Yigansan ameliorates maternal immune activation-induced autism-like behaviours by regulating the IL-17A/TRAF6/MMP9 pathway: Network analysis and experimental validation.

BACKGROUND: Maternal immune activation (MIA) is a significant factor inducing to autism spectrum disorder (ASD) in offspring. The fundamental principle underlying MIA is that inflammation during pregnancy impedes fetal brain development and triggers behavioural alterations in offspring. The intricate pathogenesis of ASD renders drug treatment effects unsatisfactory. Traditional Chinese medicine has strong potential due to its multiple therapeutic targets. Yigansan, composed of seven herbs, is one of the few that has been proven to be effective in treating neuro-psychiatric disorders among numerous traditional Chinese medicine compounds, but its therapeutic effect on ASD remains unknown. HYPOTHESIS: Yigansan improves MIA-induced ASD-like behaviours in offspring by regulating the IL-17 signalling pathway. METHODS: Pregnant C57BL/6J mice were intraperitoneally injected with poly(I:C) to construct MIA models and offspring ASD models. Network analysis identified that the IL-17A/TRAF6/MMP9 pathway is a crucial pathway, and molecular docking confirmed the binding affinity between the monomer of Yigansan and target proteins. qRT-PCR and Western blot were used to detect the expression levels of inflammatory factors and pathway proteins, immunofluorescence was used to detect the distribution of IL-17A, and behavioural tests were used to evaluate the ASD-like behaviours of offspring. RESULTS: We demonstrated that Yigansan can effectively alleviate MIA-induced neuroinflammation of adult offspring by regulating the IL-17A/TRAF6/MMP9 pathway, and the expression of IL-17A was reduced in the prefrontal cortex. Importantly, ASD-like behaviours have been significantly improved. Moreover, we identified that quercetin is the effective monomer for Yigansan to exert therapeutic effects. CONCLUSION: Overall, this study was firstly to corroborate the positive therapeutic effect of Yigansan in the treatment of ASD. We elucidated the relevant molecular mechanism and regulatory pathway involved, determined the optimal therapeutic dose and effective monomer, providing new solutions for the challenges of drug therapy for ASD.

Electroacupuncture stimulation enhances the permeability of the blood-brain barrier: A systematic review and meta-analysis of preclinical evidence and possible mechanisms.

An important cellular barrier to maintain the stability of the brain's internal and external environment is the blood-brain barrier (BBB). It also prevents harmful substances from entering brain tissue through blood circulation while providing protection for the central nervous system. It should be noted, however, that the intact BBB can be a barrier to the transport of most drugs into the brain via the conventional route of administration, which can prevent them from reaching effective concentrations for the treatment of disorders affecting the central nervous system. Electroacupuncture stimulation has been shown to be effective at opening the BBB in a series of experimental studies. This study systematically analyzes the possibility and mechanism by which electroacupuncture opens the BBB. In PubMed, Web of Science, VIP Database, Wanfang Database, and the Chinese National Knowledge Infrastructure, papers have been published for nearly 22 years aimed at opening the BBB and its associated structures. A comparison of EB content between electroacupuncture and control was selected as the primary outcome. There were also results on vascular endothelial growth factor (VEGF), nerve growth factor (NGF), P-Glycoprotein (P-gp), Matrix Metalloproteinase 9 (MMP-9), and glial fibrillary acidic protein (GFAP). We utilized Review Manager software analysis to analyze correlations between studies with a view to exploring the mechanisms of similarity. Evans Blue infiltration forest plot: pooled effect size of 2.04, 95% CI: 1.21 to 2.87, P < 0.01. These results indicate that electroacupuncture significantly increases EB penetration across the BBB. Most studies have reported that GFAP, MMP-9, and VEGF were upregulated after treatment. P-gp expression decreased as well. Electroacupuncture can open the BBB, and the sparse-dense wave is currently the most effective electroacupuncture frequency for opening the BBB. VEGF plays an important role in opening the BBB. It is also important to regulate the expression of MMP-9 and GFAP and inhibit the expression of P-gp.

Ethanol extract of Cyathulae Radix inhibits osteoclast differentiation and bone loss.

Cyathulae Radix, a traditional Chinese medicine and a common vegetable, boasts a history spanning millennia. It enhances bone density, boosts metabolism, and effectively alleviates osteoporosis-induced pain. Despite its historical use, the molecular mechanisms behind Cyathulae Radix's impact on osteoporosis remain unexplored. In this study, we investigated the effects and mechanisms of Cyathulae Radix ethanol extract (CEE) in inhibiting osteoporosis and osteoclastogenesis. Eight-week-old female mice underwent ovariectomy and were treated with CEE for eight weeks. Micro-computed tomography (micro-CT) assessed histomorphometric parameters, bone tissue staining observed distal femur histomorphology, and three-point bending tests evaluated tibia mechanical properties. Enzyme-linked immunosorbent assay (ELISA) measured serum estradiol (E2), receptor activator for nuclear factor B ligand (RANKL), and osteoprotegerin (OPG) levels. Osteoclastogenesis-related markers were analyzed via Western blotting (WB) and quantitative real-time polymerase chain reaction (qRT-PCR). Additionally, CEE effects on RANKL-induced osteoclast formation and bone resorption were investigated in vitro using tartrate-resistant acid phosphatase (TRAP) staining, qRT-PCR, and WB assay. Compared with the ovariectomy (OVX) group, CEE treatment enhanced trabecular bone density, maximal load-bearing capacity, and various histomorphometric parameters. Serum E2 and OPG levels significantly increased, while Receptor activator of nuclear factor-κB (RANK) decreased in the CEE group. CEE downregulated matrix metallopeptidase 9 (MMP-9), Cathepsin K (CTSK), and TRAP gene and protein expression. In bone marrow macrophages (BMMs), CEE reduced mature osteoclasts, bone resorption pit areas, and MMP-9, CTSK, and TRAP expression during osteoclast differentiation. Compared with DMSO treatment, CEE markedly inhibited RANK, TNF receptor associated factor 6 (TRAF6), Proto-oncogene c-Fos (c-Fos), Nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) expressions, and Extracellular regulated protein kinases (ERK), c-Jun N-terminal kinase (JNK), NF-kappa B-p65 (p65) phosphorylation in osteoclasts. In conclusion, CEE significantly inhibits OVX-induced osteoporosis and RANKL-induced osteoclastogenesis, potentially through modulating the Estrogen Receptor (ER)/RANK/NFATc1 signaling pathway.

