[Mongolian medicine Naru-3 reduces neuroinflammation in maintenance stage of neuropathic pain by inhibiting astrocyte activation].

Neuropathic pain(NP) is difficult to be treated since it has similar phenotypes but different pathogenesis in different pathological stages. Targeted intervention of the core regulatory elements at different pathological stages of NP has become a new direction of drug research and development in recent years and provides the possibility for the treatment of NP. The Mongolian medicine Naru-3(NR-3) is effective in the treatment of sciatica and trigeminal neuralgia, the mechanisms of which remain unknown. On the basis of the previous study of the priming stage, this study established the mouse model of spinal nerve ligation(SNL) and measured the changes of pain thresholds by behavioral tests. The network analysis, Western blot, immunofluorescence assay, ELISA, and agonist/antagonist were employed to decipher the mechanism of NR-3 in the treatment of NP in the maintenance stage. The results showed that NR-3 increased the mechanical and thermal pain thresholds of SNL mice, while it had no significant effect on the basal pain threshold of normal mice. NR-3 may relieve the pain in the maintenance stage of NP by blocking the matrix metalloproteinase 2(MMP2)/interleukin-1ß(IL-1ß) pathway in the astrocytes of the dorsal root ganglion(DRG) and spinal cord. The findings have enriched the biological connotation of NR-3 in the treatment of the maintenance stage of NP and provide reference for the rational use of this medicine in clinical practice.

[Mechanism of Mongolian drug Naru-3 in initiation of neuroinflammation of neuropathic pain from MMP9/IL-1ß signaling pathway].

Neuropathic pain(NP) has similar phenotypes but different sequential neuroinflammatory mechanisms in the pathological process. It is of great significance to inhibit the initiation of neuroinflammation, which has become a new direction of NP treatment and drug development in recent years. Mongolian drug Naru-3 is clinically effective in the treatment of trigeminal neuralgia, sciatica, and other NPs in a short time, but its pharmacodynamic characteristics and mechanism of analgesia are still unclear. In this study, a spinal nerve ligation(SNL) model simulating clinical peripheral nerve injury was established and the efficacy and mechanism of Naru-3 in the treatment of NPs was discussed by means of behavioral detection, side effect evaluation, network analysis, and experimental verification. Pharmacodynamic results showed that Naru-3 increased the basic pain sensitivity threshold(mechanical hyperalgesia and thermal radiation hyperalgesia) in the initiation of SNL in animals and relieved spontaneous pain, however, there was no significant effect on the basic pain sensitivity threshold and motor coordination function of normal animals under physiological and pathological conditions. Meanwhile, the results of primary screening of target tissues showed that Naru-3 inhibited the second phase of injury-induced nociceptive response of formalin test in mice and reduced the expression of inflammatory factors in the spinal cord. Network analysis discovered that Naru-3 had synergy in the treatment of NP, and its mechanism was associated with core targets such as matrix metalloproteinase-9(MMP9) and interleukin-1ß(IL-1ß). The experiment further took the dorsal root ganglion(DRG) and the stage of patho-logical spinal cord as the research objects, focusing on the core targets of inducing microglial neuroinflammation. By means of Western blot, immunofluorescence, agonists, antagonists, behavior, etc., the mechanism of Naru-3 in exerting NP analgesia may be related to the negative regulation of the MMP9/IL-1ß signaling pathway-mediated microglia p38/IL-1ß inflammatory loop in the activation phase. The relevant research enriches the biological connotation of Naru-3 in the treatment of NP and provides references for clinical rational drug use.

The Construction of Sustained-Release Systems of Tanshinone IIA and Sodium Tanshinone IIA Sulfonate by SBA-15 Mesoporous Material.

