic gene (Bcl-2) renal expression, and increased autophagy as they increased LC-3 as well as Beclin-1 renal expression. Autophagy activation, inhibition of apoptosis, and renal fibrosis could be due to their inhibitory impact on miR-192 and miR-21 renal expression. AuNPs and DAPA have a protective effect on DN in rats by targeting miR-192 and miR-21 and their downstream pathways, including fibrosis, apoptosis, autophagy, and oxidative stress.

Naringenin mitigates thioacetamide-induced hepatic encephalopathy in rats: targeting the JNK/Bax/caspase-8 apoptotic pathway.

Hepatic encephalopathy (HE) is a serious neurological disorder which is related to liver dysfunction. HE was induced by thioacetamide (TAA) injection (350 mg kg-1, i.p.) for 3 consecutive days. This study was performed to investigate the prophylactic impact of naringenin against TAA-induced HE. Naringenin (100 mg kg-1) was orally administered for 7 days starting 4 days prior to TAA injection. Naringenin effectively mitigated TAA-induced behavioural, structural and functional alterations. Naringenin ameliorated TAA-induced cognitive impairment as evidenced by the increase in the fall-off time in the rotarod test, decrease in the escape latency in the Morris water maze test and increase in the time spent in the center and in the number of rearing in the open field test. Additionally, naringenin significantly decreased the serum levels of transaminases, alkaline phosphatase, gamma-glutamyl transferase, bile and ammonia. Moreover, naringenin succeeded in reducing the levels of hepatic and cerebral c-Jun N-terminal kinases (JNK) as well as hepatic SORT1 levels. In addition, naringenin successfully elevated the levels of hepatic and cerebral pro-brain-derived neurotrophic factor (pro-BDNF) and BDNF in addition to the cerebral SORT1 level. Finally, naringenin markedly decreased the expression of Bax and caspase-8 as presented by the immunohistochemical results. Collectively, the ameliorative effect of naringenin on the development of HE might be attributed to the modulation of the JNK/Bax/caspase-8 apoptotic pathway.

Hepatoprotective and cognitive-enhancing effects of hesperidin against thioacetamide-induced hepatic encephalopathy in rats.

Hepatic encephalopathy (HE) is a serious neurological disorder which might occur in both acute and chronic liver injury. AIMS: This study was carried out to explore the protective effects of hesperidin against experimentally induced HE. MAIN METHODS: Rats were sorted into four groups each of six; Normal group, TAA group: rats were administered 350 mg/kg of TAA i.p. from day 5 to day 7. TAA+ Hesp 100 group: rats were administered hesperidin 100 mg/kg/day orally for 7 days along with i.p TAA injection 350 mg/kg from day 5 to 7. TAA+ Hesp 200 group: rats were administered hesperidin 200 mg/kg/day orally for 7 days along with i.p TAA injection 350 mg/kg from day 5 to 7. Liver function, oxidative stress biomarkers, behavioral tests in addition to histopathological examination were assessed. KEY FINDINGS: Hesperidin efficiently mitigated TAA-induced HE as evidenced by significant reduction in liver enzymes, bile and ammonia levels in serum. Moreover, hesperidin restored oxidant/antioxidant balance as manifested by reduction in MDA content in both cerebral and hepatic tissues. Additionally, hesperidin improved motor and cognitive abilities besides tissues' architecture as demonstrated by behavioral tests and histopathology results, respectively. Hesperidin also decreased levels of NLRP3 and increased levels of Sirt1 and FOXO in both cerebral and hepatic tissues. Finally, hesperidin markedly decreased the expression of IL-1ß and caspase-1 as shown by immunohistochemical results. SIGNIFICANCE: Taken together, the hepatoprotective impact of hesperidin and its ameliorative effect on the progression of HE appear to be mediated by its modulatory influence on NLRP3/Sirt1/FOXO signaling.

Ameliorative effects of bromelain on aluminum-induced Alzheimer's disease in rats through modulation of TXNIP pathway.

