Jiedu Xiaozheng Yin Inhibits the Progression of Colitis Associated Colorectal Cancer by Stimulating Macrophage Polarization Towards an M1 Phenotype via the TLR4 Pathway.

To investigate the effect of Jiedu Xiaozheng Yin (JXY) on the polarization of macrophages in colitis-associated colon cancer (CAC). An orthotopic model of CAC was established to monitor changes in the pathological state of mice. Colon length, number of colon tumors were recorded, and indices for liver, spleen, and thymus were calculated. Hematoxylin and eosin (H&E) staining was employed to observe intestinal mucosal injury and tumor formation. Immunohistochemistry (IHC) staining was utilized to investigate the effect of JXY on M1 and M2 polarization of macrophages in the colonic mucosa of CAC mice. For in vitro experiments, RT-qPCR (Reverse Transcription-quantitative PCR) and flow cytometry were used to observe the effect of JXY on various M1-related molecules such as IL-1ß, TNF-α, iNOS, CD80, CD86, and its phagocytic function as well as M2-related molecules including Arg-1, CD206, and IL-10. Subsequently, after antagonizing the TLR4 pathway with antagonists (TAK242, PDTC, KG501, SR11302, LY294002), the expression of IL-6, TNF-α, iNOS, and IL-1ß mRNA were detected by RT-qPCR. In vivo experiments, the results showed that JXY improved the pathological condition of mice in general. And JXY treatment decreased the shortening of colon length and number of tumors as compared to non-treated CAC mice. Additionally, JXY treatment improved the lesions in the colonic tissue and induced a polarization of intestinal mucosal macrophages towards the M1 phenotype, while inhibiting polarization towards the M2 phenotype. In vitro experiments further confirmed that JXY treatment promoted the activation of macrophages towards the M1 phenotype, leading to increased expression of IL-1ß, TNF-α, iNOS, CD80, CD86, as well as enhanced phagocytic function. JXY treatment concomitantly inhibited the expression of M2-phenotype related molecules Arginase-1 (Arg-1), CD206, and IL-10. Furthermore, JXY inhibited M1-related molecules such as IL-6, TNF-α, iNOS, and IL-1ß after antagonizing the TLR4 pathway. Obviously, JXY could exhibit inhibitory effects on the development of colon tumors in mice with CAC by promoting M1 polarization through TLR4-mediated signaling and impeding M2 polarization of macrophages.

Effects of moxibustion on intestinal barrier function and TLR4/NF-κB p65 signaling pathway in obese rats. / è‰¾ç¸å¯¹è‚¥èƒ–å¤§é¼ è‚ é“å±éšœåŠŸèƒ½åŠTLR4/NF-κB p65信号通路的影响.

OBJECTIVES: To observe the effects of moxibustion on intestinal barrier function and Toll-like receptor 4 (TLR4)/nuclear factor-κB p65 (NF-κB p65) signaling pathway in obese rats and explore the mechanism of moxibustion in the intervention of obesity. METHODS: Fifty-five Wistar rats of SPF grade were randomly divided into a normal group (10 rats) and a modeling group (45 rats). In the modeling group, the obesity model was established by feeding high-fat diet. Thirty successfully-modeled rats were randomized into a model group, a moxibustion group, and a placebo-control group, with 10 rats in each one. In the moxibustion group, moxibustion was applied at the site 3 cm to 5 cm far from the surface of "Zhongwan" (CV 12), with the temperature maintained at (46±1 ) ℃. In the placebo-control group, moxibustion was applied at the site 8 cm to 10 cm far from "Zhongwan" (CV 12), with the temperature maintained at (38±1) ℃. The intervention was delivered once daily for 8 weeks in the above two groups. The body mass and food intake of the rats were observed before and after intervention in each group. Using ELISA methool, the levels of serum triacylglycerol (TG), total cholesterol (TC) and lipopolysaccharide (LPS) were detected and the insulin resistance index (HOMA-IR) was calculated. HE staining was used to observe the morphology of colon tissue. The mRNA expression of zonula occludens-1 (ZO-1), Occludin, Claudin-1, TLR4 and NF-κB p65 in the colon tissue was detected by quantitative real-time PCR; and the protein expression of ZO-1, Occludin, Claudin-1, TLR4 and NF-κB p65 was detected by Western blot in the rats of each group. RESULTS: Compared with the normal group, the body mass, food intake, the level of HOMA-IR, and the serum levels of TC, TG and LPS were increased in the rats of the model group (P<0.01); those indexes in the moxibustion group were all reduced when compared with the model group and the placebo-control group respectively (P<0.01, P<0.05). Compared with the normal group, a large number of epithelial cells in the mucosa of colon tissue was damaged, shed, and the inflammatory cells were infiltrated obviously in the interstitium in the rats of the model group. When compared with the model group, in the moxibustion group, the damage of the colon tissue was recovered to various degrees and there were few infiltrated inflammatory cells in the interstitium, while, the epithelial injury of the colon tissue was slightly recovered and the infiltrated inflammatory cells in the interstitium were still seen in the placebo-control group. The mRNA and protein expressions of ZO-1, Occludin and Caudin-1 were decreased in the model group compared with those in the normal group (P<0.01). When compared with the model group and the placebo-control group, the mRNA and protein expressions of these indexes were increased in the moxibustion group (P<0.01, P<0.05). In the model group, the mRNA and protein expressions of TLR4 and NF-κB p65 were increased when compared with those in the normal group (P<0.01), and the mRNA and protein expressions of these indexes were reduced in the moxibustion group when compared with those in the model group and the placebo-control group (P<0.01). CONCLUSIONS: Moxibustion can reduce the body mass and food intake, regulate the blood lipid and improve insulin resistance in the rats of obesity. It may be related to alleviating inflammatory response through improving intestinal barrier function and modulating the intestinal TLR4/NF-κB p65 signaling pathway.

[Essential oil of Cinnamomum camphora mitigates depression-like behavior in mice by regulating Nrf2/HO-1 pathway and inhibiting inflammatory cytokines].