The basic problems of the low dissolution rate of Tanshinone IIA (TSN) and the instability and precipitation of sodium tanshinone IIA sulfonate (STS) injection limit their usage in clinical. For these facts, the study aims to improve the dissolution rate of TSN and to enhance the sustained release effects of TSN and STS by using SBA-15 mesoporous molecular sieve as a drug carrier. Furthermore, controlling the pore size of SBA-15 and using different loaded methods to achieve expectations and provide a novel scheme for existing Danshen formulations. The effect of loading methods on drug loading efficiency (DL%), as well as the influence of the pore size of SBA-15s, the drug polarities and release mediums on drug loading and release behaviors were analyzed. It was found that the DL% was enhanced with the enlargement of the pore size, and was higher of TSN than STS. The in vitro tests of drug-loaded SBA-15s confirmed that the dissolution rate of TSN was improved obviously as compared with pure TSN. Moreover, SBA-15s prolonged the release times up to 12 h of TSN and 60 h of STS and promoted the sustained-release behaviors by decreasing the pore size. To ascertain the kinetic mechanisms of these samples, the Korsmeyer-Peppas release model was employed and the fitted results indicated that TSN/SBA-15s followed Fickian diffusion and non-Fickian transport was the predominant kinetic release mechanisms for STS/SBA-15s.

Identification of NR0B1 as a novel androgen receptor co-repressor in mouse Sertoli cells.

Nuclear receptor subfamily 0 group B member 1 (Nr0b1) is an atypical member of the nuclear receptor family that is predominantly expressed in mouse Sertoli cells (SCs). Mutations of NR0B1 in humans cause adrenal failure and hypogonadotropic hypogonadism. The targeted mutagenesis of Nr0b1 in mice has revealed a primary gonadal defect characterized by the overexpression of aromatase and cellular obstruction of the seminiferous tubules and efferent ductules, leading to germ cell death and infertility. The transgenic expression of Nr0b1 under the control of the Müllerian-inhibiting substance promoter (MIS-Nr0b1), which is selectively expressed in SCs, improves fertility. Testicular androgen receptor (AR) was also expressed in SCs. Many genes are directly regulated by androgen and its AR, which are involved in spermatogenesis and male infertility. As the association between NR0B1 and AR remains unclear in mouse SCs, we decided to further explore the relationship between them. In the present study, we have identified NR0B1 as a novel AR co-repressor in mouse SCs. Using RT­qPCR and immunofluorescence, we determined that NR0B1 was mainly expressed in mouse SCs in an age-dependent manner from 2-8 weeks of age postnatally. The inhibition of the effects of AR on AR target genes by NR0B1, in an androgen­dependent manner, was further demonstrated by western blot analysis and RT-qPCR in TM4 cells, a mouse Sertoli cell line. Finally, in vitro luciferase and co-immunoprecipitation assays validated that NR0B1, as an AR co-repressor, significantly inhibited the transcriptional activation of its target genes. These results suggest that novel inhibitory mechanisms underlie the effects of NR0B1 in modulating androgen-dependent gene transcription in mouse SCs.

Identification and characteristics of the testes-specific gene, Ccdc38, in mice.

Distinguishing the testes-specific genes in different species may disclose key genes associated with testes-specific functions and provide sufficient information for the study and treatment of male infertility. A testes­specific gene, coiled-coil domain containing 38 (Ccdc38), was identified by screening UniGene libraries. Systematic bioinformatics analysis demonstrated that the CCDC38 protein was conserved in various mammalian species. It was determined that CCDC38 was exclusively expressed in testes and its expression increased from 2­8 weeks of age. Additional immunohistochemical analysis indicated that CCDC38 was mainly expressed in spermatogonia and spermatocytes. It is of note that, immunofluorescence and co-immunoprecipitation assays demonstrated that CCDC38 interacted with ubiquitinated histone H2A in mouse testes. Therefore, these results suggest that Ccdc38 is a testes-specific gene, which may be important for mouse spermatogenesis.

[Expression of structural protein VP1 of enterovirus 71 in E.coli and preparation of anti-VP1 monoclonal antibodies].

AIM: To express recombinant structural protein VP1 of enterovirus 71 (EV71) in E.coli and prepare anti-VP1 monoclonal antibodies (mAbs). METHODS: With P1 gene as a template, EV71 VP1 gene was amplified by PCR and cloned into the expression vector pET-32a(+). After transformation into E.coli TG1, the positive clones were screened and sequenced. The recombinant plasmid was then transformed into BLgold (DE3) and the recombinant protein in inclusion bodies was induced by IPTG and detected by SDS-PAGE. After the inclusion bodies were solubilized with 6 mol/L guanidine hydrochloride, we purified VP1 protein using Ni-NTA affinity chromatography, and then immunized the mice with it to prepare the mAbs against VP1 protein. RESULTS: Recombinant VP1 protein was expressed in E.coli. We obtained totally 24 VP1 monoclonal hybridoma cell strains, of which one was EV71 positive determined by Western blotting and five were positive for IFA. CONCLUSION: These mAbs are valuable reagents for the development of new vaccines and detection kits for EV71.