ZeXieYin formula alleviates atherosclerosis by inhibiting the MAPK/NF-κB signaling pathway in APOE-/- mice to attenuate vascular inflammation and increase plaque stability.

ETHNOPHARMACOLOGICAL RELEVANCE: Zexieyin formula (ZXYF), a traditional Chinese herbal formula recorded in the Huangdi Neijing to have efficacy in relieving spleen dampness and heat accumulation syndrome, which is also the key pathogenesis of atherosclerosis (AS). The efficacy has demonstrated by our previous studies. However, the intrinsic mechanism of ZXYF for treating vascular inflammation and the effect of inflammatory response on plaque are not known. Currently, plaque stabilization is crucial for the prognosis of AS. AIM OF THE STUDY: Our study mainly focused on the therapeutic effects of ZXYF on high-fat diet (HFD)-induced vascular inflammation and vulnerable plaques (VP) in mice and explored its underlying mechanism. METHODS AND MATERIALS: Male apolipoprotein E knockout (APOE-/-) mice were fed HFD for 8 weeks to establish a VP model. During this period, the mice were also administered ZXYF, while atorvastatin (ATO) was used as a positive control. Aortic plaque area and morphology were detected by oil red staining and HE staining. Aortic plaque collagen content was detected by Masson staining. M1/M2 type macrophages were detected using immunofluorescence (IF). The study analyzed the levels of inflammation-related cytokines (IL-1ß, IL-10, IL-6), MAPK/NF-κB pathway proteins, and NLRP3 inflammasomes (NLRP3, Caspase-1) using Western blot. Additionally, the levels of matrix metalloproteinase (MMP)-2 and MMP-9 and α-smooth muscle actin (α-SMA) in the aorta were analyzed using immunohistochemistry (IHC). The plaque instability index was calculated for each group using the vulnerable plaque formula. RESULTS: In this study, APOE-/- mice were fed high-fat diet for 8 weeks. The results of oil-red and HE staining indicated a significant increase in the aortic plaque area of the mice, which exhibited a typical VP phenotype. ZXYF and ATO significantly improved AS plaques and prevented plaque rupture. HFD exacerbated vascular inflammation, stimulated macrophage conversion to M1-type through the MAPK/NF-κB signaling pathway, and released pro-inflammatory factors such as interleukin (IL)-1ß, IL-1α, and IL-6. These factors activated NLRP3 inflammasome, leading to cellular death. However, ZXYF could reverse this trend and promote the conversion of macrophages to the anti-inflammatory M2 type. The anti-inflammatory effect of ATO was not significant. Moreover, HFD promoted the release of MMP-2 and MMP-9 from macrophages, which degraded plaque collagen, and induced a decrease in plaque SMC content, resulting in a thinning of the plaque fibrous cap. In contrast, ZXYF inhibited the decomposition of plaque collagen and increased the content of plaque smooth muscle cells (SMC) by reducing macrophage secretion of MMPs, thereby stabilizing plaques. Although ATO could reverse the decrease in plaque collagen and SMC content, its effect on MMPs was not significant. Finally, we calculated the vulnerability index to assess the overall risk of the plaque vulnerability phenotype. In line with these findings, ZXYF and ATO were able to effectively reverse the increase in the vulnerability index caused by HFD and lower the risk of adverse cardiovascular events. CONCLUSION: Our results suggested that ZXYF could reduce inflammation and increase plaque stability by inhibiting the MAPK/NF-κB signaling pathway, which provided a theoretical basis for clinical application and subsequent research.

[Trametenolic acid inhibits migration and invasion of hepatocellular carcinoma HepG2.2.15 cells via RhoC/ROCK1 pathway].

This study investigated the effect of trametenolic acid(TA) on the migration and invasion of human hepatocellular carcinoma HepG2.2.15 cells by using Ras homolog gene family member C(RhoC) as the target and probed into the mechanism, aiming to provide a basis for the utilization of TA. The methyl thiazolyl tetrazolium(MTT) assay was employed to examine the proliferation of HepG2.2.15 cells exposed to TA, and scratch and Transwell assays to examine the cell migration and invasion. The pull down assay was employed to determine the impact of TA on RhoC GTPase activity. Western blot was employed to measure the effect of TA on the transport of RhoC from cytoplasm to cell membrane and the expression of RhoC/Rho-associated kinase 1(ROCK1)/myosin light chain(MLC)/matrix metalloprotease 2(MMP2)/MMP9 pathway-related proteins. RhoC was over-expressed by transient transfection of pcDNA3.1-RhoC. The changes of F-actin in the cytoskeleton were detected by Laser confocal microscopy. In addition, the changes of cell migration and invasion, expression of proteins in the RhoC/ROCK1/MLC/MMP2/MMP9 pathway, and RhoC GTPase activity were detected. The subcutaneously transplanted tumor model of BALB/c nude mice and the low-, medium-, and high-dose(40, 80, and 120 mg·kg~(-1), respectively) TA groups were established and sorafenib(20 mg·kg~(-1)) was used as the positive control. The tumor volume and weight in each group were measured, and the expression of related proteins in the tumor tissue was determined by Western blot. The results showed that TA inhibited the proliferation of HepG2.2.15 cells in a concentration-dependent manner, with the IC_(50) of 66.65 and 23.09 µmol·L~(-1) at the time points of 24 and 48 h, respectively. The drug administration groups had small tumors with low mass. The tumor inhibition rates of sorafenib and low-, medium-and high-dose TA were 62.23%, 26.48%, 55.45%, and 62.36%, respectively. TA reduced migrating and invading cells and inhibited RhoC protein expression and RhoC GTPase activity in a concentration-dependent manner, dramatically reducing RhoC and membrane-bound RhoC GTPase. The expression of ROCK1, MLC, p-MLC, MMP2, and MMP9 downstream of RhoC can be significantly inhibited by TA, as confirmed in both in vitro and in vivo experiments. After HepG2.2.15 cells were transfected with pcDNA3.1-RhoC to overexpress RhoC, TA down-regulated the protein levels of RhoC, ROCK1, MLC, p-MLC, MMP2, and MMP9 and decreased the activity of RhoC GTPase, with the inhibition level comparable to that before overexpression. In summary, TA can inhibit the migration and invasion of HepG2.2.15 cells. It can inhibit the RhoC/ROCK1/MLC/MMP2/MMP9 signaling pathway by suppressing RhoC GTPase activity and down-regulating RhoC expression. This study provides a new idea for the development of autophagy modulators targeting HSP90α to block the proliferation and inhibit the invasion and migration of hepatocellular carcinoma cells via multiple targets of active components in traditional Chinese medicines.