This study aims to investigate the essential oil(EOL) of Cinnamomum camphora regarding its anti-depression effect and mechanism in regulating inflammatory cytokines and the nuclear factor erythroid 2-related factor 2(Nrf2)/heme oxygenase-1(HO-1) pathway. A mouse model of depression was established by intraperitoneal injection of lipopolysaccharide(LPS). Open field, elevated plus maze, and forced swimming tests were carried out to examine mouse behaviors. Western blot and qRT-PCR were employed to determine the expression of proteins and genes in the Nrf2/HO-1 pathway in the hippocampus. The levels of tumor necrosis factor(TNF)-α, interleukin(IL)-6, and IL-1ß in the serum were measured by enzyme-linked immunosorbent assay(ELISA). The changes of apoptosis in mouse brain were detected by Tunel staining. Compared with the blank control group, the model group showed shortened distance travelled and time spent in the central zone and reduced number of entries in the central zone in the open field test. In the elevated plus maze test, the model group showed reduced open arm time(OT%) and open arm entries(OE%). In the force swimming test, the model group showed extended duration of immobility compared with the blank control group. Compared with the model group, the treatment with EOL significantly increased the distance travelled and time spent in the central zone and increased the number of entries in the central zone in the open field test. In addition, EOL significantly increased the OT% and OE% in the elevated plus maze and shor-tened the immobility duration in the forced swimming test. The model group showed lower expression levels of Nrf2 and HO-1 and hig-her levels of TNF-α, IL-6, and IL-1ß than the blank control group. Compared with the model group, the treatment with EOL up-regulated the expression levels of Nrf2 and HO-1 and lowered the levels of TNF-α, IL-6, and IL-1ß. The Tunel staining results showed that the apoptosis rate in the brain tissue of mice decreased significantly after the treatment with EOL. To sum up, EOL can mitigate the depression-like behaviors of mice by up-regulating the expression of Nrf2 and HO-1 and preventing hippocampal inflammatory damage. The findings provide empirical support for the application of EOL and aromatherapy in the treatment of depression.

[Effect and mechanism of Maxing Shigan Decoction on reducing inflammatory response in rats with cough variant asthma via TLR4/MyD88/NF-κB and p38 MAPK signaling pathways].

This study aims to investigate the effect and mechanism of Maxingshigan Decoction on inflammation in the rat model of cough variant asthma(CVA). The SPF-grade SD rats of 6-8 weeks were randomized into normal, model, Montelukast sodium, and low-, medium-, and high-dose Maxing Shigan Decoction groups, with 8 rats in each group. The CVA rat model was induced by ovalbumin(OVA) and aluminum hydroxide sensitization and ovalbumin stimulation. The normal group and model group were administrated with equal volume of normal saline by gavage, and other groups with corresponding drugs by gavage. After the experiment, the number of white blood cells in blood and the levels of interleukin-6(IL-6), interleukin-10(IL-10), and tumor necrosis factor-α(TNF-α) in the serum were measured. The lung tissue was stained with hematoxylin-eosin(HE). Western blot was employed to determine the protein levels of nuclear factor-κB(NF-κB), Toll-like receptor 4(TLR4), myeloid differentiation protein(MyD88), and mitogen-activated protein kinase(MAPK) in the lung tissue. Real-time PCR was carried out to measure the mRNA levels of TLR4 and MyD88 in the lung tissue. Compared with the normal group, the model group showed increased white blood cells, elevated IL-6 and TNF-α levels(P<0.01), lowered IL-10 level(P<0.01), up-regulated protein levels of TLR4, MyD88, p-p65/NF-κB p65, and p-p38 MAPK/p38 MAPK(P<0.01) and mRNA levels of TLR4 and MyD88(P<0.01) in the lung tissue. HE staining showed obvious infiltration of inflammatory cells around the airway and cell disarrangement in the model group. Compared with the model group, Montelukast sodium and high-dose Maxing Shigan Decoction reduced the white blood cells, lowered the IL-6 and TNF-α levels(P<0.01), and elevated the IL-10 level(P<0.01). Moreover, they down-regulated the protein levels of TLR4, MyD88, p-p65/NF-κB p65, p-p38 MAPK/p38 MAPK in the lung tissue(P<0.01) and the mRNA levels of TLR4 and MyD88 in the lung tissue(P<0.01). HE staining showed that Montelukast sodium and high-dose Maxing Shigan Decoction reduced inflammatory cell infiltration and cell disarrangement. The number of white blood cells, the levels of IL-10 and TNF-α in the serum, the protein levels of TLR4, MyD88, p-p65/NF-κB p65, and p-p38 MAPK/p38 MAPK, and the mRNA levels of TLR4 and MyD88 in the lung tissue showed no significant differences between the Montelukast sodium group and high-dose Maxing Shigan Decoction group. Maxing Shigan Decoction can inhibit airway inflammation in CVA rats by inhibiting the activation of TLR4/MyD88/NF-κB and p38 MAPK signaling pathways.

[Buzhong Yiqi Decoction ameliorates spleen deficiency syndrome by regulating gut microbiota].