The effect of QuYuHuaTanTongLuo Decoction on the non-alcoholic steatohepatitis.

UNLABELLED: Non-alcoholic steatohepatitis (NASH) is the most common cause of cryptogenic cirrhosis, is becoming more prevalent in China. However, there is as yet no clearly established therapy for reversing fatty liver. Our aim is to explore the effect of traditional Chinese herbs QuYuHuaTanTongLuo Decoction (QYHTTLD) on non-alcoholic steatohepatitis. Sixty-nine non-alcoholic steatohepatitis patients were randomly divided into two groups. One group of 35 patients were treated by QYHTTLD, another group of 34 patients were treated by Ursodeoxycholic acid (UDCA). The TNF-alpha, IL-8, MDA level, SOD activity and liver function, as well as B ultrasonic image were detected before and after being treated. The results showed: after 6 months treatment, MBI of the treatment group was obviously decreased (p<0.05). The levels of TC, TG and LDL-C were significantly decreased whereas the level of HDL-C increased (p<0.01, p<0.05, p<0.05, and p<0.05, respectively) in the treatment group, the levels of TC, TG, LDL-C and HDL-C had no significant difference in the control group (p>0.05). The levels of TNF-alpha, IL-8 and MDA were significantly decreased whereas SOD activity was significantly increased (p<0.01, p<0.05, p<0.01, and p<0.01, respectively) in the treatment group, the level of MDA was significantly decreased in the control group (p<0.05). B ultrasonic images were ameliorated in different degree (p<0.01 and p<0.01, respectively). Both QYHTTLD and UDCA had the effect in improving the scores of symptoms and signs of patients, however, the difference value of the scores in treatment group were significantly higher than that in control group after being treated for 6 months (p<0.05). CONCLUSION: QYHTTLD is effective for treating non-alcoholic steatohepatitis, and its effect seems to relate with the ways of QYHTTL down-regulating inflammation cytokine IL-8 level and relieving lipid peroxidation of liver.

Induction of rejection after small-for-size liver transplantation: size matters.

BACKGROUND: Reduced-size liver transplantation is associated with liver regeneration. This study was designed to analyze the influence of graft size on liver rejection and liver regeneration. METHODS: Reduced-size liver transplantations were performed in the rejecting ACI to Lewis and the graft acceptance BN to Lewis strain combination. The BN to Lewis control group was treated with the immunosuppressive drug FK506. RESULTS: An accelerated liver rejection in the ACI to Lewis strain combination was found in small-for-size partial liver grafts. Graft weight to recipient liver weight ratio (GW/RLW) showed a positive correlation with survival time. In the BN to Lewis strain combination, lethal rejection was seen in small-for-size partial liver grafts. A critical immunologic GW/RLW of 33% was calculated. In rats dying from lethal rejection, GW/RLW and survival time showed a positive correlation. However, GW/RLW showed a negative correlation with hepatocellular proliferation. In regenerating livers, MHC II upregulation was also observed in the control group. All control animals survived small-for-size liver transplantation. CONCLUSIONS: The relative graft size seems to be a decisive factor influencing the kinetic of liver rejection and the induction of liver rejection. Relative critical immunologic liver mass was determined to be 33%.

Effect of seasonal variation on the clinical course of chronic hepatitis B.

