Higenamine inhibits acute and chronic inflammatory pain through modulation of TRPV4 channels.

Pain is the cardinal symptom of many debilitating diseases and results in heavy health and economic burdens worldwide. Asarum (Asarum sieboldii Miq.) is a commonly used analgesic in Chinese medicine. However, the analgesic components and mechanisms of asarum in acute and chronic pain mice model remain unknown. In this study, we first generated asarum water extract and confirmed strong analgesic properties in mice in both the acute thermal and mechanical pain models, as well as in the complete Freund's adjuvant (CFA) induced chronic inflammatory pain model. Second, we identified higenamine as a major component of asarum and found that higenamine significantly inhibited thermal and mechanical induced acute pain and CFA induced chronic inflammatory pain. Then, using Trpv4-/- mice, we found that TRPV4 is necessary for CFA induced thermal and mechanical allodynia, and demonstrated that higenamine analgesia in the CFA model is partly through TRPV4 channel inhibition. Finally, we found that GSK1016790A, a TRPV4 agonist, induced calcium response was significantly inhibited by higenamine in both cultured DRG neurons and TRPV4 transfected HEK293 cells. Consistent with calcium imaging results, higenamine pretreatment also dose-dependently inhibited GSK1016790A induced acute pain. Taken together, our behavior and calcium imaging results demonstrate that the asarum component higenamine inhibits acute and chronic inflammatory pain by modulation of TRPV4 channels.

Mitochondrial proteomic analysis of ecdysterone protection against oxidative damage in human lens epithelial cells.

AIM: To investigate the protective effects of the natural medicinal monomer ecdysterone (ECR) with estrogenic activity against oxidative damage in human lens epithelial cells B3 (HLE-B3) caused by hydrogen peroxide 21(H2O2) and to pursue the possible mitochondrial proteomic regularity of the protective effects. METHODS: HLE-B3 cells were treated with H2O2 (300µmol/L), ß-estuarial (E2; 10(-8)mol/L) and H2O2, ECR (10(-6)mol/L) and H2O2, or left untreated. Altered expression of all mitochondrial proteins was analyzed by protein array and surface-enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS). The mass/charge (M/Z) ratios of each peak were tested by the Kruskal-Wallis rank sum test, and the protein peak value of the M/Z ratio for each treatment by pair comparison was analyzed with the Nemenyi test. RESULTS: H2O2 up-regulated expression of two protein spots (with M/Z of 6 532 and 6 809). When E2 mitigated the oxidative damage, the expression of one protein spot (M/Z 6 532) was down-regulated. In contrast, ECR down-regulated both of protein spots (M/Z 6 532 and 6 809). CONCLUSION: ECR could effectively inhibite H2O2 induced oxidative damage in HLE-B3 cells. The protein spot at M/Z of 6 532 might be the target spot of ECR against oxidative damage induced by H2O2.

Mitochondrial proteomic analysis of isopsoralen protection against oxidative damage in human lens epithelial cells.

OBJECTIVE: To investigate the protective effects of the natural medicinal monomer isopsoralen (ISR) with estrogenic activity against oxidative damage in human lens epithelial cells B3 (HLE-B3) caused by hydrogen peroxide (H(2)O(2)) and to pursue the possible mitochondrial proteomic regularity of the protective effects. METHODS: HLE-B3 cells were treated with H(2)O(2) (300 µ mol/L), ß-estradiol (E(2): 10(-8) mol/L) and H(2)O(2), ISR (10(-5) mol/L) and H(2)O(2), or left untreated. Altered expressions of all mitochondrial proteins were analyzed by protein array and surfaceenhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS). The mass/charge (m/z) ratios of each peak were tested by the Kruskal-Wallis rank sum test, and the protein peak value of the m/z ratio for each treatment by pair comparison was analyzed with the Nemenyi test. RESULTS: H(2)O(2) up-regulated the expressions of two protein spots (with m/z of 6532 and 6809). E(2) mitigated the oxidative damage, and the expression of one protein spot (m/z 6532) was down-regulated. In contrast, ISR down-regulated both of protein spots (m/z 6532 and 6809). CONCLUSIONS: ISR could effectively inhibit H(2)O(2)-induced oxidative damage in HLE-B3 cells. The protein spot at m/z of 6532 might be the target spot of ISR against oxidative damage induced by H(2)O(2).

Curcumin inhibits proliferation of human lens epithelial cells: a proteomic analysis.