This study aims to decipher the mechanism of Buzhong Yiqi Decoction(BZYQD) in the treatment of spleen deficiency syndrome via gut microbiota. The mouse models of spleen deficiency syndrome were established by fecal microbiota transplantation(FMT, from patients with spleen deficiency syndrome) and administration of Sennae Folium(SF, 10 g·kg~(-1)), respectively, and treated with BZYQD for 5 d. The pseudosterile mice(administrated with large doses of antibiotics) and the mice transplanted with fecal bacteria from healthy human were taken as the controls. The levels of IgA, interleukin(IL)-2, IL-1ß, interferon(IFN)-γ, tumor necrosis factor-alpha(TNF-α), and 5-hydroxytryptamine(5-HT) in the intestinal tissue of two models were measured by enzyme-linked immunosorbent assay, and the CD8~+/CD3~+ ratio was determined by flow cytometry. The composition and changes of the gut microbiota were determined by 16S rRNA high-throughput sequencing and qPCR. Furthermore, the correlation analysis was performed to study the mediating role of gut microbiota in the treatment. The results showed that BZYQD elevated the IgA level, lowered the IL-1ß, TNF-α, and 5-HT levels, and decreased the CD8~+/CD3~+ ratio in the intestinal tissue of the two models. Moreover, BZYQD had two-way regulatory effects on the levels of IL-2 and IFN-γ. BZYQD inhibited the overgrowth and reduced the richness of gut microbiota in the SF model, and improved the gut microbiota structure in the two models. Algoriphagus, Mycobacterium, and CL500＿29＿marine＿group were the common differential genera in the two models compared with the control. Acinetobacter, Parabacteroides, and Ruminococcus were the differential genera unique to the FMT model, and Sphingorhabdus, Lactobacillus, and Anaeroplasma were the unique differential genera in the SF model. BZYQD was capable of regulating all these genera. The qPCR results showed that BZYQD increased the relative abundance of Akkermansia muciniphila and decreased that of Bacteroides uniformis in the two models. The correlation analysis revealed that the levels of above intestinal cytokines were significantly correlated with characteristic gut microorganisms in different mo-dels. The IL-1ß level had a significantly positive correlation with Acinetobacter and CL500＿29＿marine＿group in the two models, while the different levels of IL-2 and IFN-γ in the two models may be related to its different gut microbiota structures. In conclusion, BZYQD could regulate the disordered gut microbiota structure in different animal models of spleen deficiency syndrome to improve the intestinal immune status, which might be one of the mechanisms of BZYQD in treating spleen deficiency syndrome.

Folic acid supplementation on inflammation and homocysteine in type 2 diabetes mellitus: systematic review and meta-analysis of randomized controlled trials.

BACKGROUND: The beneficial effects of folate have been observed under different conditions, but the available evidence on inflammation and reduction of cardiovascular disease (CVD) in type 2 diabetes mellitus (T2DM) is limited. The study aimed to explore the effects of folate on inflammation and homocysteine amongst individuals with T2DM. METHODS: PubMed, Scopus, and Cochrane Library were used to search for evidence. A random-effect model meta-analysis through Review Manager (version 5.4) and metaHun was performed. Results were reported as standardized mean differences (SMD) and 95% confidence intervals graphically using forest and funnel plots. RESULTS: Data from 9 trials with 426 patients living with T2DM were analyzed. Folic acid supplementation significantly revealed a large effect size on homocysteine levels compared to placebo, SMD = -1.53, 95%CI (-2.14,-0.93), p < 0.05. Additionally, we observed a medium marginal effect size on C-reactive protein (SMD = -0.68, 95%CI (-1.34, -0.01), p = 0.05). However, no significant effect on tumor necrosis factor-α (SMD = -0.86, 95%CI (-2.65, 0.93), p = 0.34), and interleukin-6 (SMD = -0.04, 95%CI (-1.08, 1.01), p = 0.95) was observed. CONCLUSION: Evidence analyzed in this study suggests that folic acid supplementation in T2DM reduces homocysteine and may mitigate CVDs. However, its effect on inflammation is inconclusive.

Kaempferol 3-O-Rutinoside, a Flavone Derived from Tetrastigma hemsleyanum Diels et Gilg, Reduces Body Temperature through Accelerating the Elimination of IL-6 and TNF-α in a Mouse Fever Model.

Fever is a serious condition that can lead to various consequences ranging from prolonged illness to death. Tetrastigma hemsleyanum Diels et Gilg (T. hemsleyanum) has been used for centuries to treat fever, but the specific chemicals responsible for its antipyretic effects are not well understood. This study aimed to isolate and identify the chemicals with antipyretic bioactivity in T. hemsleyanum extracts and to provide an explanation for the use of T. hemsleyanum as a Chinese herbal medicine for fever treatment. Our results demonstrate that kaempferol 3-rutinoside (K3OR) could be successfully isolated and purified from the roots of T. hemsleyanum. Furthermore, K3OR exhibited a significant reduction in rectal temperature in a mouse model of fever. Notably, a 4 µM concentration of K3OR showed more effective antipyretic effects than ibuprofen and acetaminophen. To explore the underlying mechanism, we conducted an RNA sequencing analysis, which revealed that PXN may act as a key regulator in the fever process induced by lipopolysaccharide (LPS). In the mouse model of fever, K3OR significantly promoted the secretion of IL-6 and TNF-α during the early stage in the LPS-treated group. However, during the middle to late stages, K3OR facilitated the elimination of IL-6 and TNF-α in the LPS-treated group. Overall, our study successfully identified the chemicals responsible for the antipyretic bioactivity in T. hemsleyanum extracts, and it answered the question as to why T. hemsleyanum is used as a traditional Chinese herbal medicine for treating fever. These findings contribute to a better understanding of the therapeutic potential of T. hemsleyanum in managing fever, and they provide a basis for further research and development in this field.

Protective Effect of Mesenchymal Stem Cell Active Factor Combined with Alhagi maurorum Extract on Ulcerative Colitis and the Underlying Mechanism.

Ulcerative colitis (UC) is a relapsing and reoccurring inflammatory bowel disease. The treatment effect of Alhagi maurorum and stem cell extracts on UC remains unclear. The aim of the present study was to investigate the protective role of Alhagi maurorum combined with stem cell extract on the intestinal mucosal barrier in an intestinal inflammation mouse model. Sixty mice were randomly divided into a control group, model group, Alhagi group, MSC group, and MSC/Alhagi group. MSC and Alhagi extract were found to reduce the disease activity index (DAI) scores in mice with colitis, alleviate weight loss, improve intestinal inflammation in mice (p < 0.05), preserve the integrity of the ileal wall and increase the number of goblet cells and mucin in colon tissues. Little inflammatory cell infiltration was observed in the Alhagi, MSC, or MSC/Alhagi groups, and the degree of inflammation was significantly alleviated compared with that in the model group. The distribution of PCNA and TNF-alpha in the colonic tissues of the model group was more disperse than that in the normal group (p < 0.05), and the fluorescence intensity was lower. After MSC/Alhagi intervention, PCNA and TNF-alpha were distributed along the cellular membrane in the MSC/Alhagi group (p < 0.05). Compared with that in the normal control group, the intensity was slightly reduced, but it was still stronger than that in the model group. In conclusion, MSC/Alhagi can alleviate inflammatory reactions in mouse colonic tissue, possibly by strengthening the protective effect of the intestinal mucosal barrier.