Alzheimer's disease (AD) is known as "type 3 diabetes". As thioredoxin binding protein (TXNIP) has been shown to be involved in brain insulin resistance, the present study evaluated the roles of TXNIP, phospho-insulin receptor substrate 1 (P-IRS-1), and phosphatidyl inositol-3 kinase (PI3K) in the pathogenesis of AD. The potential ameliorative effect of bromelain compared to donepezil was evaluated in an aluminum chloride (AlCl3)-induced AD in rats. Behavioral tests demonstrated similar improvements in exploratory activity, cognitive and spatial memory functions, anxiety, and depression levels between rats treated with bromelain and donepezil. Donepezil was superior to bromelain in improving locomotor activity. Histopathological examinations demonstrated neuronal degeneration in the AlCl3 group that was almost normalized by bromelain and donepezil. Moreover, there was deposition of amyloid plaques in the AlCl3 group that was improved by bromelain and donepezil. Acetylcholine esterase levels were significantly increased in rats treated with AlCl3 group and significantly decreased in rats treated with bromelain and donepezil. Furthermore, AlCl3 group showed a significantly increased TXNIP and P-IRS1 and a significantly reduced PI3K levels. These effects were ameliorated by bromelain and donepezil treatment. The present study demonstrates a previously unreported modulatory effect of bromelain on the TXNIP/P-IRS-1/PI3K axis in AD model.

The hepatoprotective effects of n3-polyunsaturated fatty acids against non-alcoholic fatty liver disease in diabetic rats through the FOXO1/PPARα/GABARAPL1 signalling pathway.

AIM: We compared the efficacy of n3-polyunsaturated fatty acids (n3-PUFAs) and metformin in halting the progression of non-alcoholic fatty liver disease (NAFLD) developed in the milieu of insulin deficiency. MAIN METHODS: NAFLD was induced by a chronic high-fat diet (HFD) in male Sprague Dawley rats, rendered diabetic by a low dose streptozotocin (STZ). Diabetic rats were treated with n3-PUFAs (300 mg/kg/d) or metformin (150 mg/kg/d) for 8 weeks. Improvements in the NAFLD score and hepatic insulin resistance (IR) were addressed and correlated to changes in the hepatic expression of Forkhead box protein O1 (FOXO-1), microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B) and gamma-aminobutyric acid receptor-associated protein-like 1 (GABARAPL1) genes. Hepatic peroxisome proliferator-activated receptor alpha (PPAR-α), and B-cell lymphoma 2 (Bcl-2) protein expression was also assessed. KEY FINDINGS: Driven by insulin deficiency and HFD, the FOXO-1 gene along with its downstream targets, MAP1LC3B and GABARAPL1, were highly expressed in the liver tissue of the HFD/STZ group. Meanwhile, hepatic expression of PPAR-α and Bcl-2 was markedly decreased. These abnormalities coincided with a marked increase in the hepatic IR and NAFLD activity. Comparable to metformin, n3-PUFAs were able to rearrange hepatic PPAR-α and FOXO-1 expression in HFD/STZ rats, resulting in improved diabetic/steatotic liver phenotype. SIGNIFICANCE: Along with the enhancement of PPAR-α expression, inhibition of FoxO1/GABARAPL1/MAP1LC3B transcription is suggested as a core mechanism for the protective effects of n3-PUFAs on hepatic IR and NAFLD. Under conditions of insulin deficiency, n3-PUFAs retain their potential as a safe and promising approach for the control of NAFLD.

Antidiabetic effects of quercetin and liraglutide combination through modulation of TXNIP/IRS-1/PI3K pathway.