OBJECTIVE: The incidence of after-cataracts [also known as posterior capsular opacification (PCO)] is between 30% and 50% three years following cataract surgery. Suppressing the proliferation of lens epithelial cells (LECs) is a primary goal in preventing PCO. Here, we investigated the proteomic regulation of the inhibitory effects of curcumin (Cur) on the proliferation of human lens epithelial B3 (HLE-B3) cells. METHODS: Recombinant human basic fibroblast growth factor (rhbFGF) was used to induce proliferation of HLE-B3 cells, which were incubated with 20 mg/L Cur in a CO(2) incubator for 24 h. RESULTS: We found that the absorbance (A) value of rhbFGF group was significantly higher than the A value of the control group. Furthermore, the A value of the Cur group was significantly lower compared to the rhbFGF group, with an inhibition of 53.7%. Five different protein spots were obtained from proliferative HLE-B3 cells induced by rhbFGF. Eight different protein spots were obtained in HLE-B3 cells incubated with Cur. There were the common variational protein spots at mass/charge (m/z) ratios of 8093 and 13767 between rhbFGF group and control group as well as between the Cur group and rhbFGF group. CONCLUSIONS: These results show that Cur effectively inhibited HLE-B3 cell proliferation induced by rhbFGF. The protein spots at m/z of 8093 and 13767 may be the targets of Cur-induced inhibition of HLE-B3 cell proliferation. Cur may be a reliable and effective drug for prevention and treatment of polymerase chain reaction (PCR).

[Effects of ecdysterone on the expression of NF-kappaB p65 in H2O2 induced oxidative damage of human lens epithelial cells].

OBJECTIVE: To study the effects of ecdysterone (ECR) on the expression of nuclear factor (NF)-kappaB in H2O2 induced oxidative damage of human lens epithelial cells (HLECs). METHODS: The cultured HLECs were divided into 5 groups, i.e., the control group, the H2O2 group, the beta-estradiol (E2) group, the ECR group, and the pyrrolidine dithiocarbamate group (PDTC) group. The expression rate of NF-kappaB p65 in the HLECs were detected by flow cytometer (FCM). RESULTS: The expression of NF-kappaB p65 occurred in normal HLECs (9. 53%). The expression rate of NF-kappaB p65 in the H2O2 group obviously increased (39.87%, P < 0.01). The expression rate of NF-kappaB p65 in the PDTC group obviously decreased (5.90%, P < 0.01). The expression rates of NF-kappaB p65 in the ECR group (13.99%) and the E2 group (25.18%) ranged between the control group and the H2O2 group, but still lower than that of the H2O2 group (P < 0.01). CONCLUSIONS: The activation of NF-kappaB in the HLECs could be induced by H2O2 ECR with the estrogenic activity could effectively inhibit the activation of NF-kappaB.

Mechanisms of inhibition of elemene on human lens epithelial cell proliferation in vitro.

AIM: To study the effects of elemene (Ele) on proliferation and cell cycle of human lens epithelial cells B3 (HLE-B3) and the mechanisms of its signal transduction. METHODS: Recombinant human basic fibroblast growth factor (rhbFGF) was used to induce proliferation of HLE-B3 cells, which were incubated with 80mg/L Ele for 24 hours. The inhibitory effects of Ele on the proliferation of HLE-B3 cells were evaluated by MTT method. The effect of Ele on HLE-B3 cell cycle was analyzed by flow cytometry(FCM). The expressions of protein kinase A (PKA) and protein kinase G (PKG) of HLE-B3 were also analyzed by FCM. RESULTS: Ele altered the cell cycle of HLE-B3 and effectively inhibited HLE-B3 cell proliferation induced by rhbFGF. Ele up-regulated PKA and down-regulated the expression of PKG in HLE-B3 cell. CONCLUSION: Ele inhibits HLE-B3 proliferation, making it an attractive potential agent in regimens to treat after-cataracts.

[Study on proteomics of inhibitory effects of elemene on proliferation of human lens epithelial cell].

