Oral administration of fasudil hydrochloride suppresses development of experimental autoimmune encephalomyelitis in mice

AIM:To explore the therapeutic effect of fasudil hydrochloride by the oral route on the development of experimental autoimmune encephalomyelitis (EAE) in mice and its possible mechanism. METHODS:The EAE model in female C57BL/6 mice was established by myelin oligodendrocyte glycoprotein 35-55 peptide(MOG35-55) immunization and the immunized mice were randomly divided into saline control group and fasudil intervention group, in which saline and fasudil were administered by the oral route once every day from post-immunization (PI) day 3 to day 27. Clinical score and body weight were recorded every other day. On PI day 28, the spinal cords were obtained for HE and myelin staining. The splenocytes were isolated and the expression of CD16/32, CD206 and interleukin (IL)-10 was analyzed by flow cytometry. The levels of IL-1β and tumor necrosis factor α (TNF-α) were detected by ELISA. RESULTS:Oral administration of fasudil delayed the onset of EAE, and attenuated the myelinoclasis of the model animals and the severity of EAE, accompanied by the phenotypic switch from M1 to M2 macrophages, the inhibition of the proinflammatory cytokine (IL-1β and TNF-α) production and the increase in IL-10 release. CONCLUSION: Oral administration of fasudil exhibits therapeutic effect on the development of EAE possibly through switching M1 macrophages to M2 phenotype and inhibiting inflammatory responses in mice.     

Calreticulin expression is necessary for protective immune effect of T-cell vaccine in EAE mice

AIM:To study the role of cell membrane ectopic calreticulin (CALR) expression on the protective immunie effect of T-cell vaccine (TCV) on experimental autoimmune encephalomyelitis (EAE). METHODS:EAE model was established by myelin oligodendrocyte glycoprotein 35-55 (MOG_35-55) immunization in C57BL/6 mice, and the mice were immunized with MOG_35-55-specific CALR⁺ and CALR^- T-lymphocytes. Symptomatic scores were compared at the maximum of the disease. On the 15th day after immunization, the proportion of CD4⁺ CD25⁺ Foxp3⁺ regulatory T cells (Treg) in the spleen, and the expression of interferon-γ (IFN-γ), interleukin-4 (IL-4), IL-10 and IL-17A in the serum were measured. RESULTS:Increased expression of CALR in activated T cells after γ-irradiation was observed. Blockade of CALR on the vaccinating T-cell surface reduced the protective effect of TCV. Furthermore, blockade of CALR reduced the number of Treg in the spleen and up-regulated pro-inflammatory cytokines. CONCLUSION:CALR expression in the T cells is necessary for the protective immunity induced by TCV in EAE mice.     

Immunoregulatory effect of Buyang-Huanwu decoction on monocyte-macrophages in mice with experimental autoimmune encephalomyelitis

AIM: To explore the therapeutic effect of Buyang-Huanwu decoction (BYHWD) on experimental autoimmune encephalomyelitis (EAE) and its immunoregulatory effect on monocyte-macrophages.METHODS: Chronic EAE was induced by myelin oligodendrocyte glycoprotein peptide fragment 35-55 (MOG_35-55) in the female C57BL/6 mice, which were randomly divided into saline group and BYHWD group. On day 3 after immunization, the mice in BYHWD group were orally administrated with BYHWD, while normal saline was given to the control mice. The clinical score and body mass were recorded every other day. At day 17 after immunization, the mice were sacrificed and spinal cords were obtained for HE staining and myelin staining. The M1 and M2 macrophage phenotypes of splenic cells were detected by flow cytometry and immunofluorescence staining. The protein expression of iNOS, TNF-α, arginase and IL-10 in the spinal cord macrophages was determined by Western blotting.RESULTS: BYHWD delayed the onset of EAE, reduced the clinical scores of EAE, inhibited the inflammatory cell infiltration and demyelination in the spinal cord, and promoted the conversion of M1 macrophages into M2 phenotype in the spinal cord and spleen.CONCLUSION: BYHWD intervention attenuates the behavioral and pathological changes in the EAE mice, and its mechanism may be related to the macrophage conversion.     