The Difference of Milk-Derived Extracellular Vesicles from Cow Colostrum and Mature Milk on miRNAs Expression and Protecting Intestinal Epithelial Cells against Lipopolysaccharide Damage.

Intestinal epithelial cells (IECs) play crucial roles in forming an essential barrier, providing host defense against pathogens and regulating nutrients absorption. Milk-derived extracellular vesicles (EVs) within its miRNAs are capable of modulating the recipient cell function. However, the differences between colostrum and mature milk EVs and their biological function in attenuating intestinal epithelial cell injury remain poorly understood. Thus, we carried out the present study to characterize the difference between colostrum and mature milk-derived miRNA of EVs and the effect of colostrum and mature milk EVs on the proliferation, apoptosis, proinflammatory cytokines and intestinal epithelial barrier related genes in IEC-6 induced by LPS. Differential expression of 329 miRNAs was identified between colostrum and mature milk EVs, with 185 miRNAs being downregulated and 144 upregulated. In addition, colostrum contains a greater number and protein concentration of EVs than mature milk. Furthermore, compared to control, EVs derived from colostrum significantly inhibited the expression of apoptosis- (Bax, p53, and caspase-3) and proinflammatory-related genes (TNFα, IL6, and IL1ß). EVs derived from mature milk did not affect expression of apoptosis-related genes (Bax, p53, bcl2, and caspase-3). The EVs derived from mature milk significantly inhibited the expression of proinflammatory-related genes (TNFα and IL6). Western blot analysis also indicated that colostrum and mature milk EVs significantly decreased the apoptosis of IEC-6 cells. The EdU assay results showed that colostrum and mature milk EVs significantly increased the proliferation of IEC-6 cells. The expression of intestinal barrier-related genes (TJP1, CLDN1, OCLN, CDX2, MUC2, and IGF1R) was significantly promoted in IEC-6 cells after colostrum and mature milk EVs addition. Importantly, colostrum and mature milk EVs significantly relieved the LPS-induced inhibition of proliferation and intestinal barrier-related genes expression and attenuated apoptosis and proinflammatory responses induced by LPS in IEC-6 cells. Flow cytometry and Western blot analysis also indicated that colostrum and mature milk EVs significantly affect the apoptosis of IEC-6 cells induced by LPS. The results also indicated that EVs derived from colostrum had better effects on inhibiting the apoptosis- and proinflammatory cytokines-related genes expression. However, the EVs derived from mature milk exhibited beneficial effects on intestinal epithelial barrier protection. The present study will provide a better understanding of the role of EVs derived from colostrum and milk in dairy cows with different responses in the regulation of intestinal cells function, and also presents new evidence for the change of EVs cargos during various stages of lactation.

Garcinia dulcis Extract Alleviates Inflammation in Kidney and Liver of the 2-Kidney-1-Clip Hypertensive rat.

Garcinia dulcis (GD) extract possesses anti-hypertensive property that are poorly characterized. This study aimed to investigate an anti-inflammatory effect of GD flower extract in the 2-kidney-1-clip (2K1C) hypertensive compared to sham operative (SO) rat. Male Wistar rats were divided into 2 groups; the 2K1C group in which a silver clip was placed around renal artery to induce hypertension, and the SO normotensive group. Four weeks later, each group of rats were further divided into 2 subgroups, each subgroup was orally gavaged of either corn oil (vehicle) or 50 mg/kg BW GD extract daily for 4 weeks. The malondialdehyde (MDA) levels in serum, liver, and kidney were determined. Hematoxylin and eosin staining was carried out for histological examination, Periodic acid - Schiff staining for glomerular injury, Masson's trichrome staining for renal fibrosis, and immunohistochemistry for either tumor necrosis factor alpha (TNF-α) or endothelial nitric oxide synthase (eNOS) investigation. Taken together, our results demonstrated that GD flower extract decreased the MDA level in both serum and liver and kidney tissue and suppressed the expression of TNF-α in both liver and kidney of 2K1C hypertensive rats. Mesangial cell proliferation, expansion of mesangial matrix, widening Bowman's capsule space, congestion of glomerular capillary and vessel, cloudy swelling of renal tubular epithelial cell, and renal fibrosis were observed in the kidneys of 2K1C rats. Therefore, we concluded that GD flower extract can alleviate liver and kidney inflammation in which partially attenuates the glomerular injury in the 2K1C rat.

Protective effects of emodin on subchondral bone and articular cartilage in osteoporotic osteoarthritis rats: A preclinical study.

BACKGROUND: Osteoporotic osteoarthritis (OP-OA) is a severe pathological form of OA, urgently requiring precise management strategies and more efficient interventions. Emodin (Emo), an effective ingredient found in the traditional Chinese medicine rhubarb, has been dEmonstrated to promote osteogenesis and inhibit extracellular matrix degradation. In this study, we aimed to investigate the interventional effects of Emo on the subchondral bone and cartilage of the knee joints in OP-OA model rats. METHODS: Thirty-two SD rats were randomly and equally divided into sham, OP-OA, Emo low-dose, and Emo high-dose groups. Micro-CT scanning was conducted to examine the bone microstructure of the rat knee joints. H&E and Safranin O and Fast Green staining (SO&FG) were performed for the pathomorphological evaluation of the rat cartilage tissues. ELISA was used to estimate the rat serum expression levels of inflammatory factors, including interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α). Additionally, the CCK-8 assay was utilized for determining the viability of Emo-treated BMSCs. Western blot and real-time PCR analyses were also employed to measure the bone formation indexes and cartilage synthesis and decomposition indexes. Lastly, the osteogenic and chondrogenic differentiation efficiency of the BMSCs was investigated via Alizarin Red and Alcian Blue staining. RESULTS: Emo intervention alleviated the bone microstructural disruption of the subchondral bone and articular cartilage in the OP-OA rats and up-regulated the expression of bone and cartilage anabolic metabolism indicators, decreased the expression of cartilage catabolism indicators, and diminished the expression of inflammatory factors in the rat serum (P<0.05). Furthermore, Emo reversed the decline in the osteogenic and chondrogenic differentiation ability of the BMSCs (P<0.05). CONCLUSION: Emo intervention mitigates bone loss and cartilage damage in OP-OA rats and promotes the osteogenic and chondrogenic differentiation of BMSCs.