BACKGROUND: Seasonal variation in immunity has been found in healthy individuals and in association with some diseases. It is still unknown whether seasonal variation affects the clinical course of chronic hepatitis B. Our aim in this study was to explore the effect of seasonal variation on the clinical course of chronic hepatitis B. METHODS: The flare and remission time of chronic hepatitis B were observed in patients with hepatitis B virus (HBV) infection. All patients enrolled were followed up at least every 3 months for a mean follow-up time of 24.0 (range, 12-60) months. Seasonal decomposition was employed to analyze the relationship between seasonal variation and flares, remission, and hepatitis B e antigen (HBeAg) seroconversion in chronic hepatitis B patients during follow-up. RESULTS: A total of 2238 patients were observed in our study. Flare and HBeAg seroconversion were seldom seen in 1076 patients (48.08%) with alanine aminotransferase (ALT) levels of less than 2.0 x upper limit of normal (ULN) during follow-up (mean, 36 months). The remaining 1162 patients (51.92%) (766, HBeAg positive; 387 anti-HBeAg positive; 9 negative for both HBeAg and anti-HBeAg) with ALT levels >or=2.0 x ULN were followed longitudinally for 12 months to judge flare, remission, and HBeAg seroconversion. Flare, remission, and HBeAg seroconversion in patients with ALT levels >or=2.0 x ULN showed clear seasonal patterns (P < 0.001), with high peaks during spring, summer, and summer, respectively. An autocorrelation correlogram showed that flares, remission, and HBeAg seroconversion occurred with distinct periodicity in winter, spring, summer, and autumn. CONCLUSIONS: Seasonal variation might affect the clinical course of chronic hepatitis B. The role of seasonal triggering factors should be further investigated.

Administration of granulocyte colony stimulating factor after liver transplantation leads to an increased incidence and severity of ischemic biliary lesions in the rat model.

AIM: Recently it has been reported that granulocyte colony stimulating factor (G-CSF) can induce hypercoagulability in healthy bone marrow donors. It is conceivable that the induction of a prothrombotic state in a recipient of an organ graft with already impaired perfusion might cause further deterioration in the transplanted organ. This study evaluated whether G-CSF treatment worsens liver perfusion following liver transplantation in the rat model. METHODS: A non-arterialized rat liver transplantation model was employed to evaluate the effect of G-CSF treatment on the liver in a syngeneic and allogeneic strain combination. Study outcomes included survival time and liver damage as investigated by liver enzymes and liver histology. Observation times were 1 d, 1 wk and 12 wk. RESULTS: Rats treated with G-CSF had increased incidence and severity of biliary damage following liver transplantation. In these animals, hepatocellular necrosis was accentuated in the centrilobular region. These lesions are indicative of impaired perfusion in G-CSF treated animals. CONCLUSION: G-CSF should be used with caution in recipients of liver transplantation, as treatment might enhance preexisting, undetected perfusion problems and ultimately lead to ischemia induced biliary complications.

Influence of stem cell mobilization and liver regeneration on hepatic parenchymal chimerism in the rat.

BACKGROUND: Despite the newly discovered plasticity of hematopoietic stem cells, their capacity to "transdifferentiate" into parenchymal cells and the regulation of this process is not yet elucidated. The present study was designed to investigate the effect of stem cell mobilization and liver regeneration on this process using a syngeneic rat female-to-male liver transplantation model. METHODS: Rate and frequency of Y-chromosome containing hepatocytes was determined by fluorescence-in situ hybridization (FISH) using a rat Y-chromosome specific probe. Rats were subjected to full-size or partial liver transplantation with and without stem cell mobilization using granulocyte colony stimulating factor (G-CSF). The effect of stem cell mobilization was confirmed by assessing the white blood count and by evaluating the migration of granulopoietic precursor cells to the liver. RESULTS: Treatment with G-CSF induced a twofold increase of peripheral white blood cells and a strong influx of granulopoietic precursor cells into the liver. Transplantation of a partial liver graft was followed by a 90% recovery of the original liver weight within a week, demonstrating the strong regenerative stimulus. Irrespective of the experimental model, the incidence and rate of Y-chromosome containing hepatocytes never exceeded 0.77%. CONCLUSION: Neither stem cell mobilization nor liver regeneration enhanced the incidence or rate of stem cell derived hepatocytes in a liver graft with unimpaired regenerative potential.

He Jie Tang in the treatment of chronic hepatitis B patients.