Effect of idazoxan on permeability of blood-brain barrier and expression of MMP-9/TIMP-1 in mouse experimental autoimmune encephalomyelitis

AIM：To study the effect of idazoxan (IDA) on the permeability of blood-brain barrier (BBB) and the expression of matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1) in mouse experimental autoimmune encephalomyelitis (EAE).METHODS：Female C57BL/6 mice (n=36) were randomly divided into control group, EAE group and IDA group, with 12 mice in each group. EAE was induced by myelin oligodendrocyte glycoprotein 35-55 (MOG35-55). IDA (2 mg/kg, ip, bid) was administered for 15 d after immunization. The neurological defects of the mice were observed daily and scored. The pathological changes were observed under microscope with HE staining and LFB myelin staining. The BBB permeability was detected by Evans blue extravasation. The expression of MMP-9 and TIMP-1 in the brain of EAE mice was determined by Western blotting.RESULTS：Compared with EAE group, the score of neurological defects in IDA group was decreased, the inflammation was relieved, the BBB permeability was reduced, and the expression MMP-9 and the ratio of MMP-9/TIMP-1 were decreased (P<0.05).CONCLUSION：The neuroprotective effect of IDA on mouse EAE might be related to the down-regulation of MMP-9 and the ratio of MMP-9/TIMP-1, thus reducing the degradation of BBB and the permeability of BBB, and ameliorating the pathologic process of EAE.     

Effects of Astragalus injection combined with puerarin injection on expression of BMP-7 and TGF-β1 in type 2 diabetic KKAy mouse kidney

AIM: To observe the effects of Astragalus injection combined with puerarin injection on the expression of transforming growth factor beta 1 (TGF-β₁) and bone morphogenetic protein 7 (BMP-7) in the kidney of type 2 diabetic KKA^y mouse. METHODS: The male KKA^y mice of 14 weeks old were randomly divided into model group and Astragalus injection combined with puerarin injection treatment (Astragalus+puerarin) group. The age-matched male C57BL/6J mice were selected as normal group. The general conditions and body weight of the mice were observed. Blood glucose (BG), triglyceride (TG), cholesterol (TC) and serum creatinine (SCr) were examined at the 20th, 24th and 28th week. The protein expression of renal TGF-β₁ was determined by immunohistochemical method. The mRNA expression of BMP-7 and TGF-β₁ was detected by RT-PCR. RESULTS: Compared with normal group, the body weight, BG, TG, TC and SCr increased significantly in model group. TGF-β₁ expression at protein and mRNA levels was increased, while mRNA expression of BMP-7 was decreased in KKA^y mice. Compared with model group, the body weight, BG, TG, TC and SCr reduced in Astragalus+puerarin group. The mRNA expression of BMP-7 in the renal tissues was higher, and TGF-β₁ expression at mRNA and protein levels was significantly lower in Astragalus+puerarin group than those in model group. CONCLUSION: Astragalus injection combined with puerarin injection has renal protective effects on type 2 diabetic KKA^y mice. The mechanism may be related to restoring BMP-7 expression and reducing the overexpression of TGF-β₁ in renal tissues.     

Attenuation of periventricular leukomalacia in neonatal rats by fasudil

AIM To investigate the effect of fasudil on neonatal rats with periventricular leukomalacia （PVL） and its mechanism. METHODS Three-day-old neonatal rat PVL model was established by hypoxia and ischemia. The newborn SD rats （3-day-old, n=225） were randomly divided into 3 groups： sham group, PVL group and PVL+fasudil group. The rats in these 3 groups were further divided into 12 h, 24 h, 48 h,72 h and 30 d subgroups, with 15 neonatal rats each. The neonatal rats in the subgroups were rapidly decapitated and their brains were removed after treatment for 72 h. The pathological changes of brain tissues were observed by HE staining. The expression level of ROCK2 was assessed by RT-qPCR. The protein levels of ROCK2 and NF-κB P65 were analyzed by Western blot. Neurobehavior was observed after 30 d. RESULTS （1） Growth rates of body weight of the rats in PVL group and PVL+fasudil group were significantly lower than that in sham group after ischemia （P<0.05）. （2） The results of HE staining showed significant pathological changes at 72 h in PVL group and PVL+fasudil group. But the pathological changes in PVL+fasudil group were relatively mild as compared with PVL group. （3） The expression of ROCK2 was significantly increased in PVL group compared with PVL+fasudil group and sham group （P<0.05）. （4） The expression of NF-κB P65 was increased in PVL group compared with other groups at 24 h and 48 h（P<0.05）. CONCLUSION Fasudil reduces the pathological damage of brain, and has a long-term neuroprotective effect, which improves neurological prognosis in neonatal rats with PVL by inhibiting ROCK pathway, reducing the activation of NF-κB P65 and attenuating inflammatory damage.     

