BACKGROUND: The application of programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) antibodies has greatly improved the clinical outcomes of lung cancer patients. Here, we retrospectively analyzed the efficacy of PD-1 antibody therapy in locally advanced non-surgical or metastatic lung cancer patients, and preliminarily explored the correlation between peripheral blood biomarkers and clinical responses. METHODS: We conducted a single center study that included 61 IIIA-IV lung cancer patients who received PD-1 antibody treatment from March 2020 to December 2021, and collected the medical record data on PD-1 antibody first-line or second-line treatment. The levels of multiple Th1 and Th2 cytokines in the patient's peripheral blood serum, as well as the phenotype of peripheral blood T cells, were detected and analyzed. RESULTS: All the patients completed at least 2 cycles of PD-1 monoclonal antibody treatment. Among them, 42 patients (68.9%) achieved partial response (PR); 7 patients (11.5%) had stable disease (SD); and 12 patients (19.7%) had progressive disease (PD). The levels of peripheral blood interferon gamma (IFN-γ) (P=0.023), tumor necrosis factor α (TNF-α) (P=0.007) and interleukin 5 (IL-5) (P=0.002) before treatment were higher in patients of the disease control rate (DCR) (PR+SD) group than in the PD group. In addition, the decrease in absolute peripheral blood lymphocyte count after PD-1 antibody treatment was associated with disease progression (P=0.023). Moreover, the levels of IL-5 (P=0.0027) and IL-10 (P=0.0208) in the blood serum after immunotherapy were significantly increased compared to baseline. CONCLUSIONS Peripheral blood serum IFN-γ, TNF-α and IL-5 in lung cancer patients have certain roles in predicting the clinical efficacy of anti-PD-1 therapy. The decrease in absolute peripheral blood lymphocyte count in lung cancer patients is related to disease progression, but large-scale prospective studies are needed to further elucidate the value of these biomarkers.
Humans ; Lung Neoplasms/metabolism* ; Interleukin-5/therapeutic use* ; Tumor Necrosis Factor-alpha/therapeutic use* ; Retrospective Studies ; Programmed Cell Death 1 Receptor ; Biomarkers ; Immunotherapy ; Disease Progression ; B7-H1 Antigen

This study explores the effect of total flavonoids of Rhododendra simsii(TFR) on middle cerebral artery occlusion(MCAO)-induced cerebral injury in rats and oxygen-glucose deprivation/reoxygenation(OGD/R) injury in PC12 cells and the underlying mechanism. The MCAO method was used to induce focal ischemic cerebral injury in rats. Male SD rats were randomized into sham group, model group, and TFR group. After MCAO, TFR(60 mg·kg~(-1)) was administered for 3 days. The content of tumor necrosis factor-α(TNF-α), interleukin-1(IL-1), and interleukin-6(IL-6) in serum was detected by enzyme-linked immunosorbent assay(ELISA). The pathological changes of brain tissue and cerebral infarction were observed based on hematoxylin and eosin(HE) staining and 2,3,5-triphenyltetrazolium chloride(TTC) staining. RT-qPCR and Western blot were used to detect the mRNA and protein levels of calcium release-activated calcium channel modulator 1(ORAI1), stromal interaction molecule 1(STIM1), stromal intera-ction molecule 2(STIM2), protein kinase B(PKB), and cysteinyl aspartate specific proteinase 3(caspase-3) in brain tissues. The OGD/R method was employed to induce injury in PC12 cells. Cells were randomized into the normal group, model group, gene silencing group, TFR(30 μg·mL~(-1)) group, and TFR(30 μg·mL~(-1))+gene overexpression plasmid group. Intracellular Ca~(2+) concentration and apoptosis rate of PC12 cells were measured by laser scanning confocal microscopy and flow cytometry. The effect of STIM-ORAI-regulated store-operated calcium entry(SOCE) pathway on TFR was explored based on gene silencing and gene overexpression techniques. The results showed that TFR significantly alleviated the histopathological damage of brains in MCAO rats after 3 days of admini-stration, reduced the contents of TNF-α, IL-1, and IL-6 in the serum, down-regulated the expression of ORAI1, STIM1, STIM2, and caspase-3 genes, and up-regulated the expression of PKB gene in brain tissues of MCAO rats. TFR significantly decreased OGD/R induced Ca~(2+) overload and apoptosis in PC12 cells. However, it induced TFR-like effect by ORAI1, STIM1 and STIM2 genes silencing. However, overexpression of these genes significantly blocked the effect of TFR in reducing Ca~(2+) overload and apoptosis in PC12 cells. In summary, in the early stage of focal cerebral ischemia-reperfusion injury and OGD/R-induced injury in PC12 cells TFR attenuates ischemic brain injury by inhibiting the STIM-ORAI-regulated SOCE pathway and reducing Ca~(2+) overload and inflammatory factor expression, and apoptosis.