Structure characteristics, protective effect and mechanisms of ethanol-fractional polysaccharides from Dendrobium officinale on acute ethanol-induced gastritis.

Gastritis is a common disease characterized by gastric ulcers and severe bleeding. Excessive daily alcohol consumption can cause acute gastritis, impacting individuals' quality of life. This study aims to explore the protective effects of different ethanol-fractional polysaccharides of Dendrobium officinale (EPDO) on acute alcohol-induced gastric injury in vivo. Results showed that EPDO-80, identified as a ß-glucan, exhibited significant anti-inflammatory properties in pathology. It could reduce the area of gastric mucosal injury and cell infiltration. EPDO-80 had a dose-effect relationship in reducing the levels of malondialdehyde and cyclooxygenase-2 and decreasing the levels of inflammation mediators such as tumor necrosis factor α. More extensively, EPDO-80 could inhibit the activation of the TNFR/IκB/NF-κB signaling pathway, reducing the production of TNF-α mRNA and cell apoptosis in organs. Conversely, EPDO-80 could promote changes in the gut microbiota structure. These findings suggest that EPDO-80 could have great potential in limiting oxidative stress and inflammation mediated by inhibiting the NF-κB signaling pathway, which is highly related to its ß-glucan structure and functions in gut microbiota.

Effects of Reishimmune-S, a Fungal Immunomodulatory Peptide Supplement, on the Quality of Life and Circulating Natural Killer Cell Profiles of Patients With Early Breast Cancer Receiving Adjuvant Endocrine Therapy.

OBJECTIVES: To evaluate the effects of Reishimmune-S, a fungal immunomodulatory peptide, on the quality of life (QoL) and natural killer (NK) cell subpopulations in patients receiving adjuvant endocrine therapy (ET) for breast cancer (BC). METHODS: Patients who received adjuvant ET for stage I-III hormone receptor-positive BC without active infection were enrolled in this prospective pilot study. Reishimmune-S was administered sublingually daily for 6 months. QoL scores, circulating immune cell levels, including lymphocyte/NK cell subpopulations, and plasma levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α were measured at baseline and every 4 weeks. Data were analyzed using linear mixed-effect regression models. RESULTS: Nineteen participants were included in the analyses. One patient with underlying asthma did not complete the study owing to the occurrence of skin rashes 15 days after the initiation of Reishimmune-S. No other adverse events were reported. Reishimmune-S supplementation significantly improved the cognitive function at 3 months and significantly decreased the fatigue and insomnia levels at 3 and 6 months, respectively. There was no significant change in the global health/QoL score between baseline and week 4 of treatment. The proportion of CD19+ lymphocytes was significantly higher at 3 and 6 months, and that of NKG2A+ and NKp30+ NK cells was significantly lower at 6 months than at baseline. In addition, fatigue positively correlated with the proportion of NKp30+ NK cells (ß ± standard error: 24.48 ± 8.75, P = .007 in the mixed-effect model). CONCLUSIONS: Short-term supplementation with Reishimmune-S affected the circulating immune cell composition and exerted positive effects on cognitive function, fatigue, and insomnia in patients with BC undergoing adjuvant ET, providing a potential approach for the management of treatment-related adverse reactions in this patient population.

[Ligusticum cycloprolactam inhibits IL-1ß-induced apoptosis and inflammation of rat chondrocytes via HMGB1/TLR4/NF-κB signaling pathway].

Chondrocytes are unique resident cells in the articular cartilage, and the pathological changes of them can lead to the occurrence of osteoarthritis(OA). Ligusticum cycloprolactam(LIGc) are derivatives of Z-ligustilide(LIG), a pharmacodynamic marker of Angelica sinensis, which has various biological functions such as anti-inflammation and inhibition of cell apoptosis. However, its protective effect on chondrocytes in the case of OA and the underlying mechanism remain unclear. This study conducted in vitro experiments to explore the molecular mechanism of LIGc in protecting chondrocytes from OA. The inflammation model of rat OA chondrocyte model was established by using interleukin-1ß(IL-1ß) to induce. LIGc alone and combined with glycyrrhizic acid(GA), a blocker of the high mobility group box-1 protein(HMGB1)/Toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB) signaling pathway, were used to intervene in the model, and the therapeutic effects were systematically evaluated. The viability of chondrocytes treated with different concentrations of LIGc was measured by the cell counting kit-8(CCK-8), and the optimal LIGc concentration was screened out. Annexin V-FITC/PI apoptosis detection kit was employed to examine the apoptosis of chondrocytes in each group. The enzyme-linked immunosorbent assay(ELISA) was employed to measure the expression of cyclooxygenase-2(COX-2), prostaglandin-2(PGE2), and tumor necrosis factor-alpha(TNF-α) in the supernatant of chondrocytes in each group. Western blot was employed to determine the protein levels of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), caspase-3, HMGB1, TLR4, and NF-κB p65. The mRNA levels of HMGB1, TLR4, NF-κB p65, and myeloid differentiation factor 88(MyD88) in chondrocytes were determined by real-time fluorescent quantitative PCR(RT-qPCR). The safe concentration range of LIGc on chondrocytes was determined by CCK-8, and then the optimal concentration of LIGc for exerting the effect was clarified. Under the intervention of IL-1ß, the rat chondrocyte model of OA was successfully established. The modeled chondrocytes showed increased apoptosis rate, promoted expression of COX-2, PGE2, and TNF-α, up-regulated protein levels of Bax, caspase-3, HMGB1, TLR4, and NF-κB p65 and mRNA levels of HMGB1, TLR4, NF-κB p65, and MyD88, and down-regulated protein level of Bcl-2. However, LIGc reversed the IL-1ß-induced changes of the above factors. Moreover, LIGc combined with GA showed more significant reversal effect than LIGc alone. These fin-dings indicate that LIGc extracted and derived from the traditional Chinese medicine A. sinensis can inhibit the inflammatory response of chondrocytes and reduce the apoptosis of chondrocytes, and this effect may be related to the HMGB1/TLR4/NF-κB signaling pathway. The pharmacological effect of LIGc on protecting chondrocytes has potential value in delaying the progression of OA and improving the clinical symptoms of patients, and deserves further study.