Isorhamnetin Downregulates MMP2 and MMP9 to Inhibit Development of Rheumatoid Arthritis through SRC/ERK/CREB Pathway.

OBJECTIVE: To investigate the effect of isorhamnetin on the pathology of rheumatoid arthritis (RA). METHODS: Tumor necrosis factor (TNF)- α -induced fibroblast-like synoviocytes (FLS) was exposed to additional isorhamnetin (10, 20 and 40 µ mol/L). Overexpression vectors for matrix metalloproteinase-2 (MMP2) or MMP9 or SRC were transfected to explore their roles in isorhamnetin-mediated RA-FLS function. RA-FLS viability, migration, and invasion were evaluated. Moreover, a collagen-induced arthritis (CIA) rat model was established. Rats were randomly divided to sham, CIA, low-, medium-, and high-dosage groups using a random number table (n=5 in each group) and administed with normal saline or additional isorhamnetin [2, 10, and 20 mg/(kg·day)] for 4 weeks, respectively. Arthritis index was calculated and synovial tissue inflammation was determined in CIA rats. The levels of MMP2, MMP9, TNF-α, interleukin-6 (IL-6), and IL-1 ß, as well as the phosphorylation levels of SRC, extracellular regulated kinase (ERK), and cyclic adenosine monophosphate response element-binding (CREB), were detected in RA-FLS and synovial tissue. Molecular docking was also used to analyze the binding of isorhamnetin to SRC. RESULTS: In in vitro studies, isorhamnetin inhibited RA-FLS viability, migration and invasion (P<0.05). Isorhamnetin downregulated the levels of MMP2, MMP9, TNF-α, IL-6, and IL-1 ß in RA-FLS (P<0.05). The overexpression of either MMP2 or MMP9 reversed isorhamnetin-inhibited RA-FLS migration and invasion, as well as the levels of TNF-α, IL-6, and IL-1 ß (P<0.05). Furthermore, isorhamnetin bound to SRC and reduced the phosphorylation of SRC, ERK, and CREB (P<0.05). SRC overexpression reversed the inhibitory effect of isorhamnetin on RA-FLS viability, migration and invasion, as well as the negative regulation of MMP2 and MMP9 (P<0.05). In in vivo studies, isorhamnetin decreased arthritis index scores (P<0.05) and alleviated synovial inflammation. Isorhamnetin reduced the levels of MMP2, MMP9, TNF-α, IL-6, and IL-1 ß, as well as the phosphorylation of SRC, ERK, and CREB in synovial tissue (P<0.05). Notably, the inhibitory effect of isorhamnetin was more pronounced at higher concentrations (P<0.05). CONCLUSION: Isorhamnetin exhibited anti-RA effects through modulating SRC/ERK/CREB and MMP2/MMP9 signaling pathways, suggesting that isorhamnetin may be a potential therapeutic agent for RA.

Withania somnifera root extract inhibits MGO-induced skin fibroblast cells dysfunction via ECM-integrin interaction.

ETHNOPHARMACOLOGICAL RELEVANCE: Withania somnifera (L.) Dunal, known as Ashwagandha, has long been used in traditional medicine in Ayurveda, India, a representative adaptogen. The main active constituents of W. somnifera are withanolides, and the root is often used as a medicine with a wide range of pharmacological activities, which can be used to treat insomnia, neurasthenia, diabetes mellitus and skin cancer. AIM OF THE STUDY: Whole-component qualitative and quantitative analyses were performed on W. somnifera. We explored the ameliorative effect of the adaptogen representative plant W. somnifera on the senescence events of MGO-injured fibroblasts and its action mechanism and verified the hypotheses that WS can inhibit the accumulation of AGEs and regulate the dynamic balance among the components of the ECM by modulating the expression of integrin ß1 receptor; as a result, WS maintains cellular behavioural and biological functions in a normal range and retards the aging of skin from the cellular level. MATERIALS AND METHODS: In this study, the components of WS were first qualitatively and quantitatively analysed by HPLC fingerprinting and LC-MS detection. Second, a model of MGO-induced injury of CML-overexpressing fibroblasts was established. ELISA was used to detect CML expression and the synthesis of key extracellular matrix ECM protein components COL1, FN1, LM5 and TNC synthesis; CCK-8 was used to detect cell viability; EDU was used to detect cell proliferation capacity; fluorescence was used to detect cell adhesion capacity; and migration assay were used to detect cell migration capacity; qRT-PCR was used to detect the regulatory pathway TGF-ß1 and MMP-2, MMP-9 in ECMs; immunofluorescence was used to detect the expression of ITGB1; and WB was used to detect the expression of COL1, FN1, LM5, Tnc, TGF-ß1, MMP-2, MMP-9 and ITGB1. RESULTS: In total, 27 active ingredients were analysed from WS, which mainly consisted of withanolide components, such as withaferin A and withanolide A. Based on the model of MGO-induced fibroblast senescence injury, WS significantly inhibited CML synthesis. By up-regulating the expression of integrin ß1, it upregulated the expression of the TGF-ß1 gene, which is closely related to the generation of ECMs, downregulated the expression of the MMP-2 and MMP-9 genes, which are closely related to the degradation of ECMs, maintained the dynamic balance of the four types of ECMs, and improved cell viability as well as proliferation, migration and adhesion abilities. CONCLUSIONS: WS can prevent cellular behavioural dysfunction and delay skin ageing by reducing the accumulation of CML, upregulating the expression of the ITGB1 receptor, maintaining the normal function of ECM-integrin receptor interaction and preventing an imbalance between the production and degradation of protein components of ECMs. The findings reported in this study suggest that WS as a CML inhibitor can modulate ECM-integrin homeostasis and has great potential in the field of aging retardation.