Effects of tripterygium glycosides combined with dexamethasone on TLRs/NF-κB signaling pathway in EAE rats

AIM: To investigate the role of TLRs/NF-κB pathway in experimental allergic encephalomyelitis (EAE) rats treated with tripterygium glycosides (TG) + dexamethasone (DX). METHODS: Lewis rats were used in the study and divided into control group, EAE model group, therapy 1 group (EAE rats treated with DX) and therapy 2 group (EAE rats treated with DX+TG). The mean clinical score of the rats was determined. The expression of TLR4 and TLR9 at mRNA and protein levels was detected by the methods of real-time quantitative RT-PCR and immunohistochemistry. The protein level of NF-κB p65 was also measured. The levels of TNF-α, IL-1β and IL-6 were assayed by ELISA. RESULTS: The mean clinical scores at 5th, 16th and 20th day were lower in therapy 1 group and therapy 2 group than that in EAE model group. The mean clinical score in therapy 2 group was even lower than that in therapy 1 group. At the 16th day (the peaking period), the mRNA expression of TLR4 and TLR9 in therapy 1 group and therapy 2 group were obviously lower than that in EAE model group. The protein levels of TLR4, TLR9 and NF-κB p65 were also significantly lower in therapy 1 group and therapy 2 group than those in EAE model group at peak stage of EAE. The levels of TNF-α, IL-1β and IL-6 were lower in therapy1 group and therapy2 group than those in EAE model group. The significant differences of the mean clinical score, the mRNA expression of TLR4 and TLR9, the positive ratio of NF-κB p65 and the levels of TNF-α, IL-1β and IL-6 between therapy 1 group and therapy 2 group were found. The result of orthogonal factorial analysis of variance indicated that the difference of therapeutic effect between DX and DX+TG was significant (F=75.749, P<0.01). CONCLUSION: The TLRs/NF-κB pathway takes part in the pathological process of EAE. TG combined with DX alleviates the symptoms of EAE by suppressing inflammatory and immunological reactions of EAE.     

Fasudil ameliorates myocardial fibrosis by regulating polarization of macrophages in diabetic mice

AIM: To investigate the effect of hydroxyl fasudil (HF) on myocardial fibrosis and macrophage polarization in the diabetic (D) mice. METHODS: C57BL/6 mice (n=60) were randomly divided into normal saline group (NS group), normal+hydroxyl fasudil group (N+HF group), diabetes group (D+NS group), diabetes+low dose of HF group (D+LHF group), diabetes+middle dose of HF group (D+MHF group) and diabetes+high dose of HF group (D+HHF group). A mouse model of type 1 diabetes mellitus was established by intraperitoneal injection of streptozotocin (STZ). The mice in treatment groups received different doses of fasudil through intraperitoneal injection for 8 weeks. At the end of the study, the effects of fasudil at different doses on the body weight and blood glucose were observed. The histopathological changes of the cardiac tissues were observed by HE staining. The myocardial collagen volume fraction (CVF) was calculated by Masson staining. Immumohistochemical staining was used to test the macrophage polarization and protein expression of interleukin-6 (IL-6), tumor necrosis factor-α(TNF-α) and IL-10 and Western blot was applied to determine the protein levels of p-MYPT1 Thr853, inducible nitric oxide synthase (iNOS) and Arginase-1 (Arg-1).RESULTS: Compared with NS group, the body weight of the mice in D+NS group was decreased and the blood glucose was increased significantly(P<0.05). However, no statistically difference of blood glucose and body weight between the treatment groups and D+NS group was observed. Compared with NS group, CVF, the number of M1-type macrophages and the protein levels of IL-6, TNF-α, p-MYPT1 Thr853 and iNOS were increased markedly, while M2-type macrophages and the expression of IL-10 and Arg-1 were decreased in D+NS group (P<0.05). Compared with D+NS group, CVF, the number of M1-type macrophages, and the protein levels of IL-6 and TNF-α were relatively decreased, conversely the number of M2-type macrophages and the protein level of IL-10 was increased in treatment groups (P<0.05). Moreover, the protein levels of p-MYPT1 Thr853 and iNOS were reduced and the protein level of Arg-1 was increased in D+MHF and D+HHF group compared with D+NS group (P<0.05). No statistical difference in above mentioned indexes between NS group and N+HF group was observed. CONCLUSION: Fasudil significantly attenuates the myocardial fibrosis of diabetic cardiomyopathy in mice, which is possibly related to increased polarization of M2-type macrophages, decreased polarization of M1-type macrophages and inflammation.     