[Mechanism of Yuxuebi Tablets in treating synovial inflammation in rheumatoid arthritis based on transcriptomics].

This study investigated the mechanism of Yuxuebi Tablets(YXB) in the treatment of synovial inflammation in rheumatoid arthritis(RA) based on transcriptomic analysis. Transcriptome sequencing technology was employed to analyze the gene expression profiles of joint tissues from normal rats, collagen-induced arthritis(CIA) rats(an RA model), and YXB-treated rats. Common diffe-rentially expressed genes(DEGs) were subjected to Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analyses. RA synovial inflammation-related target genes were retrieved from the OMIM and GeneCards databases. Venny 2.1 software was used to identify the intersection of YXB target genes and RA synovial inflammation-related target genes, and GO and KEGG enrichment analyses were performed on the intersecting target genes. Immunohistochemistry was used to assess the protein expression levels of the inflammatory factors interleukin-1ß(IL-1ß) and tumor necrosis factor-α(TNF-α) in rat joint tissues. Western blot analysis was employed to measure the expression levels of key proteins in the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt) signaling pathway. A total of 2 058 DEGs were identified by intersecting the genes from the normal group vs model group and the model group vs YXB treatment group. A search in OMIM and GeneCards databases yielded 1 102 RA synovial inflammation-related target genes. After intersecting with the DEGs in the YXB treatment group, 204 intersecting target genes were identified, primarily involving biological processes such as immune response, signal transduction, and inflammatory response; cellular components including plasma membrane, extracellular space, and extracellular region; molecular functions like protein binding, identical protein binding, and receptor binding. These target genes were mainly enriched in signaling pathways such as PI3K/Akt, cytokine-cytokine receptor interaction, and Janus kinase/signal transducer and activator of transcription(JAK/STAT). Western blot results showed that YXB at low, medium, and high doses could significantly inhibit the expression levels of key proteins in the PI3K/Akt signaling pathway in rat joint tissues in a dose-dependent manner. Immunohistochemistry further confirmed these findings, showing that YXB not only suppressed the protein expression levels of the inflammatory factors IL-1ß and TNF-α in the joint synovial tissues of CIA rats, but also inhibited p-Akt protein expression. In conclusion, this study used transcriptomic analysis to uncover the key mechanisms of YXB in inhibiting synovial inflammation and alleviating the progression of RA, with a focus on its role in suppressing the PI3K/Akt signaling pathway.

[Mechanism of aqueous extract of Strychni Semen in improving pain in rats with rheumatoid arthritis based on TLR4/TNF-α/MMP-9 signaling pathway].

This study aims to explore the mechanism of aqueous extract of Strychni Semen(SA) in relieving pain in the rat model of rheumatoid arthritis(RA) via Toll-like receptor 4(TLR4)/tumor necrosis factor-α(TNF-α)/matrix metalloproteinase-9(MMP-9) signaling pathway. Firstly, the main chemical components of Strychni Semen were searched against TCMSP, TCMID, ETCM, and related literature, and the main targets of the chemical components were retrieved from TargetNet and SwissTargetPrediction. The main targets of RA and pain were searched against GeneCards, Online Mendelian Inheritance in Man(OMIM), and Therapeutic Target Database(TTD). Venny 2.1.0 was used to obtain the common targets shared by Strychni Semen, RA, and pain, and STRING and Cytoscape 3.6.1 were used to build the protein-protein interaction network. Then, molecular docking was carried out in AutoDock Vina. Finally, the rat model of type Ⅱ collagen-induced arthritis(CIA) was established. The up-down method and acetone method were employed to examine the mechanical pain threshold and cold pain threshold of rats, and the pain-relieving effect of SA on CIA rats was evaluated comprehensively. Hematoxylin-eosin(HE) staining was employed to evaluate the histopathological changes of joints in CIA rats. The expression levels of key target proteins was determined by immunohistochemistry and Western blot, and the mRNA levels of key targets were determined by real-time fluorescence quantitative polymerase chain reaction(real-time PCR). The results of network prediction showed that Strychni Semen may act on the TLR4/TNF-α/MMP-9 signaling pathway to exert the pain-relieving effect. The results of molecular docking showed that brucine, the main active component of SA, had strong binding ability to TLR4, TNF-α, and MMP-9. The results of animal experiments showed that SA improved the mechanical and cold pain sensitivity(P<0.05, P<0.01) and reduced the joint histopathological score of CIA rats(P<0.01). In addition, medium and high doses of SA down-regulated the protein and mRNA levels of TNF-α, TLR4, and MMP-9(P<0.05,P<0.01). In conclusion, SA alleviated the mechanical pain sensitivity, cold pain sensitivity, and joint histopathological changes in CIA rats by inhibiting the over activation of TLR4/TNF-α/MMP-9 signaling pathway.

[Mechanism of Fushen Granules treat peritoneal dialysis-related peritonitis by regulating TLR4/NF-κB pathway:based on network pharmacology and animal experiments].