Animals ; Male ; Rats ; Apoptosis ; Brain Ischemia/metabolism* ; Caspase 3 ; Interleukin-1 ; Interleukin-6 ; Rats, Sprague-Dawley ; Reperfusion Injury/metabolism* ; Tumor Necrosis Factor-alpha/genetics* ; Flavonoids/pharmacology* ; Rhododendron/chemistry*

The aim of this study was to explore the effects of Huangqin Tang(HQT) on the NLRP3/Caspase-1 signaling pathway in mice with DSS-induced ulcerative colitis(UC). C57BL/6J mice were randomly divided into a blank group, a model group(DSS group), and low-, medium-and high-dose HQT groups(HQT-L, HQT-M, and HQT-H), and western medicine mesalazine group(western medicine group). The UC model was induced in mice. Subsequently, the mice in the HQT-L, HQT-M, HQT-H groups, and the western medicine group were given low-, medium-, high-dose HQT, and mesalazine suspension by gavage, respectively, while those in the blank and DSS groups were given an equal volume of distilled water by gavage. After 10 days of administration, the body weight, DAI scores, and colonic histopathological score of mice in each group were determined. The levels of IL-6, IL-10, IL-1β, and TNF-α in serum were determined by ELISA. The mRNA expression of NLRP3 and Caspase-1 in colon tissues was determined by RT-qPCR. The protein expression of NLRP3 and Caspase-1 in colon tissues was detected by immunohistochemistry. The results showed that compared with the blank group, the DSS group showed decreased body weight of mice and increased DAI scores and intestinal histopathological score. Compared with the DSS group, the HQT groups and the western medicine group showed improved DAI scores, especially in the HQT-M, HQT-H, and the western medicine groups(P<0.05). The intestinal histopathological scores of the HQT groups and the western medicine group significantly decreased, especially in the HQT-M, HQT-H, and the western medicine groups(P<0.05). In addition, compared with the blank group, the DSS group showed elevated expression of NLRP3 and Caspase-1 in colon tissues, increased serum levels of IL-6, IL-1β, and TNF-α, and decreased IL-10 level. Compared with the DSS group, the HQT groups and the western medicine group displayed decreased expression of NLRP3 and Caspase-1 in colon tissues, reduced serum levels of IL-6, IL-1β, and TNF-α, and increased IL-10 level. The improvement was the most significant in the HQT-H group and the western medicine group(P<0.01). In conclusion, HQT may reduce the expression of NLRP3 and Caspase-1 in colon tissues, reduce the se-rum levels of IL-6, IL-1β, and TNF-α, and increase the expression of IL-10 by regulating the classic pyroptosis pathway of NLRP3/Caspase-1, thereby improving the symptoms of intestinal injury and inflammatory infiltration of intestinal mucosa in DSS mice to achieve its therapeutic effect.