The study was designed to assess the possible augmented antidiabetic effects of combining quercetin and liraglutide in a type 1 diabetes model, with emphasis on the contribution of hepatic thioredoxin interacting protein (TXNIP)/insulin receptor substrate 1 (IRS-1)/phosphatidyl inositol-3 kinase (PI3K) pathway. The wound-healing effects were also examined. Diabetes was induced by a single i.p STZ injection (55 mg/kg). Diabetic rats were treated with either quercetin (100 mg/kg/day, orally) or liraglutide (0.3 mg/kg/twice daily, S.C.) or their combination. Drugs were also applied topically on the wound. Blood glucose levels, serum albumin, total protein, total cholesterol and triglycerides were measured. Histopathological examination of the liver, pancreas and skin tissues was performed using haematoxylin and eosin staining. The hepatic malondialdehyde level was measured spectrophotometrically. Hepatic TXNIP and PI3K levels were measured by enzyme-linked immunsorbent assay (ELISA). Tissue expression of IRS-1 and phospho-IRS-1 (Ser 616) was assessed by immunohistochemistry. Quercetin, liraglutide and their combination effectively decreased blood glucose levels, improved lipid profile, upregulated albumin and total protein serum levels and reduced hepatic oxidative stress with the combination being most effective. Moreover, the combination group showed enhanced wound-healing effects and almost normalized hepatic and pancreatic histopathology. Quercetin and/or liraglutide significantly decreased TXNIP levels and serine phosphorylation of IRS-1 and increased PI3K levels compared to the diabetic untreated group. Interestingly, only the combination therapy normalized hepatic IRS-1 expression. The combination of quercetin and liraglutide showed enhanced antidiabetic effects, possibly through lowering hepatic TXNIP levels, with the resultant up-regulation of the IRS-1/PI3K pathway.

The ameliorative effect of bromelain on STZ-induced type 1 diabetes in rats through Oxi-LDL/LPA/LPAR1 pathway.

AIMS: Diabetes, a serious worldwide problem, is modulated via inflammation and oxidative stress. Bromelain, a natural compound, recently attracts interest due to its anti-inflammatory effects, while its mode of action remains not properly understood. Thus, investigating the antidiabetic effect of bromelain is promising. MATERIALS AND METHODS: Rats were randomized into normal group, STZ group (were administrated single intraperitoneal (i.p) injection of 55 mg/kg streptozotocin (STZ)) and STZ + Bro group (were administrated single i.p injection of STZ, 72 h later were i.p administrated 10 mg/kg/day bromelain for 15 days). Wound healing ability was investigated for different groups. Spectrophotometry, ELISA, histopathological and immunohistochemical techniques were applied. KEY FINDINGS: Bromelain significantly decreased fasting blood glucose, serum triglycerides and cholesterol and hepatic malondialdehyde levels compared with STZ group. Moreover, Bromelain significantly increased serum albumin and total protein levels and percentage of wound healing compared with STZ group. These results were confirmed through the histopathological examination of liver, pancreas, and skin tissues. Investigating the molecular mechanism underlying these effects, STZ injection caused significant increase in hepatic oxidized-LDL (Oxi-LDL) and lysophosphatidic acid (LPA) levels and hepatic lysophosphatidic acid receptor 1 (LPAR1), and beta secretase (BACE1) protein tissue expressions, while bromelain significantly aborted these effects. Thus, STZ caused upregulation of Oxi-LDL/LPA/LPAR1/BACE1 pathway, while bromelain significantly ameliorated these effects. SIGNIFICANCE: To our best knowledge, this study represents the 1st study investigating Oxi-LDL/LPA/LPAR1/BACE1 pathway in STZ-induced diabetes in rats, in addition to the promising ameliorative effect of bromelain in STZ-induced diabetes in rats.

Effects of omega-3 fatty acids and metformin combination on diabetic cardiomyopathy in rats through autophagic pathway.

Diabetic cardiomyopathy is a primary cause of increased morbidity and mortality in diabetics. Evidence has suggested a pivotal role for interrupted mitochondrial dynamics and quality control machinery in the onset and development of diabetic cardiomyopathy. Sequestosome 1 (SQSTM1) is a major reporter of selective autophagic activity. Other than controlling the expression of genes involved in mitochondrial biogenesis, recently peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) was reported to directly affect SQSTM1 gene expression. Calcineurin, a pivotal mediator of cardiac hypertrophy, has been also linked to enhanced expression of SQSTM1. This study aimed to test the cardioprotective effects of adding ω-3 polyunsaturated fatty acids (PUFAs) to metformin in a rat model of type 2 diabetes mellitus and to evaluate the molecular mechanisms underlying their effects on mitochondrial quality. Diabetes was induced in male Sprague Dawley rats by a high-fat diet for 6 weeks, followed by a low-dose streptozotocin (35 mg/kg). Diabetic rats were either treated with metformin (150 mg/kg/d), ω-3 PUFAs (300 mg/kg/d), or their combination in the same doses for further 8 weeks. Along with metabolic and pathological derangements, we report that correlating with electron microscopic evidence of mitochondrial degeneration, gene expression of the autophagic indicators SQSTM1, PGC-1α, and calcineurin were decreased in the hearts of diabetic rats. Independent of its anti-hyperglycemic effects, metformin successfully preserved mitochondrial integrity and upregulated myocardial PGC-1α, calcineurin, and SQSTM1 gene expression. ω-3 PUFAs possess synergistic cardioprotection when added to metformin, suggested by improvements in myocardial ultrastructure, autophagic activity, and SQSTM1 gene expression.