Osmundacetone Inhibits Angiogenesis of Infantile Hemangiomas through Inducing Caspases and Reducing VEGFR2/MMP9.

AIM: This study aims to explore the potential of Osmundacetone (OSC) as a new treatment for infantile hemangiomas (IH), the most common benign tumors in infancy. Currently, propranolol serves as the primary treatment for IH, but its effectiveness is limited, and it poses challenges of drug resistance and side effects. Therefore, there is a pressing need to identify alternative therapies for IH. METHODS: The effects of OSC on the proliferation and apoptosis of HemECs (endothelial cells from hemangiomas) were assessed using CCK-8 assay, colony formation assay, HOCHEST 33342 staining, and flow cytometry. Western blot analysis was performed to investigate OSC's influence on Caspases and angiogenesis-related proteins. Animal models were established using HemECs and BALB/c mice, and histological and immunohistochemical staining were conducted to evaluate the impact of OSC on mouse hemangiomas, VEGFR2, and MMP9 expression. RESULTS: OSC treatment significantly reduced HemECs' viability and colony-forming ability, while promoting apoptosis, as indicated by increased HOCHEST 33342 staining. OSC upregulated the protein expression of Bax, PARP, Caspase9, Caspase3, AIF, Cyto C, FADD, and Caspase8 in HemECs. In animal models, OSC treatment effectively reduced hemangioma size and improved histopathological changes. OSC also suppressed VEGFR2 and MMP9 expression while elevating Caspase3 levels in mouse hemangiomas. CONCLUSION: OSC demonstrated promising results in inhibiting HemECs' proliferation, inducing apoptosis, and ameliorating pathological changes in hemangiomas in mice. Moreover, it influenced the expression of crucial caspases and angiogenesis-related proteins. These findings suggest that OSC holds potential as a novel drug for clinical treatment of IH.

Cotransplantation of NSCs and ethyl stearate promotes synaptic plasticity in PD rats by Drd1/ERK/AP-1 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine views kidney shortage as a significant contributor to the aetiology of Parkinson's disease (PD), a neurodegenerative condition that is closely linked to aging. In clinical, patients with Parkinson's disease are often treated with Testudinis Carapax et Plastrum (Plastrum Testudinis, PT), a traditional Chinese medication that tonifies the kidney. Previous research has demonstrated that ethyl stearate (PubChem CID: 8122), an active component of Plastrum Testudinis Extracted with ethyl acetate (PTE), may encourage neural stem cells (NSCs) development into dopaminergic (DAergic) neurons. However, the effectiveness and mechanism of cotransplantation of ethyl stearate and NSCs in treating PD model rats still require further investigation. AIM OF THE STUDY: PD is a neurodegenerative condition marked by the loss and degradation of dopaminergic neurons in the substantia nigra of the midbrain. Synaptic damage is also a critical pathology in PD. Because of their self-renewal, minimal immunogenicity, and capacity to differentiate into dopaminergic (DAergic) neurons, NSCs are a prospective treatment option for Parkinson's disease cell transplantation therapy. However, encouraging transplanted NSCs to differentiate into dopaminergic neurons and enhancing synaptic plasticity in vivo remains a significant challenge in improving the efficacy of NSCs transplantation for PD. This investigation seeks to examine the efficacy of cotransplantation of NSCs and ethyl stearate in PD model rats and its mechanism related to synaptic plasticity. MATERIALS AND METHODS: On 6-hydroxydopamine-induced PD model rats, we performed NSCs transplantation therapy and cotransplantation therapy involving ethyl stearate and NSCs. Rotating behavior induced by apomorphine (APO) and pole climbing tests were used to evaluate behavioral changes. Using a variety of methods, including Western blotting (WB), immunofluorescence analysis, enzyme-linked immunosorbent assay, and quantitative real-time polymerase chain reaction (qRT-PCR), we examined the function and potential molecular mechanisms of ethyl stearate in combined NSCs transplantation therapy. RESULTS: In the rat PD model, cotransplantation of ethyl stearate with NSCs dramatically reduced motor dysfunction, restored TH protein levels, and boosted dopamine levels in the striatum, according to our findings. Furthermore, the expression levels of SYN1 and PSD95, markers of synaptic plasticity, and BDNF, closely related to synaptic plasticity, were significantly increased. Cotransplantation with ethyl stearate and NSCs also increased the expression levels of Dopamine Receptor D1 (Drd1), an important receptor in the dopamine neural circuit, accompanied by an increase in MMP9 levels, ERK1/2 phosphorylation levels, and c-fos protein levels. CONCLUSIONS: According to the results of our investigation, cotransplantation of ethyl stearate and NSCs significantly improves the condition of PD model rats. We found that cotransplantation of ethyl stearate and NSCs may promote the expression of MMP9 by regulating the Drd1-ERK-AP-1 pathway, thus improving synaptic plasticity after NSCs transplantation. These findings provide new experimental support for the treatment of PD with the kidney tonifying Chinese medicine Plastrum Testudinis and suggest a potential therapeutic strategy for PD based on cotransplantation therapy.