Animals ; Mice ; Caspase 1/genetics* ; Colitis, Ulcerative/genetics* ; Colon ; Dextran Sulfate/adverse effects* ; Disease Models, Animal ; Interleukin-10/genetics* ; Interleukin-6/genetics* ; Mesalamine/pharmacology* ; Mice, Inbred C57BL ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Scutellaria baicalensis/chemistry* ; Tumor Necrosis Factor-alpha/metabolism* ; Drugs, Chinese Herbal/pharmacology*

Objective: To investigate the role of Keap1/Nrf2/HO-1 signaling pathway in liver injury induced by neodymium oxide (Nd(2)O(3)) in mice. Methods: In March 2021, forty-eight SPF grade healthy male C57BL/6J mice were randomly divided into control group (0.9% NaCl), low dose group (62.5 mg/ml Nd(2)O(3)), medium dose group (125.0 mg/ml Nd(2)O(3)), and high dose group (250.0 mg/ml Nd(2)O(3)), each group consisted of 12 animals. The infected groups were treated with Nd(2)O(3) suspension by non-exposed tracheal drip and were killed 35 days after dust exposure. The liver weight of each group was weighed and the organ coefficient was calculated. The content of Nd(3+) in liver tissue was detected by inductively coupled plasma mass spectrometry (ICP-MS). HE staining and immunofluorescence was used to observe the changes of inflammation and nuclear entry. The mRNA expression levels of Keap1, Nrf2 and HO-1 in mice liver tissue were detected by qRT-PCR. Western blotting was used to detect the protein expression levels of Keap1 and HO-1. The contents of catalase (CAT), glutathione peroxidase (GSH-Px) and total superoxide dismutase (T-SOD) were detected by colorimetric method. The contents of interleukin 1β (IL-1β), interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) were determined by ELISA. The data was expressed in Mean±SD. Two-independent sample t-test was used for inter-group comparison, and one-way analysis of variance was used for multi-group comparison. Results: Compared with the control group, the liver organ coefficient of mice in medium and high dose groups were increased, and the Nd(3+) accumulation in liver of mice in all dose groups were significantly increased (P<0.05). Pathology showed that the structure of liver lobules in the high dose group was slightly disordered, the liver cells showed balloon-like lesions, the arrangement of liver cell cords was disordered, and the inflammatory exudation was obvious. Compared with the control group, the levels of IL-1β and IL-6 in liver tissue of mice in all dose groups were increased, and the levels of TNF-α in liver tissue of mice in high dose group were increased (P<0.05). Compared with the control group, the mRNA and protein expression levels of Keap1 in high dose group were significantly decreased, while the mRNA expression level of Nrf2, the mRNA and protein expression levels of HO-1 were significantly increased (P<0.05), and Nrf2 was successfully activated into the nucleus. Compared with the control group, the activities of CAT, GSH-Px and T-SOD in high dose group were significantly decreased (P<0.05) . Conclusion: A large amount of Nd(2)O(3) accumulates in the liver of male mice, which may lead to oxidative stress and inflammatory response through activation of Keap1/Nrf2/HO-1 signal pathway. It is suggested that Keap1/Nrf2/HO-1 signal pathway may be one of the mechanisms of Nd(2)O(3) expose-induced liver injury in mice.
Mice ; Male ; Animals ; NF-E2-Related Factor 2/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Kelch-Like ECH-Associated Protein 1/metabolism* ; Interleukin-6/metabolism* ; Mice, Inbred C57BL ; Oxidative Stress ; Liver/metabolism* ; Metals, Rare Earth ; Signal Transduction ; Superoxide Dismutase/metabolism* ; RNA, Messenger/metabolism*

OBJECTIVE: To investigate whether circular RNA circRSF1 regulates radiation-induced inflammatory phenotype of hepatic stellate cells (HSCs) by binding to HuR protein and repressing its function. METHODS: Human HSC cell line LX2 with HuR overexpression or knockdown was exposed to 8 Gy X-ray irradiation, and the changes in the expression of inflammatory factors (IL-1β, IL-6 and TNF-α) were detected by qRT-PCR. The expressions of IκBα and phosphorylation of NF-κB were detected with Western blotting. The binding of circRSF1 to HuR was verified by RNA pull-down assay and RNA-binding protein immunoprecipitation (RIP). The expressions of inflammatory factors, IκBα and the phosphorylation of NF-κB were detected after modifying the interaction between circRSF1 and HuR. RESULTS: Knockdown of HuR significantly up- regulated the expressions of IL-1β, IL-6 and TNF-α, decreased IκBα expression and promoted NF-κB phosphorylation in irradiated LX2 cells, whereas overexpression of HuR produced the opposite changes (P < 0.05). Overexpression or knockdown of circRSF1 did not significantly affect the expression of HuR. RNA pull-down and RIP experiments confirmed the binding between circRSF1 and HuR. Overexpression of circRSF1 significantly reduced the binding of HuR to IκBα and down-regulated the expression of IκBα (P < 0.05). Overexpression of circRSF1 combined with HuR overexpression partially reversed the up-regulation of the inflammatory factors, down-regulated IκBα expression and increased phosphorylation of NFκB in LX2 cells, while the opposite effects were observed in cells with knockdown of both circRSF1 and HuR (P < 0.05). CONCLUSION circRSF1 reduces IκBα expression by binding to HuR to promote the activation of NF-κB pathway, thereby enhancing radiation- induced inflammatory phenotype of HSCs.