OBJECTIVE: To investigate the inhibitory effects of natural medicinal monomer elemene (Ele) on proliferation of human lens epithelial cells B3 (HLE-B3) inducing by recombinant human basic fibroblast growth factor(rhbFGF) and to pursue the proteomics regularity of the inhibitory effects of Ele on proliferation of HLE-B3. METHODS: Experimental study. This study is divided into three group: control group, rhbFGF group and Ele group. Using 10 microg/L rhbFGF to induce proliferation of HLE-B3. Proliferative HLE-B3 were incubated with 80 mg/L Ele in CO2 incubator for 24 hours. Then the inhibitory effects of Ele on proliferation of HLE-B3 was detected by methyl thiazolyl tetrazolium (MTT). The change of expressions of all protein in HLE-B3 was assayed and analyzed by protein array and surface-enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS) proteomics technology. RESULTS: MTT test showed that the A values of rhbFGF (0.599+/-0.053) group were higher than that of control group (0.409+/-0.042) remarkably. The A values of Ele group (0.450+/-0.061) decreased obviously compared to rhbFGF group, the inhibition rates were 24.90% (F=28.886, P=0.000). Five different protein spots were obtained in proliferative HLE-B3 induced by rhbFGF. The expressions were up-regulated in two of the five protein spots at the ratios of mass/charge (m/z) of 8093 and 9516, while the expressions were down-regulated in three of the five protein spots at m/z of 5361, 9666 and 13 767. Ten different protein spots were obtained in HLE-B3 incubated with Ele. The expressions were up-regulated in four of the ten protein spots at m/z of 2487, 4392, 8566 and 11 600, while the expressions were down-regulated in six of the ten protein spots at m/z of 3679, 4826, 6861, 9516, 9557 and 9672. CONCLUSIONS: Ele could effectively inhibit HLE-B3 proliferation induced by rhbFGF. The protein spot at m/z of 9516 might be the target of proliferative inhibition in HLE-B3 by Ele.

[Inhibition effects of compound leech eye drops on apoptosis of lens epithelial cells and expressions of Bcl-2 and Bax genes in rats].

OBJECTIVE: To investigate the inhibition effects of compound leech eye drops (Co-SZ) on apoptosis of lens epithelial cells (LECs) induced by hydrogen peroxide (H2O2) and the expressions of apoptosis-related genes Bcl-2 and Bax in rats. METHODS: All fresh transparent LECs in SD rats were bathed in culture medium with H2O2 in vitro, meanwhile Co-SZ were added in the culture medium. All LECs were incubated for 24 hours. The apoptosis rate of LECs was determined by terminal deoxynucleotidyl transferase mediated biotin-dUTP nick end labeling method (TUNEL). The changes of LEC ultrastructure and the formation of apoptotic body were observed by transmission electron microscopy. The expressions of apoptosis-related genes Bcl-2 and Bax were detected by streptavdin-peroxidase-biotin method. RESULTS: The apoptosis rate of LECs in the Co-SZ-treated group was significantly lower than that in the H2O2-treated group. The changes of apoptotic LEC ultrastructure in the Co-SZ-treated group were less than those in the H2O2-treated group. The expression of Bcl-2 protein was up-regulated and the expression of Bax protein was down-regulated in the Co-SZ-treated group as compared with the H2O2-treated group. CONCLUSION: The LEC apoptosis induced by H2O2 can be inhibited by Co-SZ. The molecular mechanisms of Co-SZ in inhibiting LEC apoptosis may be related to regulating the expressions of apoptosis-related genes Bcl-2 and Bax.

[Effect of Rhizoma Curcumae and arsenite trioxide on proliferation and signal transduction molecule of lens epithelial cell].

OBJECTIVE: To investigate the effect of Rhizoma Curcumae (RC), arsenite trioxide (As2O3) on proliferation ana signal transduction molecule in lens epithelial cell (LEC), in order to provide experiment evidence for prevention and treatment of after cataract. METHOD: Proliferation of cultured bovine LEC were induced by induced by recombinant human basic fibroblast growth factor (rhbFGF); Inhibitory rates of LEC proliferation induced by RC, As2O3 were detected by methyl thiazolyl tetrazolium (MTT); Inhibitory effects of expression of proliferating cell nuclear antigen (PCNA) induced by RC, As2O3 in LEC were assayed via flow cytometer (FCM); Concentrations of LEC calcium ([Ca2+]i) were determined by spectrofluoremeter, intracellular concentrations of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) of LEC were measured by radioimmunoassay. RESULT: Inhibitory rates of RC, As2O3 on LEC proliferation induced by rhbFGF increased significantly, showing dose-dependent (P < 0.01). PCNA expression of LEC proliferation induced by rhbFGF were down regulated obviously by RC, As2O3, showing dose-dependent (P < 0.01). Concentrations of [Ca2+]and cAMP increased and cGMP decreased significantly in LEC of proliferation inhibited by RC, As2O3 (P < 0.01). CONCLUSION: RC, As2O3 can inhibit LEC proliferation obviously. Signal transductions of [Ca2+]i, cAMP, cGMP may be the important molecular mechanism. There are broad prospect for RC, As2O3 on prevention and treatment of after cataract.

[Apoptosis of lens epithelial cell induced by curcumin and its mechanism].