Omega-3 fatty acids protect against acetaminophen-induced hepatic and renal toxicity in rats through HO-1-Nrf2-BACH1 pathway.

Although acetaminophen (APAP) is a commonly used analgesic antipyretic drug, hepatotoxicity and nephrotoxicity are common after the overdose. The main mechanism of APAP toxicity is oxidative stress based. Stress may induce the production of heme oxygenase 1 (HO)-1 which is regulated by interleukin (IL)-10 and inhibit the production of tumor necrosis factor-alpha (TNF-α). HO-1 expression is further regulated by nuclear factor erythroid 2-related factor 2 (Nrf2) and the transcription factor BTB and CNC homology 1 (BACH1). Drug-induced toxicity can be relieved by several natural products, which are preferred due to their dietary nature and less adverse reactions. Of these natural products, omega-3 (ω-3) fatty acids are known for anti-inflammatory and antioxidant actions. However, effects of ω-3fatty acids on APAP-induced hepatic and renal toxicity are not well addressed. We designed this study to test the potential protecting actions of ω-3 fatty acids (270 mg/kg Eicosapentaenoic acid and 180 mg/kg docosahexaenoic acid, orally, for 7 days) in hepatotoxicity and nephrotoxicity induced by APAP (2 g/kg, once orally on day 7) in rats. Moreover, we focused on the molecular mechanism underlying APAP hepatotoxicity and nephrotoxicity. Pre-treatment with ω-3 fatty acids enhanced liver and kidney functions indicated by decreased serum aminotransferases activities and serum creatinine and urea concentrations. These results were further confirmed by histopathological examination. Moreover, ω-3 fatty acids showed antioxidant properties confirmed by decreased malondialdehyde level and increased total antioxidant capacity. Antioxidant Nrf2, its regulators (HO-1 and BACH1) and the anti-inflammatory cytokine (IL-10) were up-regulated by APAP administration as a compensatory mechanism and they were normalized by ω-3 fatty acids. ω-3 fatty acids showed anti-inflammatory actions through down-regulating nuclear factor kappa B (NF-ĸB) and its downstream TNF-α. Moreover, Western blot analysis showed that ω-3 fatty acids promoted Nrf2 translocation to the nucleus; BACH1 exit from the nucleus and inhibited NF-ĸB nuclear translocation. These findings suggested the protecting actions of ω-3 fatty acids against APAP-induced hepatic and renal toxicity through regulation of antioxidant Nrf2 and inflammatory NF-ĸB pathways.

The molecular mechanism underlining the preventive effect of vitamin D against hepatic and renal acute toxicity through the NrF2/ BACH1/ HO-1 pathway.

AIM: Drug-induced liver and kidney injuries are worldwide problems that cause restrictions in the use of drugs. The injury is highly mediated by oxidative stress and inflammation pathways. So, demonstrating the role of the natural compound (Vit.D) on the prevention of acetaminophen (APAP) overdose toxicity and the molecular mechanism through NrF2/BACH1/HO-1 pathway is promising. EXPERIMENTAL: Male Sprague Dawley rats (40 rats) were divided randomly into 4 groups: Normal, APAP, APAP+Vit.D (500 IU/kg) and APAP+Vit.D (1000 IU/kg). The APAP toxicity caused by 2 g/kg (orally) on day 7. KEY FINDINGS: Vit D decreased significantly liver and kidney functions: serum ALT and AST activities (P < 0.0005); creatinine and urea (P < 0.0005) concentrations; liver and kidney histopathological scores. Furthermore, Vit.D ameliorated APAP-caused oxidative stress through the liver malondialdehyde concentration's decrease and the total antioxidant capacity's increase (P < 0.0005). The molecular mechanism of Vit.D may include the prevention of high deteriorating increase of oxidative stress mediators: hepatic and renal NrF2 and BACH1 tissue expression in addition to serum HO-1 (P < 0.0005); the increase of inflammatory mediators; hepatic and renal NF-κB tissue expression, serum interleukin-10 (P < 0.0005) and TNF-α (P < 0.05). The 500 IU/kg Vit.D administration caused better protection results especially on the histopathological and immunohistochemical results than the 1000 IU/kg Vit.D administration. SIGNIFICANCE: Vit.D ameliorates APAP-induced liver and kidney injury that may be attributed to its ability to moderately increase antioxidant status to counteract the toxicity without the massive destructive increase in the anti-oxidant pathway (NrF2/HO-1/BACH1). So, this work represents a great prophylactic role of Vit.D against drug-induced liver and kidney injury.