AIM: To explore the effect of He Jie Tang (decoction for medication) on serum levels of T lymphocyte subsets, NK cell activity and cytokines in chronic hepatitis B patients. METHODS: Eighty-five patients with chronic hepatitis B were divided randomly into two groups. Fifty patients in group I were treated with He Jie Tang (HJT) and 35 patients in group II were treated with combined medication. The levels of T-lymphocyte subsets (CD(3)(+), CD(4)(+), CD(8)(+)), NK cell activity, cytokines (TNF-alpha, IL-8, sIL-2R) were observed before and after the treatment. Another 20 normal persons served as group 3. RESULTS: The level of CD(4)(+) cells and NK cell activity were lower, whereas the level of CD(8)(+) cells in patients was higher than that in normal persons (t = 2.685, 3.172, and 2.754 respectively; P<0.01). The levels of TNF-alpha, IL-8, and sIL-2R in chronic hepatitis B patients were higher than those in normal persons (t = 3.526, 3.170, and 2.876 respectively; P<0.01). After 6 months of treatment, ALT, AST, and TB levels in the two groups were obviously decreased (t = 3.421, 3.106, and 2.857 respectively; P<0.01). The level of CD(4)(+) cells and NK cell activity were increased whereas the level of CD(8)(+) cells decreased (t = 2.179, 2.423, and 2.677 respectively; P<0.05) in group I. The levels of TNF-alpha, IL-8, and sIL-2R in group I were decreased significantly after the treatment (t = 2.611, 2.275, and 2.480 respectively; P<0.05) but had no significant difference in group II after the treatment (t = 1.906, 1.833, and 2.029 respectively; P>0.05). The total effective rate had no significant difference between the two groups (c2 = 2.882, P>0.05) but the markedly effective rate was significantly different between the two groups (c2 = 5.340, P<0.05). CONCLUSION: HJT is effective in treating chronic hepatitis B. HJT seems to exert its effect by improving the cellular immune function and decreasing inflammatory cytokines in chronic hepatitis B patients. The function of HJT in protecting liver function in the process of eliminating virus needs to be further studied.

Synergistic effects of a novel nanoporous stent coating and tacrolimus on intima proliferation in rabbits.

To overcome the problem of in-stent restenosis, the concept of local delivery of antiproliferative or immunosuppressive drugs has been introduced into interventional cardiology. Local drug delivery can be achieved by drug-eluting stents coated with polymer surfaces used for controlled drug release. However, several polymer coatings have shown an induction of inflammatory response and increased neointima formation. In the present study, the effect of a new inorganic ceramic nanoporous aluminum oxide (Al(2)O(3)) coating on neointima proliferation and its suitability as a carrier for the immunosuppressive drug tacrolimus have been investigated. 316 L stainless steel coronary stents were coated with a 500 nm thin nanoporous aluminum oxide layer. This ceramic nanolayer was used as a carrier for tacrolimus. Bare stents (n = 6), ceramic coated stents (n = 6), and ceramic coated stents loaded with 60 (n = 7) and 120 mug (n = 6) tacrolimus were implanted in the common carotid artery of New Zealand rabbits. The ceramic coating caused no significant reduction of neointimal thickness after 28 days. Loading the ceramic stents with tacrolimus led to a significant reduction of neointima thickness by 52% for 60 mug (P = 0.047) and 56% for 120 mug (P = 0.036) as compared to the bare stents. The ceramic coating alone as well as in combination with tacrolimus led to a reduced infiltration of lymphocytes and macrophages in the intima in response to stent implantation. Ceramic coating of coronary stents with a nanoporous layer of aluminum oxide in combination with tacrolimus resulted in a significant reduction in neointima formation and inflammatory response. The synergistic effects of the ceramic coating and tacrolimus suggest that this new approach may have a high potential to translate into clinical benefit.

Improved renal transplantation in the rat with a nonsplinted ureteroureterostomy.

Orthotopic renal transplantation in the rat is a widely used model in immunology and transplantation-related research. Although numerous modifications of the surgical technique of ureteric reconstruction were evaluated, ureter complications following this reconstruction still occurred frequently. Instead of dividing the ureter in the middle between kidney and bladder (method 1), the anastomosis was performed close to the renal pelvis after cutting the ureter obliquely (method 2), which enlarged the diameter of the ureteral anastomosis 2-fold. The incidence of stenosis of ureteric anastomosis was 12.5% (3/24) using method 1, whereas this complication was avoided completely (0/45) using method 2. Furthermore, the risk of injury to the ureter was reduced, as isolation of the ureter was limited. These modifications improved the last delicate step in the procedure of rat kidney transplantation.