Gilaburu (Viburnum opulus L.) fruit extract has potential therapeutic and prophylactic role in a rat model of acetic acid-induced oxidant colonic damage.

ETHNOPHARMACOLOGICAL RELEVANCE: Ulcerative colitis (UC) which has a global impact on the health care system with its recurrent and incompletely curable characteristics, affects the patients' quality of life. Gilaburu (GB; Viburnum opulus L.) is a fruit with rich polyphenol ingredient which is used ethnobotanically in Türkiye for medicinal purposes (for example, to pass kidney stones, to treat stomach, heart, and liver diseases, hemorrhages, hypertension, ulcers, common cold, tuberculosis, rheumatic and menstrual pain, and diabetes). On the other hand, the effects of GB in the experimental UC model have not been studied. AIM OF THE STUDY: This study aimed to explore the potential antioxidant and anti-inflammatory effects of GB fruit extract in improving acetic acid (AA)-induced UC. MATERIALS AND METHODS: Starting immediately after (AA + GB group) or 1 week before (GB + AA + GB group) the colitis induced by intrarectal AA (5%; v/v) administration, the rats orally received GB (100 mg/kg) once per day for 3 days. The control and AA groups were administered orally saline (1 ml), while the AA + SS group were administered sulfasalazine (SS; 100 mg/kg; orally) as a positive control once per day for 3 days. Distal colonic tissue specimens were obtained for the histological and biochemical [myeloperoxidase (MPO), malondialdehyde (MDA), glutathione (GSH), chemiluminescence (CL), caspase-3, 8-hydroxy-2'-deoxyguanosine (8-OHdG), matrix metalloproteinase (MMP)-9, transforming growth factor (TGF)-ß1, smad-3 and cytokine (tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, IL-8, interferon (IFN)-γ), measurements] evaluations on the 3rd day. RESULTS: Elevated macroscopic and microscopic damage scores, high tissue wet weight values, increased tissue-associated MPO, MDA, CL, caspase-3, 8-OHdG, cytokines (TNF-α, IL-1ß, IL-6, IL-8), MMP-9, TGF-ß1, smad-3 levels, and decreased GSH values of the AA group were all reversed by GB treatments (AA + GB and GB + AA + GB groups) (p < 0.05-0.001). However, sulfasalazine treatment (AA + SS group) did not change the IL-8, 8-OHdG, MMP-9, and TGF-ß1 measurements significantly. CONCLUSIONS: Gilaburu shows both anti-inflammatory and antioxidant effects against AA-induced colonic damage by suppressing neutrophil infiltration, regulating inflammatory mediators, inhibiting reactive species production, lipid peroxidation, and apoptosis, conserving endogenous antioxidant glutathione, and ameliorating oxidative DNA damage. Since the current ulcerative colitis drugs display limited benefits and adverse side effects, potential therapeutic and/or prophylactic role of gilaburu can be evaluated in ulcerative colitis.

Ameliorative effects of androstenediol against acetic acid-induced colitis in male wistar rats via inhibiting TLR4-mediated PI3K/Akt and NF-κB pathways through estrogen receptor ß activation.

5-androstenediol (ADIOL) functions as a selective estrogen receptor ß (ERß) ligand with a protective effect against many diseases. So, we conducted a novel insight into its role in acetic acid (AA)-induced colitis and investigated its effect on TLR4-Mediated PI3K/Akt and NF-κB Pathways and the potential role of ERß as contributing mechanisms. METHODS: Rats were randomized into 5 Groups; Control, Colitis, Colitis + mesalazine (MLZ), Colitis + ADIOL, and Colitis + ADIOL + PHTPP (ER-ß antagonist). The colitis was induced through a rectal enema of acetic acid (AA) on the 8th day. At the end of treatment, colons were collected for macroscopic assessment. Tissue levels of malondialdehyde (MDA), superoxide dismutase (SOD), nuclear factor kappa b (NF-κB), toll-like receptor (TLR4), and phosphorylated Protein kinase B (pAKT) were measured. Besides, Gene expression of interleukin-1beta (IL-1ß), metalloproteases 9 (Mmp9), inositol 3 phosphate kinase (PI3K), Neutrophil gelatinase-associated lipocalin (NGAL), ERß and NLRP6 were assessed. Histopathological and immunohistochemical studies were also investigated. RESULTS: Compared to the untreated AA group, the disease activity index (DAI) and macroscopic assessment indicators significantly decreased with ADIOL injections. Indeed, ADIOL significantly decreased colonic tissue levels of MDA, TLR4, pAKT, and NF-κB immunostainig while increased SOD activity and ß catenin immunostainig. ADIOL mitigated the high genetic expressions of IL1ß, NGAL, MMP9, and PI3K while increased ERß and NLRP6 gene expression. Also, the pathological changes detected in AA groups were markedly ameliorated with ADIOL. The specific ERß antagonist, PHTPP, largely diminished these protective effects of ADIOL. CONCLUSION: ADIOL could be beneficial against AA-induced colitis mostly through activating ERß.

Spent hops extract (Humulus Lupulus L.) attenuates inflammation and angiogenesis of the retina via the nuclear factor-kappaB and protein kinase B/extracellular signal-regulated kinase pathways.