Network pharmacology was employed to probe into the mechanism of Fushen Granules in treating peritoneal dialysis-rela-ted peritonitis(PDRP) in rats. The main active components of Fushen Granules were searched against the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, and their targets were predicted. PDRP-related targets were retrieved from DisGeNET and other databases. The common targets shared by the drug and the disease were identified by the online tool, and protein-protein interaction(PPI) network of the common targets. The obtained 276 common targets were imported into DAVID for GO function enrichment and KEGG pathway enrichment. The main signaling pathway of Fushen Granules in the treatment of PDRP was predicted as Toll-like receptor 4(TLR4)/nuclear factor(NF)-κB. The rat model of uremia was induced by 5/6 nephrectomy. From two weeks after operation, the rat model of peritoneal dialysis(PD) was established by intraperitoneal injection of 20 mL dialysate with 1.25% glucose every day. The sham operation group and model group received 2 mL normal saline by gavage every day. The rats in Fushen Gra-nules groups were administrated with 2 mL solutions of low-(0.54 g·kg~(-1)), medium-(1.08 g·kg~(-1)) and high-dose(2.16 g·kg~(-1)) Fushen Granules every day. The bifico group received 2 mL(113.4 mg·kg~(-1)) of bifico solution every day. At the end of the 8th week, the levels of serum creatinine(Scr) and blood urea nitrogen(BUN) in each group were measured. The serum levels of hypersensitive C reactive protein(hs-CRP), tumor necrosis factor(TNF)-α, and interleukin(IL)-6 were measured, and the pathological changes in the colon tissue were observed by hematoxylin-eosin(HE) staining. The serum levels of lipopolysaccharide(LPS) and lipopolysaccharide-binding protein(LBP) of rats were measured, and the expression levels of LBP, TLR4, NF-κB p65, inhibitor of κB kinase α(IκBα), TNF-α, and IL-1ß in the colon tissue were determined. Compared with sham operation group, the model group had abnormal structure of all layers of colon tissue, sparse and shorter intestinal villi, visible edema in mucosal layer, wider gap, obvious local inflammatory cell infiltration, significantly decreased body weight(P<0.01), and significantly increased kidney function index(Scr, BUN) content(P<0.01). Serum levels of inflammatory cytokines(hs-CRP, TNF-α, IL-6), LPS and LBP were significantly increased(P<0.01), protein expressions of LBP, TLR4, NF-κB p65, TNF-α and IL-1ß were significantly increased(P<0.01), and protein expressions of IκBα were significantly decreased(P<0.01). Compared with model group, intestinal villi damage in colonic tissue of rats in low-, medium-and high-dose Fushen Granules groups and bifico group were alleviated to different degrees, edema in submucosa was alleviated, space was narrowed, and inflammatory cell infiltration in lamina propria was reduced. The contents of renal function index(Scr, BUN) and serum inflammatory factors(hs-CRP, TNF-α, IL-6) were significantly decreased(P<0.05 or P<0.01) in medium-and high-dose Fushen Granules groups and bifico group(P<0.05 or P<0.01). Serum LPS and LBP contents in Fushen Granules group and bifico group were significantly decreased(P<0.01), protein expressions of LBP, TLR4, NF-κB p65, TNF-α and IL-1ß in Fushen Granules group were significantly decreased(P<0.05 or P<0.01), and protein expressions of IκBα were significantly increased(P<0.01). The expression of LBP protein in bifico group was significantly decreased(P<0.01). The results suggest that Fushen Granules can protect the residual renal function of PD rats, reduce the inflammatory response, and protect the colon tissue. Based on network pharmacology, TLR4/NF-κB pathway may be the main signaling pathway of Fushen granule in the treatment of PDRP. The results showed that Fushen Granules could improve intestinal inflammation and protect intestinal barrier to prevent PDRP by regulating the expression of key factors in TLR4/NF-κB pathway in colon of PD rats.

[Mechanism of Shenling Baizhu Powder on treatment of alcoholic liver disease by regulating TLR4/NLRP3 pathway].

This study aims to investigate the regulatory effects of Shenling Baizhu Powder(SBP) on cellular autophagy in alcoholic liver disease(ALD) and its intervention effect through the TLR4/NLRP3 pathway. A rat model of chronic ALD was established by gavage of spirits. An ALD cell model was established by stimulating BRL3A cells with alcohol. High-performance liquid chromatography(HPLC) was utilized for the compositional analysis of SBP. Liver tissue from ALD rats underwent hematoxylin-eosin(HE) and oil red O staining for pathological evaluation. Enzyme-linked immunosorbent assay(ELISA) was applied to quantify lipopolysaccharides(LPS), tumor necrosis factor-alpha(TNF-α), interleukin-1 beta(IL-1ß), and interleukin-18(IL-18) levels. Quantitative reverse transcription polymerase chain reaction(qRT-PCR) was conducted to evaluate the mRNA expression of myeloid differentiation factor 88(MyD88) and Toll-like receptor 4(TLR4). The effect of different drugs on BRL3A cell proliferation activity was assessed through CCK-8 analysis. Western blot analysis was performed to examine the protein expression of NOD-like receptor pyrin domain-containing 3(NLRP3), nuclear factor-kappa B P65(NF-κB P65), phosphorylated nuclear factor-kappa B P65(p-P65), caspase-1, P62, Beclin1, and microtubule-associated protein 1 light chain 3(LC3Ⅱ). The results showed that SBP effectively ameliorated hepatic lipid accumulation, reduced liver function, mitigated hepatic tissue inflammation, and reduced levels of LPS, TNF-α, IL-1ß, and IL-18. Moreover, SBP exhibited the capacity to modulate hepatic autophagy induced by prolonged alcohol intake through the TLR4/NLRP3 signaling pathway. This modulation resulted in decreased expression of LC3Ⅱ and Beclin1, an elevation in P62 expression, and the promotion of autolysosome formation. These research findings imply that SBP can substantially enhance liver function and mitigate lipid irregularities in the context of chronic ALD. It achieves this by regulating excessive autophagic responses caused by prolonged spirit consumption, primarily through the inhibition of the TLR4/NLRP3 pathway.