Humans ; Hepatic Stellate Cells/radiation effects* ; Interleukin-6 ; NF-kappa B ; NF-KappaB Inhibitor alpha ; Phenotype ; RNA ; RNA, Circular/metabolism* ; Tumor Necrosis Factor-alpha ; ELAV-Like Protein 1/metabolism*

OBJECTIVE: To investigate the role and mechanism of silent information regulator 1 (SIRT1) in regulating nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway in oxidative stress and inflammatory response to sepsis-induced liver injury. METHODS: A total of 24 male Sprague-Dawley (SD) rats were randomly divided into sham operation (Sham) group, cecal ligation and puncture (CLP) group, SIRT1 agonist SRT1720 pretreatment (CLP+SRT1720) group and SIRT1 inhibitor EX527 pretreatment (CLP+EX527) group, with 6 rats in each group. Two hours before operation, SRT1720 (10 mg/kg) or EX527 (10 mg/kg) were intraperitoneally injected into the CLP+SRT1720 group and CLP+EX527 group, respectively. Blood was collected from the abdominal aorta at 24 hours after modeling and the rats were sacrificed for liver tissue. The serum levels of interleukins (IL-6, IL-1β) and tumor necrosis factor-α (TNF-α) were detected by enzyme-linked immunosorbent assay (ELISA). The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected by microplate method. Hematoxylin-eosin (HE) staining was used to observe the pathological injury of rats in each group. The levels of malondialdehyde (MDA), 8-hydroxydeoxyguanosine (8-OHdG), glutathione (GSH) and superoxide dismutase (SOD) in liver tissue were detected by corresponding kits. The mRNA and protein expressions of SIRT1, Nrf2 and HO-1 in liver tissues were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting. RESULTS: Compared with the Sham group, the serum levels of IL-6, IL-1β, TNF-α, ALT and AST in the CLP group were significantly increased; histopathological results showed that liver cords were disordered, hepatocytes were swollen and necrotic, and a large number of inflammatory cells infiltrated; the contents of MDA and 8-OHdG in liver tissue increased, while the contents of GSH and SOD decreased; and the mRNA and protein expressions of SIRT1, Nrf2 and HO-1 in liver tissues were significantly decreased. These results suggest that sepsis rats have liver dysfunction, and the levels of SIRT1, Nrf2, HO-1 and antioxidant protein in liver tissues were decreased, while the levels of oxidative stress and inflammation were increased. Compared with the CLP group, the levels of inflammatory factors and oxidative stress were significantly decreased in the CLP+SRT1720 group, the mRNA and protein expressions of SIRT1, Nrf2 and HO-1 were significantly increased [IL-6 (ng/L): 34.59±4.21 vs. 61.84±3.78, IL-1β (ng/L): 41.37±2.70 vs. 72.06±3.14, TNF-α (ng/L): 76.43±5.23 vs. 130.85±5.30, ALT (U/L): 30.71±3.63 vs. 64.23±4.59, AST (U/L): 94.57±6.08 vs. 145.15±6.86, MDA (μmol/g): 6.11±0.28 vs. 9.23±0.29, 8-OHdG (ng/L): 117.43±10.38 vs. 242.37±11.71, GSH (μmol/g): 11.93±0.88 vs. 7.66±0.47, SOD (kU/g): 121.58±5.05 vs. 83.57±4.84, SIRT1 mRNA (2^-ΔΔCt): 1.20±0.13 vs. 0.46±0.02, Nrf2 mRNA (2^-ΔΔCt): 1.21±0.12 vs. 0.58±0.03, HO-1 mRNA (2^-ΔΔCt): 1.71±0.06 vs. 0.48±0.07, SIRT1 protein (SIRT1/β-actin): 0.89±0.04 vs. 0.58±0.03, Nrf2 protein (Nrf2/β-actin): 0.87±0.08 vs. 0.51±0.09, HO-1 protein (HO-1/β-actin): 