Aucklandiae radix targeted PKM2 to alleviate ulcerative colitis: Insights from the photocrosslinking target fishing technique.

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic and relapsing disease marked by chronic tissue inflammation that alters the integrity and function of the gut, seriously impacting patient health and quality of life. Aucklandiae Radix (AR), known as Mu Xiang in Chinese, is a traditional Chinese medicine documented in Chinese Pharmacopoeia with effects of strengthening the intestine and stopping diarrhea. However, the potential of AR in treating intestinal inflammation and its underlying mechanism have yet to be further elucidated. PURPOSE: The objective of this study was to explore the protective effect and the potential mechanism attributable to AR for treating ulcerative colitis (UC). STUDY DESIGN AND METHODS: A murine model of UC was constructed using dextran sulfate sodium (DSS) to examine the therapeutic potential of AR in alleviating inflammation and modulating the immune response. Advanced techniques such as photocrosslinking target fishing technique, click chemistry, Western blot analysis, real-time quantitative PCR, flow cytometry, immunofluorescence, and immunohistochemistry were employed to unveil the therapeutic mechanism of AR for treating IBD. RESULTS: AR decreased disease activity index (DAI) score to alleviate the course of IBD through ameliorating intestinal barrier function in DSS-induced mice. Furthermore, AR suppressed NF-κB and NLRP3 pathways to reduce the release of pro-inflammatory factors interleukin-6 and 1ß (IL-6 and IL-1ß) and tumor necrosis factor α (TNF-α), allowing to alleviate the inflammatory response. Flow cytometry revealed that AR could reduce the accumulation of intestinal macrophages and neutrophils, maintaining intestinal immune balance by regulating the ratio of Treg to Th17 cells. It was worth noting that pyruvate kinase isozyme type M2 (PKM2) served as a potential target of AR using the photocrosslinking target fishing technology, which was further supported by cellular thermal shift assay (CETSA), drug affinity target stability (DARTS), and PKM2 knockdown experiments. CONCLUSION: AR targeted PKM2 to inhibit NF-κB and NLRP3 pathways, thereby modulating the inflammatory response and immunity to alleviate DSS-induced UC. These findings suggested the potential of AR in the treatment of UC and AR as a candidate for developing PKM2 regulators.

Licochalcone A alleviates laser-induced choroidal neovascularization by inhibiting the endothelial-mesenchymal transition via PI3K/AKT signaling pathway.

Choroidal neovascularization (CNV) is a hallmark of wet age-related macular degeneration, which severely impairs central vision. Studies have shown that endothelial-mesenchymal transition (EndMT) is involved in the pathogenesis of CNV. Licochalcone A (lico A), a flavonoid extracted from the root of licorice, shows the inhibition on EndMT, but it remains unclear whether it can suppress the formation of CNV. The aim of this study is to investigate the effects of lico A on laser-induced CNV, and EndMT process in vitro and vivo. We established the model of CNV with a krypton laser in Brown-Norway rats and then intraperitoneally injected lico A. Our experimental results demonstrated that the leakage of CNV was relieved, and the area of CNV was reduced in lico A-treated rats. Cell migration and tube formation in oxidized low-density lipoprotein (Ox-LDL)-stimulated HUVECs were inhibited by lico A and promoted by PI3K activator 740Y-P. The protein expressions of snai1 and α-SMA were increased, and CD31 and VE-cadherin were decreased in the model rats of CNV, but partially reversed after treatment with lico A. The expression of CD31 was decreased and α-SMA was increased in OX-LDL-treated HUVECs, which was further strengthened by 740Y-P, while the expression of CD31 was up-regulated and α-SMA was down-regulated in lico A treated HUVECs. Our data revealed that EndMT process was alleviated by lico A. Meanwhile, PI3K/AKT signaling pathway was activated in model rat of CNV and Ox-LDL-stimulated HUVECs, which can be suppressed with treatment of lico A. Our experimental results confirmed for the first time that lico A has the potential to alleviate CNV by inhibiting the endothelial-mesenchymal transition via PI3K/AKT signaling pathway.