[Intervention effect of Erchen Decoction on methionine and choline deficient diet-induced non-alcoholic steatohepatitis in mice].

This study investigated the effect of Erchen Decoction(ECD) on the prevention of non-alcoholic steatohepatitis(NASH) in mice and explored its possible mechanism, so as to provide scientific data for the clinical application of ECD in the prevention of NASH. C57BL/6 male mice were randomly divided into normal group(methionine and choline supplement, MCS), model group(methionine and choline deficient, MCD), low-dose ECD group(ECD＿L, 6 g·kg~(-1)), medium-dose ECD group(ECD＿M, 12 g·kg~(-1)), and high-dose ECD group(ECD＿H, 24 g·kg~(-1)), with eight mice in each group. The MCS group was fed with an MCS diet, and the other groups were fed with an MCD diet. The mice in each group were given corresponding diets, but the drug intervention group was given low-, medium-, and high-dose ECD(10 mL·kg~(-1)·d~(-1)) by intragastric administration for six weeks on the basis of MCD diet feeding, and the mice could eat and drink freely during the whole experiment. At the end of the experiment, mice were fasted overnight(12 h) and were anesthetized with 20% urethane. Thereafter, the blood and liver tissue were collected. The serum was used to detect the levels of alanine aminotransferase(ALT), aspartate aminotransaminase(AST), interleukin-1ß(IL-1ß), interleukin-6(IL-6), interleukin-10(IL-10), and tumor necrosis factor-α(TNF-α). Liver tissue was processed by hematoxylin-eosin(HE) staining and used for hepatic histological analysis and detection of the expression levels of genes and proteins related to nuclear factor erythroid 2-related factor 2/glutathione peroxidase 4(Nrf2/GPX4) pathway by real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR) and Western blot analysis, respectively. The results showed that compared with the MCS group, the MCD group showed higher serum ALT and AST levels; the HE staining exhibited fat vacuoles and obvious inflammatory cell infiltration in liver tissue; serum IL-1ß, IL-6, and TNF-α levels were significantly increased, and the serum IL-10 level was significantly decreased. The mRNA expressions of fatty acid synthase(FASN), monocyte chemoattractant protein-1(MCP-1), and IL-1ß in liver tissue were significantly up-regulated, while those of GPX4, Nrf2, and NAD(P)H:quinine oxidoreductase(NQO1) were significantly down-regulated. Compared with the MCD group, the serum ALT and AST levels of ECD＿M and ECD＿H groups were significantly decreased, and the AST level in the ECD＿L group was significantly decreased. The number of fat vacuoles and the degree of inflammatory cell infiltration in liver tissue were improved; serum IL-1ß, IL-6, and TNF-α levels were significantly decreased, but the serum IL-10 level was significantly increased only in the ECD＿H group. The mRNA expressions of FASN, MCP-1, and IL-1ß in liver tissue were significantly down-regulated, and those of GPX4 and NQO1 were significantly up-regulated. The mRNA expressions of Nrf2 in ECD＿M and ECD＿H groups were significantly up-regulated. Western blot results showed that compared with the MCD group, the protein expression levels of Nrf2 and GPX4 in each group were significantly increased after ECD administration, and the protein expression level of FASN was significantly decreased; the protein expression of NQO1 was increased in ECD＿M and ECD＿H groups. In summary, ECD can reduce hepatic lipid accumulation, oxidative stress, liver inflammation, and liver injury in NASH mice, which may be related to the activation of the Nrf2/GPX4 pathway.

Green Synthesis of Zinc Oxide Nanoparticles from Althaea officinalis Flower Extract Coated with Chitosan for Potential Healing Effects on Diabetic Wounds by Inhibiting TNF-α and IL-6/IL-1ß Signaling Pathways.

Background: Diabetes Mellitus is a multisystem chronic pandemic, wound inflammation, and healing are still major issues for diabetic patients who may suffer from ulcers, gangrene, and other wounds from uncontrolled chronic hyperglycemia. Marshmallows or Althaea officinalis (A.O.) contain bioactive compounds such as flavonoids and phenolics that support wound healing via antioxidant, anti-inflammatory, and antibacterial properties. Our study aimed to develop a combination of eco-friendly formulations of green synthesis of ZnO-NPs by Althaea officinalis extract and further incorporate them into 2% chitosan (CS) gel. Method and Results: First, develop eco-friendly green Zinc Oxide Nanoparticles (ZnO-NPs) and incorporate them into a 2% chitosan (CS) gel. In-vitro study performed by UV-visible spectrum analysis showed a sharp peak at 390 nm, and Energy-dispersive X-ray (EDX) spectrometry showed a peak of zinc and oxygen. Besides, Fourier transforms infrared (FTIR) was used to qualitatively validate biosynthesized ZnO-NPs, and transmission electron microscope (TEM) showed spherical nanoparticles with mean sizes of 76 nm and Zeta potential +30mV. The antibacterial potential of A.O.-ZnO-NPs-Cs was examined by the diffusion agar method against Gram-positive (Staphylococcus aureus and Bacillus subtilis) and Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa). Based on the zone of inhibition and minimal inhibitory indices (MIC). In addition, an in-silico study investigated the binding affinity of A.O. major components to the expected biological targets that may aid wound healing. Althaea Officinalis, A.O-ZnO-NPs group showed reduced downregulation of IL-6, IL-1ß, and TNF-α and increased IL-10 levels compared to the control group signaling pathway expression levels confirming the improved anti-inflammatory effect of the self-assembly method. In-vivo study and histopathological analysis revealed the superiority of the nanoparticles in reducing signs of inflammation and wound incision in rat models. Conclusion: These biocompatible green zinc oxide nanoparticles, by using Althaea Officinalis chitosan gel ensure an excellent new therapeutic approach for quickening diabetic wound healing.