Rho kinase inhibitor fasudil improves cognitive function by promoting nerve regeneration in APP/PS1 double transgenic mice

AIM:To observe the effects of Rho kinase inhibitor fasudil on promoting nerve regeneration and improving cognitive function in amyloid precursor protein/presenilin 1 double transgenic (APP/PS1 Tg) mice, a widely used model of Alzheimer disease. METHODS:Male APP/PS1 Tg mice at 8 months old were randomly divided into 2 groups:the mice in fasudil group were intraperitoneally injected with fasudil at 25 mg/kg, while the mice in NS group were intraperitoneally injected with normal saline (NS), once daily for 2 months. Age-and sex-matched wild-type (WT) mice without treatment were used as controls. The Morris water maze (MWM) test and SMART 3.0 behavioral record system were applied to examine and analyze the spatial cognitive function of the mice. The protein levels and distribution of p-Tau, ChAT, BrdU and nestin in the hippocampal dentate gyrus (DG) and CA3 area and cerebral cortex were detected by immunohistochemistry. The protein levels of p-APP(Thr668) and p-Tau in the brain were analyzed by Western blot. RESULTS:Compared with control group, the APP/PS1 Tg mice (10 months old at the time of testing) treated with NS displayed the increase in the latency to target, and the decreases in the time and distance in SW (%) during the MWM test (SW was located in the area of the platform), indicating impaired cognition, which was reversed by fasudil treatment, indicating that the cognitive function was improved in the APP/PS1 Tg mice. In addition, compared with NS group, fasudil treatment significantly reduced the protein level of p-Tau, and increased the protein level of p-APP in the central nervous system (CNS) and the number of cholinergic neurons in the hippocampus. The neuronal axon regeneration in the hippocampus was promoted, and the endogenous neural stem cell proliferation in subgranular zone and subventricular zone of the hippocampal DG was also mobilized. CONCLUSION:Fasudil reverses spatial cognitive dysfunction in APP/PS1 Tg mice through decreasing the protein level of p-Tau, increasing the level of soluble APP, promoting the regeneration of cholinergic neurons, and mobilizating the endogenous neural stem cell proliferation in the CNS.     

Therapeutic effect of emodin on constipation induced by loperamide in mice

AIM: To study the therapeutic effect of emodin on loperamide-induced constipation in mice. METHODS: The constipation model of mice was established by lopebutamine treatment, and the effects of emodin on defecation frequency, fecal water content and intestinal transit time of the mice during the observation period were detected. Inflammatory infiltration in colon tissue of the mice was observed by HE staining. Serum nitric oxide (NO) content was detected by commercially available kit. The expression of vasoactive intestinal peptide receptor 1 (VIPR1) and 5-hydroxytryptamine type 4 receptor (5-HT₄ receptor) in mouse colon was determined by immunohistochemistry. The effects of emodin on the expression of transient receptor potential cation channel subfamily V member 1 (TRPV1), glial cell-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), nitric oxide synthase (NOS), c-Kit and their ligand stem cell factor (SCF) were detected by Western blot. RESULTS: Emodin significantly increased the number of defecation and fecal water content in the mice during the observation period, and significantly reduced the intestinal transit time and serum NO level in the mice (P<0.05). The results of HE staining showed that emodin significantly reduced the infiltration of colonic inflammation induced by loperamide. Emodin can significantly reduce the increase in VIPR1 induced by loperamine and increased the expression of 5-HT₄ receptor (P<0.01). Emodin increased the expression levels of GDNF and BDNF, reduced the expression levels of TRPV1 and NOS in the colon tissues of loperamine-induced constipation in mice, and significantly increased the expression of c-Kit and SCF (P<0.05 or P<0.01). CONCLUSION: Emodin promotes the defecation behavior of mice with loperamine-induced constipation by increasing intestinal peristalsis and activating a series of smooth muscle contraction-related factors.     