Silymarin and caffeine combination ameliorates experimentally-induced hepatic fibrosis through down-regulation of LPAR1 expression.

AIMS: Lysophosphatidic acid is a lipid mediator that is supposed to be implicated in hepatic fibrosis. Silymarin and caffeine are natural compounds known for their anti-inflammatory and antioxidant effects. Our study aimed to explore the effect of silymarin, caffeine, and their combination on lysophosphatidic acid receptor 1 (LPAR1) pathway in thioacetamide (TAA)-induced hepatic fibrosis. MAIN METHODS: Hepatic fibrosis was induced in male Sprague-Dawley rats by intraperitoneal injection of 200 mg/kg of TAA twice a week for 8 weeks. Silymarin (50 mg/kg), caffeine (50 mg/kg), and their combination (50 mg/kg silymarin + 50 mg/kg caffeine) were orally given to rats every day for 8 weeks along with TAA injection. Liver functions were measured. Histopathological examination of liver tissues was performed using hematoxylin and eosin and Masson's trichrome staining. mRNA expressions of LPAR1, transforming growth factor beta 1 (TGF-ß1), connective tissue growth factor (CTGF), and alpha smooth muscle actin (α-SMA) were measured using RT-PCR. LPAR1 tissue expression was scored using immunohistochemistry. KEY FINDINGS: Silymarin, caffeine, and their combination significantly improved liver function. They caused significant decrease in fibrosis and necro-inflammatory scores. Combination of silymain and caffeine caused a significant decrease in the necro-inflammatory score than the single treatment with silymarin or caffeine. In addition, silymarin, caffeine, and their combination significantly decreased hepatic LPAR1, TGF-ß1, CTGF, and α-SMA gene expressions and LPAR1 tissue expression. SIGNIFICANCE: Silymarin, caffeine, and their combination protect against liver fibrosis through down-regulation of LPAR1, TGF-ß1, and CTGF.

Modulating effects of omega-3 fatty acids and pioglitazone combination on insulin resistance through toll-like receptor 4 in type 2 diabetes mellitus.

Toll-like receptor 4 (TLR-4) plays important roles in innate immunity. Changes in the reduction-oxidation balance of tissues can lead to a pro-inflammatory state and insulin resistance. An action thought to be mediated by TLRs. Omega-3 fatty acids and Peroxisome Proliferator Activated Receptor gamma (PPAR-γ) agonists as pioglitazone are used for decreasing inflammation. The aim of this study is to investigate the anti-diabetic effects of combining omega -3 fatty acid with pioglitazone on type 2 diabetes, and the modifying effects on TLR-4. Type 2 diabetes was induced in male Sprague-Dawley rats by combination of high fat diet and low dose streptozotocin (35mg/kg). Diabetic rats were treated with omega-3 fatty acids (10% W/W diet), pioglitazone (20mg/kg), and their combination for 4 weeks. Omega-3 fatty acids and the combination treatment significantly decreased TLR-4 activation. Omega-3 fatty acids, pioglitazone, and their combination significantly decreased TLR-4 mRNA expression, hepatic malondialdehyde, total cholesterol and triglycerides levels, compared to diabetic group. Pioglitazone and the combination significantly decreased blood glucose levels and improved insulin resistance. In conclusion, combining omega-3 fatty acids with pioglitazone showed potential effects in lowering blood glucose levels and improving lipid profile and insulin resistance. Such effects are mediated through modulation of TLR-4.