0.93±0.14 vs. 0.54±0.12, all P < 0.05], these results indicated that SIRT1 agonist SRT1720 pretreatment could improve liver injury in sepsis rats. However, pretreatment with SIRT1 inhibitor EX527 showed the opposite effect [IL-6 (ng/L): 81.05±6.47 vs. 61.84±3.78, IL-1β (ng/L): 93.89±5.83 vs. 72.06±3.14, TNF-α (ng/L): 177.67±5.12 vs. 130.85±5.30, ALT (U/L): 89.33±9.52 vs. 64.23±4.59, AST (U/L): 179.59±6.44 vs. 145.15±6.86, MDA (μmol/g): 11.39±0.51 vs. 9.23±0.29, 8-OHdG (ng/L): 328.83±11.26 vs. 242.37±11.71, GSH (μmol/g): 5.07±0.34 vs. 7.66±0.47, SOD (kU/g): 59.37±4.28 vs. 83.57±4.84, SIRT1 mRNA (2^-ΔΔCt): 0.34±0.03 vs. 0.46±0.02, Nrf2 mRNA (2^-ΔΔCt): 0.46±0.04 vs. 0.58±0.03, HO-1 mRNA (2^-ΔΔCt): 0.21±0.03 vs. 0.48±0.07, SIRT1 protein (SIRT1/β-actin): 0.47±0.04 vs. 0.58±0.03, Nrf2 protein (Nrf2/β-actin): 0.32±0.07 vs. 0.51±0.09, HO-1 protein (HO-1/β-actin): 0.19±0.09 vs. 0.54±0.12, all P < 0.05]. CONCLUSIONS SIRT1 can inhibit the release of proinflammatory factors and alleviate the oxidative damage of hepatocytes by activating Nrf2/HO-1 signaling pathway, thus playing a protective role against CLP-induced liver injury.
Animals ; Male ; Rats ; Actins/metabolism* ; Chemical and Drug Induced Liver Injury, Chronic ; Heme Oxygenase-1/metabolism* ; Interleukin-6 ; NF-E2-Related Factor 2/metabolism* ; Rats, Sprague-Dawley ; RNA, Messenger ; Sepsis/metabolism* ; Signal Transduction ; Sirtuin 1/metabolism* ; Superoxide Dismutase/metabolism* ; Tumor Necrosis Factor-alpha/metabolism*

Objective To investigate the effect of T cell immunoreceptor with Ig and ITIM domains (TIGIT) on the function of CD8⁺ T cells in the lungs of Plasmodium infected mice. Methods The lungs of the mice infected with Plasmodium yoelii were isolated, weighed and photographed after 12 days' infection. After dissolution, lung lymphocytes were isolated, counted and stained, and then the contents of CD8⁺ and TIGIT⁺CD8⁺ T cells were detected by flow cytometry. The expressions of L selectin (CD62L), CD69, programmed death 1 (PD-1), CD25, and C-X3-C motif chemokine receptor 1 (CX3CR1) on TIGIT⁺CD8⁺ T cells were detected by flow cytometry. After stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin, the ability of TIGIT⁺CD8⁺T cells to secrete interferon γ(IFN-γ), interleukin 21 (IL-21), IL-4, IL-17, and IL-10 was detected. Results The body mass of mice with Plasmodium infection was reduced. The lungs became darker, and the ratio of the lung mass to body mass was significantly increased. Compared with the normal mice, the percentages and absolute quantity of CD8⁺ and TIGIT⁺CD8⁺ T cells in the lungs of the infected mice were significantly increased. The percentage of TIGIT⁺CD8⁺ T cells expressing CD62L in the infected group was significantly lower, while the percentage of the CD69, PD-1, and CX3CR1 cells were significantly higher than that of TIGIT⁺CD8⁺ T cells from the normal mice. The percentages of TIGIT⁺CD8⁺ T cells secreting IL-21, IL-4, IL-17 and IL-10 cells in the infected group were significantly lower. Conclusion The lung lesions from mice with Plasmodium infection are obvious, the numbers of TIGIT⁺CD8⁺ T cells increase, and these cells express a variety of activation-related molecules, but the ability to secrete cytokines is reduced.