Role of Rho kinase in blocking the return of mesenteric lymph to improve vascular calcium sensitivity in hemorrhagic shock rats

AIM: To observe the role of Rho kinase in mesenteric lymph duct ligation or mesenteric lymph drainage to improve vascular calcium sensitivity in the rats subjected to hemorrhagic shock. METHODS: Male Wistar rats were randomly divided into sham group, shock group, shock+ligation (shock plus mesenteric lymph duct ligation) group and shock+drainage (shock plus mesenteric lymph drainage) group. After induction of shock (hypotension at 40 mmHg) for 3 h, the vascular rings of superior mesenteric artery (SMA) were prepared and used to measure the response to gradient calcium ions for determining the calcium sensitivity with a wire myograph system. In shock+ligation group and shock+drainage group, the vascular rings were incubated with Rho kinase agonist angiotensinⅡ or antagonist fasudil before the measurement of the response to gradient calcium ions. RESULTS: The calcium sensitivity of vascular rings in shock group was significantly lower than that in sham group, and that in shock+ligation group and shock+drainage group was significantly higher than that in shock group, but still lower than that in sham group. AngⅡ elevated the contractile activity of the vascular rings in response to gradient calcium ions and the pD₂, and fasudil significantly decreased the response to gradient calcium ions and E_max in shock+ligation group and shock+drainage group. At the same time, fasudil decreased the pD₂ in shock+ligation group. CONCLUSION: Rho kinase plays an important role in blocking shock mesenteric lymph return that improves calcium sensitivity.     

Preventive effects of anti-IGFBPrP1 antibody on hepatic fibrosis in mice

AIM: To investigate the preventive effect and mechanism of anti-insulin-like growth factor binding protein related protein 1(IGFBPrP1) antibody on hepatic fibrosis induced by thioacetamide (TAA) in mice.METHODS: Twenty-four male C57BL/6 wild-type mice were randomly divided into 3 groups (n= 8 in each group): normal control group, TAA group (4 weeks) and TAA+anti-IGFBPrP1 antibody group (4 weeks). The morphological changes of liver tissues were observed. The expression levels of α-smooth muscle actin (α-SMA), transforming growth factor beta 1 (TGF-β₁), Smad3, phosphorylated Smad2/3 (p-Smad2/3), fibronectin (FN), collagen I, collagen Ⅲ and IGFBPrP1 were detected by the methods of immunohistochemistry and Western blotting.RESULTS: In TAA group (4 weeks), obvious injury of liver was observed, and the expression levels of α-SMA, TGF-β₁, Smad3, p-Smad2/3, FN, collagen Ⅰ, collagen Ⅲ and IGFBPrP1 were significantly increased as compared with normal control group (P<0.01). Compared with TAA group (4 weeks), the injury of the liver was alleviated and the expression levels of the proteins above were decreased in TAA+anti-IGFBPrP1 antibody group (4 weeks, P<0.01). IGFBPrP1 was positively correlated with TGF-β₁, Smad3, p-Smad2/3, FN and collagen I (P<0.01). CONCLUSION: Anti-IGFBPrP1 antibody prevents TAA-induced hepatic fibrosis in mice by inhibiting the activation of hepatic stellate cells, reducing the expression of p-Smad2/3 and inhibiting the TGF-β₁/ Smad3 signal transduction, thereby depressing the deposition of extracellular matrix in liver tissues.     

Establishment of experimental autoimmune encephalomyelitis model in mice induced by MOG and its effect on CD4+CD25+ T cells