Preparation of naringenin/ ß-cyclodextrin complex and its more potent alleviative effect on choroidal neovascularization in rats.

Choroidal neovascularization (CNV) is characterized by abnormal blood vessels growing from the choroid. Current remedies for CNV have not shown favorable therapeutic efficacy. It is urgent to identify and develop more safe and potent anti-CNV agents via multiple technologies. We previously showed that the natural product naringenin attenuated CNV. However, naringenin has poor water solubility and low bioavailability. Here, we prepared the ß-cyclodextrin (ß-CD) complex of naringenin and characterized it using infrared spectra and X-ray diffraction analyses. Determination of content and solubility in the complex showed that naringenin accounted for 20.53% in the complex and its solubility was increased by more than 10-fold. Using a laser-induced CNV model in rats we demonstrated that naringenin/ß-CD complex more significantly reduced CNV area than naringenin alone in rats. Furthermore, naringenin and its ß-CD complex significantly inhibited the mRNA and protein expression of VEGF, COX-2, PI3K, p38MAPK, MMP-2, and MMP-9 in retina and choroid tissues. Naringenin/ß-CD complex showed more significant inhibitory effect on VEGF and COX-2 expression than naringenin. These results collectively indicated that naringenin/ß-CD complex could be a promising therapeutic option for CNV and that the beneficial effects could be linked to the anti-inflammatory properties of naringenin.

[Prevention and treatment of age-related macular degeneration by extract of Fructus lycii and its constituents lutein/zeaxanthin: an in vive and in vitro experimental research].

OBJECTIVE: To investigate the in vivo inhibition of extract of Fructus lycii (FL) on the expressions of cathepsin B (Cat B) and cystatin C (Cys C) in high-fat diet and hydroquinone (HQ) induced model mice with age-related macular degeneration (AMD), and to explore the in vitro effects of lutein and zeaxanthin on hydrogen peroxide (H2O2,) induced expressions of matrix metalloproteinase 2 (MMP-2) and tissue inhibitor of metalloproteinase 2 (TIMP-2) on ARPE-19 cells. METHODS: Fifty female 8-month-old C57BL/6 mice were recruited in this research. Ten mice fed with regular diet was taken as the age control group. The rest 40 mice were fed with high fat diet for 6 months, followed by adding HQ (0. 8%) in the drinking water for 3 consecutive months. Then the modeled mice were randomly divided into the model control group (n =10), the high (at the daily dose of 3.75 g/kg), middle (at the daily dose of 2.50 g/kg), and low dose (at the daily dose of 1.25 g/kg) FL groups, 10 in each group. The extract of FL at each dose was respectively administered to mice by gastrogavage for 3 successive months. By the end of the experiment, the mice were killed and their eyeballs were removed. The protein expressions of Cat B and Cys C were observed by immunohistochemical assay. The mRNA and protein expressions of Cat B and Cys C were detected by real-time PCR and Western blot respectively. The drug concentrations of H2O2, lutein, and zeaxanthin were screened and detected using the activity of cell proliferation. The protein expressions of MMP-2 and TIMP-2 were detected using Western blot. RESULTS: Compared with the age control group, the mRNA and protein expressions of Cat B and Cys C were significantly higher in the in vivo model control group (P <0.05, P <0.01). The mRNA expressions of Cat B and Cys C were weaker in the middle and high dose FL groups than in the model control group (P <0. 05, P <0. 01). In in vitro cells, lutein and zeaxanthin could down-regulate the protein expressions of MMP-2 and TIMP-2 in H202 induced ARPE-19 cells (P <0. 05, P <0. 01). CONCLUSIONS: Extract of FL could down-regulate the high protein expressions of Cat B and Cys C in high-fat diet and HQ induced model mice. Lutein and zeaxanthin could down-regulate the protein expressions of MMP-2 and TIMP-2 in H202 induced ARPE-19 cells.