Spent hops extract (SHE) is a plant extract containing compounds with proven anti-inflammatory and anti-angiogenic activities. However, extract may exert synergic effects compared to its individual polyphenol components. Inflammatory diseases of the retina may lead to visual impairment, a reduction of the comfort of life, and even blindness due to the formation of new pathological blood vessels. More effective therapeutic options are being sought. The goal of the present study was to investigate the anti-inflammatory and anti-angiogenic potentials of SHE on human retinal pigment epithelial cells (ARPE-19) stimulated by lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-α). The SHE (250 µg/mL) was found to downregulate the gene expression of interleukin 6 (IL-6) to 33% in LPS-triggered cells; it also reduced both matrix metalloproteinase 2 (MMP-2) and 9 (MMP-9) mRNA expression to 13% and 43% respectively, and their activity to 82% (MMP-2) and 57% (MMP-9), compared to TNF-α-stimulated cells. Also, SHE modulated the TNF-α-induced expression of vascular endothelial growth factor (VEGF) and endothelial growth factor receptor 2 (VEGFR2). It is possible that SHE inhibited retinal inflammation and angiogenesis by suppressing the nuclear factor kappa B (NF-κB), protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) pathways. Our results demonstrate that SHE has anti-inflammatory and anti-angiogenic potential against retinal diseases. This is the first such study to report on the efficacy of SHE on retinal inflammatory diseases.

[Effect of acetylalkannin from Arnebia euchroma on proliferation, migration, and invasion of human melanoma A375 cells].

This study aimed to explore the effect and mechanism of acetylalkannin from Arnebia euchroma on the proliferation, migration, and invasion of human melanoma A375 cells. A375 cells were divided into a blank group, and low-, medium-, and high-dose acetylalkannin groups(0.5, 1.0, and 2.0 µmol·L~(-1)). The MTT assay was used to detect cell proliferation. Cell scratch and transwell migration assays were used to detect cell migration ability, and the transwell invasion assay was used to detect cell invasion ability. Western blot was used to detect the protein expression of migration and invasion-related N-cadherin, vimentin, matrix metalloproteina-se-9(MMP-9), and Wnt/ß-catenin pathway-related Wnt1, Axin2, glycogen synthase kinase-3ß(GSK-3ß), phosphorylated GSK-3ß(p-GSK-3ß), ß-catenin, cell cycle protein D_1(cyclin D_1), and p21. Real-time fluorescence-based quantitative polymerase chain reaction(real-time PCR) was used to detect the mRNA expression of E-cadherin, matrix metalloproteinase-2(MMP-2), N-cadherin, vimentin, ß-catenin, snail-1, and CD44. MTT results showed that the cell inhibition rates in the acetylalkannin groups significantly increased as compared with that in the blank group(P<0.01). The results of cell scratch and transwell assays showed that compared with the blank group, the acetylalkannin groups showed reduced cell migration and invasion, and migration and invasion rates(P<0.05, P<0.01) and weakened horizontal and vertical migration and invasion abilities. Western blot results showed that compared with the blank group, the high-dose acetylalkannin group showed increased expression of Axin2 protein(P<0.05), and decreased expression of N-cadherin, vimentin, MMP-9, Wnt1, p-GSK-3ß, ß-catenin, cyclin D_1, and p21 proteins(P<0.05, P<0.01). The expression of GSK-3ß protein did not change significantly. PCR results showed that the overall trend of MMP-2, N-cadherin, vimentin, ß-catenin, snail-1, and CD44 mRNA expression was down-regulated(P<0.01), and the expression of E-cadherin mRNA increased(P<0.01). Acetylalkannin can inhibit the proliferation, migration, and invasion of human melanoma A375 cells, and its mechanism of action may be related to the regulation of Wnt/ß-catenin signaling pathway.

[Mechanism of Mongolian drug Naru-3 in initiation of neuroinflammation of neuropathic pain from MMP9/IL-1ß signaling pathway].

Neuropathic pain(NP) has similar phenotypes but different sequential neuroinflammatory mechanisms in the pathological process. It is of great significance to inhibit the initiation of neuroinflammation, which has become a new direction of NP treatment and drug development in recent years. Mongolian drug Naru-3 is clinically effective in the treatment of trigeminal neuralgia, sciatica, and other NPs in a short time, but its pharmacodynamic characteristics and mechanism of analgesia are still unclear. In this study, a spinal nerve ligation(SNL) model simulating clinical peripheral nerve injury was established and the efficacy and mechanism of Naru-3 in the treatment of NPs was discussed by means of behavioral detection, side effect evaluation, network analysis, and experimental verification. Pharmacodynamic results showed that Naru-3 increased the basic pain sensitivity threshold(mechanical hyperalgesia and thermal radiation hyperalgesia) in the initiation of SNL in animals and relieved spontaneous pain, however, there was no significant effect on the basic pain sensitivity threshold and motor coordination function of normal animals under physiological and pathological conditions. Meanwhile, the results of primary screening of target tissues showed that Naru-3 inhibited the second phase of injury-induced nociceptive response of formalin test in mice and reduced the expression of inflammatory factors in the spinal cord. Network analysis discovered that Naru-3 had synergy in the treatment of NP, and its mechanism was associated with core targets such as matrix metalloproteinase-9(MMP9) and interleukin-1ß(IL-1ß). The experiment further took the dorsal root ganglion(DRG) and the stage of patho-logical spinal cord as the research objects, focusing on the core targets of inducing microglial neuroinflammation. By means of Western blot, immunofluorescence, agonists, antagonists, behavior, etc., the mechanism of Naru-3 in exerting NP analgesia may be related to the negative regulation of the MMP9/IL-1ß signaling pathway-mediated microglia p38/IL-1ß inflammatory loop in the activation phase. The relevant research enriches the biological connotation of Naru-3 in the treatment of NP and provides references for clinical rational drug use.

Tanshinone â ¡A participates in the treatment of endometriosis by regulating adhesion, invasion, angiogenesis and inhibition of PI3K/Akt/mTOR signaling pathway.