AIM: To investigate the feasibility of inducing experimental autoimmune encephalomyelitis(EAE)model in C57BL/6 mice by TrxA-extracellular immunoglobulin domain of MOG(MOGIgd-TrxA)fusion protein produced by molecular cloning in our laboratory. Also to investigate the role of CD4+CD25+ T cells in the pathogenesis of EAE. METHODS: (1)The MOGIgd-TrxA fusion protein was induced and produced by molecular cloning and purified by metal chelate affinity chromatography and concentrated through ultrafiltration. The concentration of the protein was measured by Bradford method at last. (2)Animal experiment: C57BL/6 mice(12 mice in each group)were used. The mice in group MOG were immunized with MOGIgd -TrxA fusion protein. The mice in group GPSCH were received emulsion of spinal cord homogenate of guinea pigs(GPSCH), and mice in group TrxA or normal control group(group NC)were received the same volume emulsion of TrxA or saline/adjuvant, respectively. Clinical scores and histopathology were measured to value the models quality. (3)The percentages of CD4+CD25+ regulatory T cells in EAE mice were tested through flow cytometric analysis. RESULTS: (1)The purity of purified MOGIgd -TrxA fusion protein was about 98%, and its concentration was 2.3 g/L. (2)No significant difference between group MOG and group GPSCH in the clinical score was observed(P>0.05). Histologic sections of the brain and spinal cord taken from affected animals in both groups showed pathological change of different level throughout the central nervous system(CNS). (3)Percentages of CD4+CD25+ T cells in group MOG and group GPSCH were(4.71±1.61)%and(1.44±0.65)%,respectively, both of which were significantly lower than those in group NC(9.22±1.24)%and TrxA group(8.97±1.20)％(P<0.01). CONCLUSION: (1)The animal model of EAE in C57BL/6 mice induced by MOGIgd fusion protein produced through molecular cloning in our laboratory is stable and with high incidence. Thus, the author finds a good way to study the immune mechanisms of MS further and to search for the effective treatments as well. (2)The reduction of CD4+CD25+ T cells in EAE mice may have some relationship with the clinic.     

Role of P2Y1 receptor in activation of astrocytes induced by Aβ25-35

AIM: To study the role of P2Y₁ receptor in the activation of astrocytes induced by Aβ_25-35.METHODS: Astrocytes were isolated and cultured from newborn Wistar rats and divided into control group, Aβ_25-35 group, MRS2179(P2Y₁receptor inhibitor)+Aβ_25-35 group and MRS2179 group by treating the cells with the corresponding reagents. The expression levels of GFAP and P2Y₁ were determined by the methods of immunohistochemistry, immunofluorescence and Western blotting.RESULTS: No significant change of the astrocyte numbers in all groups was observed. Compared with the control cells, the fluorescence intensity of GFAP significantly increased in Aβ_25-35 group and decreased in both MRS2179+Aβ_25-35 group and MRS2179 group. The expression level of GFAP determined by Western blotting and immunofluorescence showed the similar trend of change in each group. Compared with control group, the expression of P2Y₁ in Aβ_25-35 group was significantly increased (P<0.05), and no significant change between MRS2179+Aβ_25-35 group and MRS2179 group was found (P>0.05).CONCLUSION: Aβ_25-35 activates astrocytes by activation of P2Y₁ receptor.     

Effects of new cannabis preparations O-1602 and cannabidiol on LPS-induced intestinal motility disorder in rodents

AIM: To investigate the therapeutic effects and related mechanisms of two new cannabis preparations, O-1602 and cannabidiol (CBD), on lipopolysaccharide (LPS)-induced rodent models of intestinal motility disorder in vivo and in vitro. METHODS: The animal model of intestinal motility disorder was induced by intraperitoneal injection of LPS in mice. The gastrointestinal transit was measured by gavaging charcoal marker. Western blotting was applied to evaluate the protein expression of G-protein-coupled receptor 55 (GPR55). Meanwhile, the levels of tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) were tested by ELISA to assess the inflammatory degree. Smooth muscle strips from the rat and mouse ileum were incubated with LPS in vitro to establish motility disorder, and both the spontaneous contraction and electrically-evoked contraction were recorded using the organ bath technique. The traditional intracellular microelectrode technique was used to record the changes of membrane potential of smooth muscle cells. The method of determining phosphorus content was applied to assay the Ca²⁺-ATPase activity in smooth muscle tissues. RESULTS: In vivo, LPS resulted in significant inflammation and the disorder of gut movement (P<0.01). Pretreatment with CBD decreased both the level of IL-6 (P<0.01) and the expression of GPR55 (P<0.01), and further improved the motility of gut movement (P<0.05). O-1602 and CBD selectively normalized LPS-induced spontaneous and electrically-evoked contraction disorder of intestinal smooth muscle strips of rats and mice in vitro (P<0.05 or P<0.01), but they had no effect on the membrane potential of the smooth muscle cells both in normal and pathophysiological states. CBD also decreased the elevated Ca²⁺-ATPase activity in smooth muscle tissues induced by LPS (P<0.05). CONCLUSION: In vivo, CBD shows protective effect on LPS-induced intestinal motility disorder by reducing inflammation and down-regulating GPR55 expression. O-1602 and CBD counterbalance LPS-induced intestinal motility disorder to some extent in vitro, and the possible mechanism may be involved in regulating the Ca²⁺-ATPase activity of smooth muscle tissues, but not including the change of membrane potential.     