Animals ; Mice ; CD8-Positive T-Lymphocytes ; Cytokines/metabolism* ; Interferon-gamma/metabolism* ; Interleukin-10/metabolism* ; Interleukin-17/metabolism* ; Interleukin-4/metabolism* ; Lung/metabolism* ; Malaria/metabolism* ; Plasmodium yoelii/metabolism* ; Programmed Cell Death 1 Receptor/metabolism*

Objective To explore the effect of formononetin on immunity of mice with transplanted H22 hepatocarcinoma. Methods Male C57BL/6 mice were subcutaneously inoculated with H22 cells (4×10⁵) to establish a tumor-bearing mouse model. The mice were treated with formononetin [10 mg/(kg.d)] or [50 mg/(kg.d)] for 28 days, and then the tumor inhibition rate was calculated. Carrilizumab was used as a positive control drug. The expressions of CD8, granzyme B and forkbox transcription factor 3 (FOXP3) in HCC tissues were analyzed by immunohistochemical staining. The mRNA and protein expression of programmed cell death protein 1 (PD-1) and its ligand 1 (PD-L1) in HCC tissues were detected by real-time PCR or Western blot analysis, respectively. The serum levels of interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) were detected by ELISA. Results Formononetin increased the tumor inhibition rate and the positive rate of CD8 and granzyme B staining in tumor-bearing mice. There was no significant difference in the positive rate of FOXP3 staining in tumor tissues of mice in each group. Formononetin decreased the levels of IL-10 and TGF-β in serum of tumor-bearing mice, and decreased the relative expression of mRNA and protein of PD-1 and PD-L1 in tumor tissue of tumor-bearing mice. Conclusion Formononetin can activate CD8⁺ T cells and reduce the release of immunosuppressive factors in regulatory T cells by blocking PD-1/PD-L1 pathway and play an antitumor role.