Endometriosis (EMs) is a common gynecological disorder characterized by abnormal growth of the endometrial stroma and glands outside the uterus. Tanshinone IIA, the active component of Chinese medicine Danshen (Salvia miltiorrhiza Bge.), has a number of pharmacological effects such as anti­inflammation and anti­oxidation and serves a significant role in the treatment of EMs. In the present study, network pharmacology and experimental validation were used to elucidate the potential mechanism of tanshinone IIA for treating EMs. Several databases were used to collect information on EMs and tanshinone IIA and cross­targets for tanshinone IIA and EMs finally obtained. A total of 64 common targets were found between tanshinone IIA and EMs. Subsequently, a protein­protein interaction network was constructed, a total of 14 core targets were screened for enrichment analysis. Furthermore, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were performed. The network pharmacology showed that intercellular adhesion molecule (ICAM)­1, MMP­9 and VEGF are the core targets while PI3K/AKT pathway and mTOR pathway are the main signaling pathways through which tanshinone IIA regulates relevant biological processes to intervene in EMs. Finally, the therapeutic role and mechanism of tanshinone IIA on EMs was verified in vivo. Female Sprague­Dawley rats were treated by autologous transplantation to establish EMs. Serum inflammatory factors were detected by enzyme­linked immunosorbent assay (ELISA). The expression of ICAM­1, MMP­9 and VEGF in ectopic endometrial tissues of rats was determined by immunohistochemical. The expression of PI3K/Akt/mTOR pathway­related proteins and genes was detected by western blotting and quantitative PCR. It was found that tanshinone IIA treatment significantly decreased the formation of ectopic endometrium by reducing serum levels of TNF­α and IL­1ß, and down regulating the levels of ICAM­1, MMP­9 and VEGF in ectopic uterine tissue. In addition, tanshinone IIA can also block the activation of PI3K/Akt/mTOR signaling pathway by reducing the expression of related proteins and genes. In conclusion, tanshinone IIA can regulate adhesion, invasion and angiogenesis, thereby improving the pathological morphology of ectopic endometrium and inhibiting the formation of ectopic lesions. The PI3K/Akt/mTOR signaling pathway may play a key role in controlling this process.

Effect of glycosaminoglycans on the structure and composition of articular cartilage and bone of broilers.

This study aimed to assess the influence of glycosaminoglycan (chondroitin and glucosamine sulfates) supplementation in the diet of broilers on the expression of matrix metallopeptidase 9 (MMP-9) and metallopeptidase inhibitor 2 (TIMP-2) genes, the synthesis of proteoglycans, collagen type II and chondrocytes, bone and cartilage macroscopy, bone mineral densitometry, bone breaking strength and mineral profile. A completely randomized design was carried out in a 3 × 3 factorial scheme (3 levels of chondroitin sulfate: 0.00, 0.05, and 0.10%; and 3 levels of glucosamine sulfate: 0.00, 0.15, and 0.30%), totaling 9 treatments. At 21 and 42 d of age, broilers were slaughtered, and tibias and femurs were collected for evaluation. There was an interaction (P < 0.05) of sulfates for the expression of MMP-9 and its inhibitor TIMP-2 in femur articular cartilage, as well as for the number of chondrocytes, collagen type II and proteoglycans in tibia articular cartilage, bone and cartilage macroscopy and mineral profile (P < 0.05), with better results obtained with the inclusion of chondroitin and/or glucosamine sulfates in the feed. In conclusion, chondroitin and glucosamine sulfates can be used in broiler diets in order to favor the development of the structure of the locomotor system (bones and joints), thus preventing locomotion problems.

Fenugreek Seed Extract Regulates Human Umbilical Vein Endothelial Cell Angiogenesis and Proliferation via the PI3K/Akt/Cyclin D1 Pathway.

The significance of angiogenesis in tumour progression has been widely documented. Hence, the identification of anti-angiogenic agents with fewer common side effects would be valuable in cancer therapy. In this study, we evaluated the anti-angiogenic and anti-proliferative effects of a hydro-alcoholic extract of fenugreek seed (HAEF) on human umbilical vein endothelial cells (HUVECs). Human umbilical vein endothelial cells were treated with various concentrations of HAEF and the half-maximal inhibitory concentration (IC50) value was estimated by using the MTT assay. Vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and matrix metalloproteinase enzyme (MMP-2 and MMP-9) gene expression profiles were evaluated by using quantitative RT-PCR (qRT-PCR). Moreover, MMP activities and PI3K, Akt and cyclin D1 protein expression levels were evaluated by gel zymography and Western blotting, respectively. HAEF reduced HUVEC viability, with an IC50 value of 200 µg/ml. The qRT-PCR results demonstrated that treatment with HAEF markedly reduced MMP-2/MMP-9, VEGF and bFGF gene expression, as compared to the control group. We also found that MMP-2/MMP-9 enzyme activity and PI3K/Akt/cyclin D1 protein expression were notably decreased in cells treated with HAEF. Our results suggest that HAEF can potentially inhibit angiogenesis, and also affect cellular proliferation by targeting the PI3K/Akt/cyclin D1 pathway. Thus, fenugreek seed extract merits further investigation as a source of compounds with anti-cancer properties.

Salvianolic acid B promotes the invasion and migration of HO-induced HTR-8/Svneo trophoblast cells by upregulating matrix metalloproteinase-9 the phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B pathway.