Effect of adoptive transfer of CD4+ T cells with miR-7 knockdown on acute liver injury in mice

AIM:To study the effect of adoptive transfer of CD4⁺ T cells with microRNA-7 (miR-7) knockdown (KD) on mouse acute liver injury model and to investigate its significance. METHODS:CD4⁺ CD62L⁺ T cells were purified from the spleen of normal wild-type (WT) mice and miR-7KD mice by magnetic bead sorting, and were stained with CFSE. These 2×10⁶ CFSE-labeling cells were injected into normal mice via tail vein, and then the mouse acute liver injury model was induced by intraperitoneal injection of 30 mg/kg concanavalin A. After 72 h, the appearance, weight and weight index of the liver were investigated. The pathological change of the liver tissues was observed by HE staining. Real-time PCR was used to examine the mRNA expression of Bax and P53. The expression levels of CD62L, interleukin-4 (IL-4) and interferon-γ (IFN-γ) in the CD4⁺ T cells were analyzed by flow cytometry. RESULTS:We found that the liver tissue became lighter, and the weight (P<0.01) and weight index (P<0.05) were changed significantly in miR-7KD mice compared with control group. Moreover, HE staining showed that the liver cell damage was increased in the liver of miR-7KD mice. Meanwhile, the expression levels of Bax and P53 were significantly increased in miR-7KD group (P<0.05). The percentage of CD62L in CD4⁺ T cells was significantly decreased (P<0.01) in miR-7KD mice, with high expression of IFN-γ (P<0.05) and low expression of IL-4 (P<0.01) in CD4⁺T cells. CONCLUSION:These findings suggest that miR-7 knockdown significantly promotes the pathology of CD4⁺ T cell-mediated acute liver injury, which provides a preliminary experimental basis for further exploration on the mechanism of acute liver injury occurrence.     

Study on a novel Rho kinase inhibitor WAR5 for treating EAE

AIM：To explore the therapeutic effect of a novel Rho kinase inhibitor WAR5 on the experimental autoimmune encephalomyelitis (EAE) and its possible mechanism. METHODS：Female C57BL/6 mice were randomly divided into EAE group and WAR5 group. EAE model was induced by the application of MOG35-55 peptide. WAR5 was injected intraperitoneally every other day from post-immunization (PI) day 3 to PI day 27. The clinical score and body weight were recorded every other day. On PI day 28, the animals were sacrificed and spinal cords were obtained for HE and myelin staining. The splenocytes were isolated and the expression of CD16/32 and CD206 were analyzed by flow cytometry. The protein extracts from the brains and spinal cords were collected for the measurement of inducible nitric oxide synthase (iNOS) by Western blotting. RESULTS：The administration of WAR5 delayed the onset of EAE and attenuated the clinical symptoms. The results of the pathological examination revealed that WAR5 inhibited the infiltration of inflammatory cells and improved myelination in spinal cords, accompanied with the poralization of M1 macrophages to M2 phenotype in the spleen. WAR5 inhibited the expression of iNOS in the central nervous system, especially in the spinal cords. CONCLUSION：The therapeutic effect of WAR5 on EAE may be related to the shift of M1 macrophages to M2 phenotype and inhibition of inflammation in the central nerve system.     

Combination of Treg inhibition and intratumoral transfection of Ad.GM-CSF enhances the chemotherapeutic effect of doxorubicin against hepatocellular carcinoma