Protection of carbon monoxide inhalation on lipopolysaccharide-induced multiple organ injury in rats.

OBJECTIVE: To observe the protection of carbon monoxide (CO) inhalation on lipopolysaccharide (LPS)-induced rat multiple organ injury. METHODS: Sprague-Dawley rats with multiple organ injury induced by 5 mg/kg LPS intravenous injection were exposed to room air or 2. 5 x 10(-4) (V/V) CO for 3 hours. The lung and intestine tissues of rats were harvested to measure the expression of heme oxygenase-1 (HO-1) with reverse transcription-polymerase chain reaction, the levels of pulmonary tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and intestinal platelet activator factor (PAF), intercellular adhesion molecule-1 (ICAM-1) with enzyme-linked immunosorbent assay, the content of maleic dialdehyde (MDA) and the activity of myeloperoxidase (MPO) with chemical method, the cell apoptosis rate with flow cytometry, and the pathological changes with light microscope. RESULTS: CO inhalation obviously up-regulated the expression of HO-1 in lung (5.43 +/- 0.92) and intestine (6.29 +/- 1.56) in LPS + CO group compared with (3.08 +/- 0.82) and (3.97 +/- 1.16) in LPS group (both P < 0.05). The levels of TNF-alpha, IL-6 in lung and PAF, ICAM-1 in intestine of LPS + CO group were 0.91 +/- 0.25, 0.64 +/- 0.05, 1.19 +/- 0.52, and 1.83 +/- 0.35 pg/mg, respectively, significantly lower than the corresponding values in LPS group (1.48 +/- 0.23, 1.16 +/- 0.26, 1.84 +/- 0.73, and 3.48 +/- 0.36 pg/mg, all P < 0.05). The levels of MDA, MPO, and cell apoptosis rate in lung and intestine of LPS + CO group were 1.02 +/- 0.23 nmol/mg, 1.74 +/- 0.17 nmol/mg, 7.18 +/- 1.62 U/mg, 6.30 +/- 0.97 U/mg, 1.60% +/- 0.34%, and 30. 56% +/- 6.33%, respectively, significantly lower than the corresponding values in LPS group (1.27 +/- 0.33 nmol/mg, 2.75 +/- 0.39 nmol/mg, 8.16 +/- 1.49 U/mg, 7.72 +/- 1.07 U/mg, 3.18% +/- 0.51%, and 41.52% +/- 3.36%, all P < 0.05). In addition, injury of lung and intestine induced by LPS was attenuated at presence of CO inhalation. CONCLUSION: CO inhalation protects rat lung and intestine from LPS-induced injury via anti-oxidantion, anti-inflammation, anti-apoptosis, and up-regulation of HO-1 expression.

Carbon monoxide inhalation protects lung from lipopolysaccharide-induced injury in rat.

Carbon monoxide (CO), a metabolite of heme catalysis by heme oxygenase (HO), has been proposed to have anti-oxidative, anti-inflammatory and anti-apoptotic functions. Lipopolysaccharide (LPS)-induced lung injury (LI) is characterized by oxidative stress, inflammatory reaction and excessive pulmonary cell apoptosis. So we supposed that CO might have protection against LI. LI in rats was induced by intravenous injection of LPS (5 mg/kg). To observe the effect of CO inhalation, LI rats were exposed to 2.5 x 10(-4) (V/V) CO for 3 h. CO-induced changes of lung oxidative stress parameters, inflammatory cytokines, cell apoptosis, HO-1 expression and histology were examined. Results revealed that expressions of the tumor necrosis factor-alpha (TNF-alpha) and interlukin-6 (IL-6), activities of maleic dialdehyde (MDA) and myeloperoxidase (MPO), and cell apoptosis in LPS injection + CO inhalation group were (0.91+/-0.25) pg/mg protein, (0.64+/-0.05) pg/mg protein, (1.02+/-0.23) nmol/mg protein, (7.18+/-1.62) U/mg protein and (1.60+/-0.34)%, respectively, significantly lower than the corresponding values in LI group [(1.48+/-0.23) pg/mg protein, (1.16+/-0.26) pg/mg protein, (1.27+/-0.33) nmol/mg protein, (8.16+/-1.49) U/mg protein and (3.18+/-0.51) %, P<0.05]. Moreover, CO inhalation obviously increased the expressions of HO-1 and interlukin-10 (IL-10) and activity of superoxide dismutase (SOD) [(5.43+/-0.92), (0.26+/-0.07) pg/mg protein and (60.09+/-10.21) U/mg protein in LPS injection + CO inhalation group vs (3.08+/-0.82), (0.15+/-0.03) pg/mg protein and (50.98+/-6.88) U/mg protein in LI group, P<0.05]. LI was attenuated by CO inhalation. Our study demonstrates that inhalation of low concentration of CO protects lung against LPS-induced injury via anti-oxidant, anti-inflammation, anti-apoptosis and up-regulation of HO-1 expression.