Male ; Animals ; Mice ; Carcinoma, Hepatocellular/pathology* ; Liver Neoplasms/genetics* ; Interleukin-10/genetics* ; B7-H1 Antigen ; Granzymes/genetics* ; Programmed Cell Death 1 Receptor/metabolism* ; CD8-Positive T-Lymphocytes/metabolism* ; Mice, Inbred C57BL ; Transforming Growth Factor beta/genetics* ; RNA, Messenger/metabolism* ; Forkhead Transcription Factors/genetics* ; Cell Line, Tumor

OBJECTIVE: To investigate whether ferroptosis exists in sepsis induced intestinal injury, and to verify the association between ferroptosis in sepsis induced intestinal injury and intestinal inflammation and barrier function by stimulating and inhibiting the nuclear factor E2-related factor 2/glutathione peroxidase 4 (Nrf2/GPX4) pathway. METHODS: Forty-eight SPF grade male Sprague-Darvley (SD) rats with a body weight of 220-250 g were divided into sham operation group (Sham group), sepsis group (CLP group), sepsis+iron chelating agent deferoxamine (DFO) group (CLP+DFO group) and sepsis+ferroptosis inducer Erastin group (CLP+Erastin group) using a random number table method, with 12 rats in each group. The sepsis model was established by cecal ligation and puncture (CLP). The Sham group was only performed with abdominal opening and closing operations. After modeling, the CLP+DFO group received subcutaneous injection of 20 mg/kg of DFO, the CLP+Erastin group was intraperitoneally injected with 20 mg/kg of Erastin. Each group received subcutaneous injection of 50 mg/kg physiological saline for fluid resuscitation after surgery, and the survival status of the rats was observed 24 hours after surgery. At 24 hours after model establishment, 6 rats in each group were selected. First, live small intestine tissue was taken for observation of mitochondrial morphology in smooth muscle cells under transmission electron microscopy and determination of reactive oxygen species (ROS). Then, blood was collected from the abdominal aorta and euthanized. The remaining 6 rats were sacrificed after completing blood collection from the abdominal aorta, and then small intestine tissue was taken. Western blotting was used to detect the expression of intestinal injury markers such as Claudin-1 and ferroptosis related proteins GPX4 and Nrf2. Observe the pathological changes of small intestine tissue using hematoxylin-eosin (HE) staining and complete Chiu score; Detection of tumor necrosis factor-α (TNF-α), interleukins (IL-1β, IL-6) levels in serum using enzyme-linked immunosorbent assay (ELISA). The levels of serum iron ions (Fe³⁺), malondialdehyde (MDA), and D-lactate dehydrogenase (D-LDH) were measured. RESULTS: (1) Compared with the Sham group, the 24-hour survival rate of rats in the CLP group and CLP+Erastin group significantly decreased (66.7%, 50.0% vs. 100%, both P < 0.05), while there was no significant difference in the CLP+DFO group (83.3% vs. 100%, P = 0.25). (2) Western blotting results showed that compared with the Sham group, the expressions of GPX4 and Claudin-1 in the small intestine tissue of the CLP group, CLP+DFO group, and CLP+Erastin group decreased significantly, while the expression of Nrf2 increased significantly (GPX4/β-actin: 0.56±0.02, 1.03±0.01, 0.32±0.01 vs. 1.57±0.01, Claudin-1/β-actin: 0.60±0.04, 0.96±0.07, 0.41±0.01 vs. 1.40±0.01, Nrf2/β-actin: 0.88±0.02, 0.72±0.01, 1.14±0.01 vs. 0.43±0.02, all P < 0.05). Compared with the CLP group, the expressions of GPX4 and Claudin-1 were significantly increased in the CLP+DFO group, while the expression of Nrf2 was significantly reduced. In the CLP+Erastin group, the expressions of GPX4 and Claudin-1 further decreased, while the expression of Nrf2 further increased (all P < 0.05). (3) Under the light microscope, compared with the Sham group, the CLP group, CLP+DFO group, and CLP+Erastin group showed structural disorder in the small intestinal mucosa and submucosal tissue, significant infiltration of inflammatory cells, and destruction of glandular and villous structures. The Chui score was significantly higher (3.25±0.46, 2.00±0.82, 4.50±0.55 vs. 1.25±0.45, all P < 0.05). (4) Under transmission electron microscopy, compared with the Sham group, the mitochondria in the other three groups of small intestinal smooth muscle cells showed varying degrees of volume reduction, increased membrane density, and reduced or disappeared cristae. The CLP+Erastin group showed the most significant changes, while the CLP+DFO group showed only slight changes in mitochondrial morphology. (5) Compared to the Sham group, the CLP group, CLP+DFO group, and CLP+Erastin group had serum levels of TNF-α, IL-1β, IL-6, MDA, D-LDH, and ROS in small intestine tissue were significantly increased, while the serum Fe³⁺ content was significantly reduced [TNF-α (ng/L): 21.49±1.41, 17.24±1.00, 28.66±2.72 vs. 14.17±1.24; IL-1β (ng/L): 108.40±3.09, 43.19±8.75, 145.70±11.00 vs. 24.50±5.55; IL-6 (ng/L): 112.50±9.76, 45.90±6.52, 151.80±9.38 vs. 12.89±6.11; MDA (μmol/L): 5.61±0.49, 3.89±0.28, 8.56±1.17 vs. 1.86±0.41; D-LDH (kU/L): 39.39±3.22, 25.38±2.34, 53.29±10.53 vs. 10.79±0.52; ROS (fluorescence intensity): 90 712±6 436, 73 278±4 775, 110 913±9 287 vs. 54 318±2 226; Fe³⁺ (μmol/L): 22.19±1.34, 34.05±1.94, 12.99±1.08 vs. 51.74±11.07; all P < 0.05]. Compared with CLP group, the levels of TNF-α, IL-1β, IL-6, MDA, D-LDH and ROS in CLP+Erastin group were further increased, and the content of Fe³⁺ was further decreased, the CLP+DFO group was the opposite (all P < 0.05). CONCLUSIONS Ferroptosis exists in the intestinal injury of septic rats, and stimulating or inhibiting ferroptosis through the Nrf2/GPX4 pathway can effectively intervene in the inflammatory state and intestinal mechanical barrier of the body.