OBJECTIVE: To elucidate the regulatory effects of salvianolic acid B (SalB) on trophoblast cells in preeclampsia (PE). METHODS: 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenylte-trazolium bromide (MTT) assays were used to detect the viability of human extravillous trophoblast HTR-8/Svneo cells induced by HO following treatment with different concentrations of SalB. The levels of oxidative stress-related molecules, including superoxide dismutase, glutathione-Px and malondialdehyde were detected using corresponding kits. Cell apoptosis was detected using a Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay, and the expression of apoptosis-related proteins was detected using western blot analysis. In the present study, wound healing and Transwell assays were performed to measure the levels of cell invasion and migration. Western blot analysis was also used to detect the expression levels of epithelial-mesenchymal transition-related proteins. The mechanisms underlying SalB were further investigated using reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and western blot analysis, to determine the expression levels of matrix metallopeptidase 9 (MMP-9) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B (Akt). RESULTS: SalB increased the activity of HTR-8/Svneo cells, inhibited oxidative damage and promoted the invasion and migration of trophoblast cells induced by HO. Furthermore, the expression levels of MMP-9 and members of the PI3K/Akt signaling pathway were significantly decreased. The pathway agonist, LY294002, and MMP-9 inhibitor, GM6001, reversed the effects of SalB on HO-induced cells. CONCLUSIONS: SalB promoted the invasion and migration of HO-induced HTR-8/Svneo trophoblast cells by upregulating MMP-9 the PI3K/Akt signaling pathway.

Betulinaldehyde inhibits vascular remodeling by regulating the microenvironment through the PLCγ1/Ca2+/MMP9 pathway.

BACKGROUND: Vascular remodeling plays a crucial role in the pathogenesis of several cardiovascular diseases (CVDs). Unfortunately, current drug therapies offer limited relief for vascular remodeling. Therefore, the development of innovative therapeutic strategies or drugs that target vascular remodeling is imperative. Betulinaldehyde (BA) is a triterpenoid with diverse biological activities, but its effects on vascular remodeling remain unclear. OBJECTIVE: This study aimed to investigate the role of BA in vascular remodeling and its mechanism of action, providing valuable information for future applications of BA in the treatment of CVDs. METHODS: Network pharmacology was used to predict the key targets of BA in vascular remodeling. The effect of BA on vascular remodeling was assessed in a rat model of balloon injury using hematoxylin and eosin staining, Masson staining, immunohistochemistry staining, and Western blotting. A phenotypic transformation model of vascular smooth muscle cells (VSMCs) was induced by platelet-derived growth factor-BB, and the functional impacts of BA on VSMCs were assessed via CCK-8, EdU, Wound healing, Transwell, and Western blotting. Finally, after manipulation of phospholipase C gamma1 (PLCγ1) expression, Western blotting and Ca2+ levels determination were performed to investigate the potential mechanism of action of BA. RESULTS: The most key target of BA in vascular remodeling, matrix metalloproteinase 9 (MMP9), was identified through network pharmacology screening. Vascular remodeling was alleviated by BA in vivo and its effects were associated with decreased MMP9 expression. In vitro studies indicated that BA inhibited VSMC proliferation, migration, phenotypic transformation, and downregulated MMP9 expression. Additionally, BA decreased PLCγ1 expression and Ca2+ levels in VSMCs. However, after pretreatment with a phospholipase C agonist, BA's effects on down-regulating the expression of PLCγ1 and Ca2+ levels were inhibited, while the expression of MMP9 increased compared to that in the BA treatment group. CONCLUSION: This study demonstrated the critical role of BA in vascular remodeling. These findings revealed a novel mechanism whereby BA mediates its protective effects through MMP9 regulation by inhibiting the PLCγ1/Ca2+/MMP9 signaling pathway. Overall, BA may potentially be developed into a novel medication for CVDs and may serve as a promising therapeutic strategy for improving recovery from CVDs by targeting MMP9.

The ethanol extract of Cyperus exaltatus var. iwasakii exhibits cell cycle dysregulation, ERK1/2/p38 MAPK/AKT phosphorylation, and reduced MMP-9-mediated metastatic capacity in prostate cancer models in vitro and in vivo.

BACKGROUND: Prostate cancer is the second most common cause of cancer death worldwide in men. The development of novel and highly efficient therapeutic strategies is strongly recommended to treat prostate cancer. Cyperaceae are an ecologically and economically important family of plants with several pharmacological effects. However, the biological efficacy of Cyperus exaltatus var. iwasakii (CE) is unknown. PURPOSE: This study aimed to investigate the antitumor effect of the ethanol extract of CE against prostate cancer. METHODS: In vitro antitumor efficacy of CE was explored by the MTT assay, cell counting assay, FACS analysis, immunoblot, wound-healing migration, invasion assay, zymographic assay, and EMSA in prostate cancer cells, DU145 and LNCaP. For in vivo experiments, xenograft mice were injected with LNCaP cells. Histology (H&E and Ki-67) and biochemical enzyme assay were then performed. The toxicity test was evaluated by an acute toxicity assay. The phytochemical constituents of CE were identified by spectrometric and chromatographic analyses. RESULTS: CE exerted a significant antiproliferative effect against prostate cancer cells. CE-induced antiproliferative cells were associated with cell cycle arrest at G0/G1 (cyclin D1/CDK4, cyclin E/CDK2, p21Waf1) in DU145 cells, but G2/M (ATR, CHK1, Cdc2, Cdc25c, p21Waf1, and p53) in LNCaP cells. CE stimulated the phosphorylation of ERK1/2, p38 MAPK, and AKT in DU145 cells, but only p38 MAPK phosphorylation was increased in LNCaP cells. CE treatment suppressed migration and invasion in the two types of prostate cancer cells by inhibiting MMP-9 activity through the regulation of transcription factors, such as AP-1 and NF-κB. In vivo experiments showed a reduction in tumor weight and size following oral CE administration. Histochemistry confirmed that CE inhibited tumor growth in the mouse LNCaP xenograft model. The administration of CE had no adverse effects on body weight, behavioral patterns, blood biochemistry, and histopathology findings of vital organs in mice. Finally, a total of 13 phytochemical constituents were identified and quantified in CE. The most abundant secondary metabolites in CE were astragalin, tricin, and p-coumaric acid. CONCLUSION: Our results demonstrated the antitumor efficacy of CE against prostate cancer. These findings suggest that CE might be a potential candidate for prostate cancer prevention or treatment.