[The mechanisms of carbon monoxide inhalation on the apoptosis of pulmonary cells in rats with acute lung injury induced by lipopolysaccharide].

OBJECTIVE: To investigate the effects of carbon monoxide (CO) inhalation on the apoptosis of pulmonary cells in rats with acute lung injury (ALI) induced by lipopolysaccharide (LPS) and to investigate its mechanisms. METHODS: Eighteen male SD rats were randomly divided into three groups. The ALI group received LPS 5 mg/kg intravenously, while the control group received normal saline, and the CO inhalation group received an inhalation of 2.5 x 10(-4) CO (V/V) after ALI induced by LPS 5 mg/kg. The animals were sacrificed by exsanguinations and lung tissue was harvested at 3 h of observation for determination of the heme oxygenase-1 (HO-1) mRNA with semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), the level of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and IL-10 with enzyme-linked immunosorbent assay (ELISA) and the maleic dialdehyde (MDA) concentration, myeloperoxidase (MPO) and superoxide dismutase (SOD) activity with chemical methods. The extent of cell apoptosis was observed by using the flow cytometric method. RESULTS: The change of the levels of HO-1 mRNA, TNF-alpha, IL-6, IL-10, MDA, MPO, SOD and apoptotic cells of the ALI group was significant as compared with the control group [1.002 +/- 0.004, (0.47 +/- 0.06) pg/mg prot, (0.49 +/- 0.12) pg/mg prot, (0.42 +/- 0.08) pg/mg prot, (0.79 +/- 0.14) nmol/mg prot, (6.0 +/- 1.0) U/mg prot, (74 +/- 7) U/mg prot, (0.12 +/- 0.03)%, P < 0.05 or < 0.01], and lung injury was severe. The levels of TNF-alpha, IL-6, MDA, MPO, and apoptotic cells of the CO inhalation group [(0.91 +/- 0.25) pg/mg prot, (0.64 +/- 0.05) pg/mg prot, (1.02 +/- 0.23) nmol/mg prot, (7.2 +/- 1.6) U/mg prot, (1.60 +/- 0.34)%] were decreased, while HO-1, IL-10 and SOD expression [5.433 +/- 0.921, (0.26 +/- 0.07) pg/mg prot, (60 +/- 10) U/mg prot] were increased compared with the ALI group [(1.48 +/- 0.23) pg/mg prot, (1.16 +/- 0.26) pg/mg prot, (1.27 +/- 0.33) nmol/mg prot, (8.2 +/- 1.5) U/mg prot, (3.18 +/- 0.51)%, 3.081 +/- 0.823, (0.15 +/- 0.03) pg/mg prot, (51 +/- 7) U/mg prot, P < 0.05 or < 0.01], and lung injury was attenuated. CONCLUSION: CO inhalation protects lung from injury by regulating oxidative/anti-oxidative and inflammatory/anti-inflammatory disorders, inhibiting excessive cell apoptosis and up-regulating HO-1 expression, which may play an important role in the pathogenesis of LPS-induced ALI.