Rats ; Male ; Animals ; NF-E2-Related Factor 2 ; Tumor Necrosis Factor-alpha ; Ferroptosis ; Reactive Oxygen Species ; Actins ; Claudin-1 ; Interleukin-6 ; Sepsis/metabolism* ; Iron

OBJECTIVE: To explore whether the ethanol extract of Herpetospermum caudigerum Wall (EHC), a Xizang medicinal plant traditionally used for treating liver diseases, can improve imiquimod-induced psoriasis-like skin inflammation. METHODS: Immunohistochemistry and immunofluorescence staining were used to determine the effects of topical EHC use in vivo on the skin pathology of imiquimod-induced psoriasis in mice. The protein levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin-17A (IL-17A) in mouse skin samples were examined using immunohistochemical staining. In vitro, IFN-γ-induced HaCaT cells with or without EHC treatment were used to evaluate the expression of keratinocyte-derived intercellular cell adhesion molecule-1 (ICAM-1) and chemokine CXC ligand 9 (CXCL9) using Western blotting and reverse transcription-quantitative polymerase chain reaction. The protein synthesis inhibitor cycloheximide and proteasome inhibitor MG132 were utilized to validate the EHC-mediated mechanism underlying degradation of ICAM-1 and CXCL9. RESULTS: EHC improved inflammation in the imiquimod-induced psoriasis mouse model and reduced the levels of IFN-γ, TNF-α, and IL-17A in psoriatic lesions. Treatment with EHC also suppressed ICAM-1 and CXCL9 in epidermal keratinocytes. Further mechanistic studies revealed that EHC suppressed keratinocyte-derived ICAM-1 and CXCL9 by promoting ubiquitin-proteasome-mediated protein degradation rather than transcriptional repression. Seven primary compounds including ehletianol C, dehydrodiconiferyl alcohol, herpetrione, herpetin, herpetotriol, herpetetrone and herpetetrol were identified from the EHC using ultra-performance liquid chromatography-quadrupole-time of flight-mass spectrometry. CONCLUSION Topical application of EHC ameliorates psoriasis-like skin symptoms and improves the inflammation at the lesion sites. Please cite this article as: Zhong Y, Zhang BW, Li JT, Zeng X, Pei JX, Zhang YM, Yang YX, Li FL, Deng Y, Zhao Q. Ethanol extract of Herpetospermum caudigerum Wall ameliorates psoriasis-like skin inflammation and promotes degradation of keratinocyte-derived ICAM-1 and CXCL9. J Integr Med. 2023; 21(6): 584-592.
Animals ; Mice ; Interleukin-17/metabolism* ; Intercellular Adhesion Molecule-1 ; Imiquimod/adverse effects* ; Tumor Necrosis Factor-alpha/metabolism* ; Ligands ; Psoriasis/chemically induced* ; Keratinocytes ; Inflammation/drug therapy* ; Chemokines/metabolism* ; Interferon-gamma/metabolism* ; Disease Models, Animal ; Mice, Inbred BALB C