AIM: To explore the possibility that combination of regulatory T-cell (Treg) inhibition and intratumoral transfection of Ad.GM-CSF enhances the chemotherapeutic effect of doxorubicin against hepatocellular carcinoma (HCC). METHODS: C57BL/6J subcutaneous HCC model was established. The mice were randomly divided into 6 groups with 12 mice in each group when the tumor volume reached to 100-150 mm³: control group, cyclophosphamide(CTX) group, doxorubicin (DOX) group, Ad.GM-CSF group, CTX+DOX group and CTX+DOX+Ad.GM-CSF group. Four mice in each group were sacrificed for spleen cytotoxic T-cell(CTL) activity assay and tumor tissue histological examination 5 d after the final therapy. Eight mice in each group were examined for tumor growth and survival. RESULTS: CTX enhanced the chemotherapeutic effect of doxorubicin against HCC by inhibiting the tumor growth (P<0.05) and prolonging the mice survival (62.13 d±4.21 d vs 79.88 d±9.00 d, P<0.05), which was significantly strengthened by the combined use of Ad.GM-CSF (79.88 d±9.00 d vs 106.13 d±5.23 d, P<0.01). Compared with other groups, more tumor necrosis in tumor specimens and more infiltration of CD8⁺ T-lymphocytes in CTX+DOX+Ad.GM-CSF group were observed. Furthermore, the spleen cytotoxic T-cell(CTL) activity was significantly improved (P<0.05). CONCLUSION: Doxorubicin-based chemotherapy and immunotherapy by the method of Treg inhibition combined with intratumoral transfection of Ad.GM-CSF have synergistic effect against HCC.     

Effects of Foxp3 transfection on splenocyte functions in asthma mice

AIM: To study the expression and the effects of Foxp3 on the immunologic functions by transfecting the Foxp3 eukaryotic expression plasmid into the splenocytes of the asthma mice. METHODS: The mice were sensitized and challenged by ovalbumin to make asthma model. The splenocytes were harvested and cultured. The Foxp3 expression vector pcDNA3.1(-)-Foxp3 was transfected into the splenocytes with electroporation. The splenocytes transfected with empty vector and control splenocytes (non-transfected) were also set up. The expression of Foxp3 at mRNA and protein levels was detected by RT-PCR and Western blotting, respectively. The proportion of CD4⁺CD25⁺ Treg cells/CD4⁺ cells was measured by flow cytometry. Proliferation of the splenocytes was analyzed with MTT assay. ELISA was used to determine the levels of interleukin 4 (IL-4) and interferon γ (IFN-γ) in the supernatant of the splenocytes. RESULTS: The expression of Foxp3 at mRNA and protein levels in transfection group was significantly higher than that in empty vector group and control group. The proportion of CD4⁺CD25⁺Treg cells/CD4⁺ cells in transfection group was higher than that in empty vector group and control group. The proliferation of transfected cells was markedly inhibited compared with empty vector group and control group. The levels of IL-4 and IFN-γ were significantly lower in transfection group than those in empty vector group and control group. CONCLUSION: The transfected Foxp3 gene overexpresses in the splenocytes of asthma mice. Foxp3 increases the number of CD4⁺CD25⁺ T cells and inhibits the proliferation and production of Th1/Th2 cytokines in splenocytes.     

Effects of non-ventilated lung with nitrous oxide on intrapulmonary oxygenation and lactic acid level in arterial blood during one lung anesthesia

AIM: To investigate the effects of non-ventilated lung with N₂O on systemic oxygenation and lactic acid level in arterial blood during one lung anesthesia. METHODS: Twenty-two patients, ASA Ⅰ-Ⅲ, scheduled for selective pulmonary surgery, were randomly divided into two groups: control group (group A, n=11) and observation group (group B, n=11). Group A: the non-ventilated lung was kept open to the air; group B: N₂O 2 cmH₂O through CPAP system was insufflated into the non-ventilated lung during one lung ventilation. The anesthesia was induced with intravenous midazolam (0.05 mg·kg^-1), propofol (0.5-1.0 mg·kg^-1), fentanyl (4 μg·kg^-1), and vecuronium (0.1 mg·kg^-1) and was maintained with inhaling isoflurane. Blood gas analysis and lactic acid was recorded 20 min after two-lung ventilation (TLV) in the supine position, 20 min after one-lung ventilation (OLV) in the supine position, 20 min and 40 min after OLV in the lateral position and at the end of operation and the shunt fraction was calculated. RESULTS: PaO₂ in group B was significantly higher than that in group A (P＜0.05). Qs/Qt in group B was significantly lower than that in group A (P＜0.05), and lactic acid level in group B was significantly lower than that in group A during OLV. CONCLUSION: Non-ventilated lung with N₂O (2 cmH₂O) improves systemic oxygenation, reduces intrapulmonary shunt and prevents hypoxemia during OLV.