OBJECTIVE: To investigate the effects of natural drug curcumin (Cur) on apoptosis of lens epithelial cell (LEC) in vitro and its mechanism. METHODS: The bovine LEC were cultured with Cur, the ultrastructure changes were observed under transmission electron microscope (TEM), the DNA content and mitochondrial transmembrane potential (DeltaPsim) changes were studied by flow cytometry (FCM). RESULTS: The typical morphological changes of LEC apoptosis in Cur group detected by TEM included chromatin condensation and aggregation at the periphery of the nucleons and nuclear fragmentation. The DNA content of LEC in Cur group decreased time-dependently. The DNA content was significantly lower than that of the control group (P < 0.01). The DeltaPsim of LEC in Cur group was decreased, appeared in early stage (8 hours) and reached the maximum after 72 hours. The difference of DeltaPsim of LEC between Cur group and the control group was significant (P < 0.01). CONCLUSIONS: Cur can remarkably induce apoptosis of LEC in vitro. Cur induced LEC apoptosis is caused by decrease of DNA content in LEC nucleus. Collapse of DeltaPsim in cytoplasm induced by Cur results in the irreversible apoptosis process of LEC. This is the early event of LEC apoptosis. LEC apoptosis induced by Cur may pass through two pathways: nuclear pathway and cytoplasmic pathway. The apoptosis of LEC induced by Cur may be the cellular and molecular mechanisms of reducing lens posterior capsular opacification by Cur. Cur may become an effective and low toxic medication for the prevention and treatment of after-cataract.

[Signal transduction mechanism of curcumin in inhibiting the proliferation of bovine lens epithelial cell induced by recombinant human epidermal growth factor].

OBJECTIVE: To investigate the signal transduction mechanism of curcumin in inhibiting the proliferation of bovine lens epithelial cell (LEC) induced by recombinant human epidermal growth factor (rhEGF). METHODS: There were three groups in this experiment, which were normal control group, untreated group and curcumin-treated group. Proliferation of LEC was induced by rhEGF (50 microg/L). The concentration of intracellular Ca(2+) ([Ca(2+)](i)) in LEC was measured with Fura-2/AM by spectrofluorimetry. The contents of intracellular cAMP and cGMP were assayed by radioimmunoassay. RESULTS: The [Ca(2+)]i in LEC was obviously increased in the untrated group as compared with that in the normal control group (P<0.01), and the [Ca(2+)](i) in LEC in the curcumin-treated group was highest among three groups (P<0.01). The content of intracellular cAMP in LEC was decreased while the content of intracellular cGMP was obviously increased in the untreated group as compared with those in the normal control group (P<0.01). The content of intracellular cAMP in LEC was higher in the curcumin-treated group than that in the untreated group, while the content of intracellular cGMP was lower than that in the untreated group (P<0.01). CONCLUSION: The antiproliferation effects of curcumin on LEC may relate to the regulations of multiple processes of signal transduction.

[A study on cytokine levels and nitric oxide content in rabbit aqueous humor after lens extraction and intraocular lens implantation].

OBJECTIVE: To investigate the relationship among inflammatory reaction and cytokine levels, nitric oxide (NO) content in aqueous humor after intraocular lens implantation. METHODS: Eighteen New Zealand rabbits were divided randomly into 3 groups (each 6 rabbits): (1) control group, (2) extracapsular cataract extraction group (ECCE) and (3) ECCE and posterior chamber intraocular lens implantation group (ECCE + IOL). The inflammation of all experimental rabbit eyes were observed by a zoom-photo slit-lamp microscope 0, 1, 3, 7, 14, 30 days postoperatively, including corneal edema and anterior chamber exudation. Meanwhile, aqueous humor was drawn for white blood cell (WBC) count and classification, as well as for NO(2)(-)/NO(3)(-) and cytokine assays, including interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-alpha). Statistics were taken by SPSS software. RESULTS: (1) The anterior chamber exudation was the most serious and monocyte/macrophage in aqueous humor were the highest in ECCE + IOL group in postoperative 7 - 14 days. (2) The levels of IL-2, TNF-alpha and the content of NO(2)(-)/NO(3)(-) in aqueous humor of ECCE + IOL group were higher than that in ECCE group and control group in the postoperative 1 - 14 days respectively, and they increased to their peak values at the postoperative 3 - 7 days and decreased gradually after postoperative two weeks. (3) The change regularity of IL-2, TNF-alpha, NO(2)(-)/NO(3)(-) and inflammatory reaction in each group were basically similar, i.e. the more serious the reaction, the higher the levels of the contents. CONCLUSION: The intraocular inflammation after intraocular lens implantation is closely related to the changes of cytokine levels and NO content in aqueous humor.