Glycyrrhizic acid, as an inhibitor of HMGB1, alleviates bleomycin-induced pulmonary toxicity in mice through the MAPK and Smad3 pathways.

AIM: High-mobility group box 1 (HMGB1) protein has been noticed particularly for its pivotal role in several pathologies. However, the relevance between HMGB1 and pathological progress in lung toxicity still remains unclear. In the study, we evaluated the effect of glycyrrhizic acid as an HMGB1 inhibitor on the early inflammation and late fibrosis in bleomycin-induced pulmonary toxicity in mice. METHODS: We established a bleomycin-induced pulmonary toxicity model to detect the relevance between HMGB1 and pathological changes in the early inflammatory and late fibrotic stages. RESULTS: We found that bleomycin-induced increase in inflammatory cytokines interleukin (IL)-ß1, tumor necrosis factor (TNF)-α, monocyte chemotactic protein (MCP)-1, and inflammatory lesions in lung tissue in the early stage of the model. However, markers of fibrosis such as transforming growth factor (TGF)-ß1 and α-smooth muscle actin (α-SMA) were significantly elevated on day 7 after bleomycin instillation. Interestingly, HMGB1 also began to rise on day 7, rather than in the early inflammatory phase. However, early (from day 0 to 14 after bleomycin instillation) or late (from day 14 to 28) intervention with HMGB1 neutralizing antibody or glycyrrhizic acid alleviated inflammation and fibrosis through down-regulating the inflammatory signaling mitogen-activated protein kinase (MAPK) and fibrotic signaling Smad3 pathway. CONCLUSION: Our results suggested that HMGB1 mediates both inflammation and fibrosis in this model. The development of high-potency and low-toxicity HMGB1 inhibitors may be a class of potential drugs for the treatment of pulmonary fibrosis.

Salidroside Ameliorates Diabetic Neuropathic Pain in Rats by Inhibiting Neuroinflammation.

More than half of diabetic patients suffer from intractable neuropathic pain. As inflammation plays an important role in diabetic neuropathic pain, anti-inflammatory drugs might have therapeutic potentials for neuropathic pain. Salidroside (SAL), a phenylpropanoid glucoside, modulates a variety of cell functions, including inflammation. Here, we explored anti-nociceptive and anti-inflammatory effects of SAL on Zucker diabetic fatty rats with type 2 diabetes (DM rats). DM rats were tested for mechanical and thermal hyperalgesia using von Frey filament and plantar hot box test, respectively. The anti-nociceptive effect of chronic SAL (25-100 mg/kg, per oral) treatment was tested. The expression of inflammatory cytokines (TNF-α and IL-1ß) and P2X7 receptors in spinal cord and sciatic nerve were measured with ELISA. SAL alleviated mechanical and thermal hyperalgesia and reduced TNF-α and IL-1ß in sciatic nerve and spinal cord in DM rats. Furthermore, SAL reduced P2X7 receptor upregulation in spinal cord of DM rats and directly inhibited P2X7 receptors expressed in HEK293 cells. This study provides evidence that SAL attenuated nociception in diabetic neuropathic pain rat models probably through inhibiting neuroinflammation and P2X7 receptors.

Genistein alleviates anxiety-like behaviors in post-traumatic stress disorder model through enhancing serotonergic transmission in the amygdala.

Post-traumatic stress disorder (PTSD) is a chronic psychiatric disorder, characterized by intense fear, and increased arousal and avoidance of traumatic events. The current available treatments for PTSD have limited therapeutic value. Genistein, a natural isoflavone, modulates a variety of cell functions. In this study, we tested anti-anxiety activity and underlying mechanisms of genistein in a PTSD rat model. The rats were trained to associate a tone with foot shock delivery on day 0, then fear conditioning was performed on day 7, 14 and 21. Genistein (2-8mg/kg) was injected intraperitoneally daily for 7 days. The anti-anxiety effects of genistein were measured by contextual freezing behavior and elevated plus maze. By the end of the experiments, the amygdala was extracted and subject to neurochemistry analysis. Genistein alleviated contextual freezing behavior and improved performance in elevated plus maze dose-dependently in PTSD rats. Furthermore, in these rats, genistein enhanced serotonergic transmission in the amygdala, including upregulation of tryptophan hydroxylase, serotonin, and phosphorylated (p)-CaMKII and p-CREB, as well. Genistein exerts anti-anxiety effects on a PTSD model probably through enhancing serotonergic system and CaMKII/CREB signaling pathway in the amygdala.

Contribution of Hippocampal 5-HT3 Receptors in Hippocampal Autophagy and Extinction of Conditioned Fear Responses after a Single Prolonged Stress Exposure in Rats.

One of the hypotheses about the pathogenesis of posttraumatic stress disorder (PTSD) is the dysfunction of serotonin (5-HT) neurotransmission. While certain 5-HT receptor subtypes are likely critical for the symptoms of PTSD, few studies have examined the role of 5-HT3 receptor in the development of PTSD, even though 5-HT3 receptor is critical for contextual fear extinction and anxiety-like behavior. Therefore, we hypothesized that stimulation of 5-HT3 receptor in the dorsal hippocampus (DH) could prevent hippocampal autophagy and the development of PTSD-like behavior in animals. To this end, we infused SR57227, selective 5-HT3 agonist, into the DH after a single prolonged stress (SPS) treatment in rats. Three weeks later, we evaluated the effects of this pharmacological treatment on anxiety-related behaviors and extinction of contextual fear memory. We also accessed hippocampal autophagy and the expression of 5-HT3A subunit, Beclin-1, LC3-I, and LC3-II in the DH. We found that SPS treatment did not alter anxiety-related behaviors but prolonged the extinction of contextual fear memory, and such a behavioral phenomenon was correlated with increased hippocampal autophagy, decreased 5-HT3A expression, and increased expression of Beclin-1 and LC3-II/LC3-I ratio in the DH. Furthermore, intraDH infusions of SR57227 dose-dependently promoted the extinction of contextual fear memory, prevented hippocampal autophagy, and decreased expression of Beclin-1 and LC3-II/LC3-I ratio in the DH. These results indicated that 5-HT3 receptor in the hippocampus may play a critical role in the pathogenesis of hippocampal autophagy, and is likely involved in the pathophysiology of PTSD.

SiRNA-mediated serotonin transporter knockdown in the dorsal raphe nucleus rescues single prolonged stress-induced hippocampal autophagy in rats.

The neurobiological mechanisms underlying the development of post-traumatic stress disorder (PTSD) remain elusive. One of the hypotheses is the dysfunction of serotonin (5-HT) neurotransmission, which is critically regulated by serotonin transporter (SERT). Therefore, we hypothesized that attenuation of SERT gene expression in the hippocampus could prevent hippocampal autophagy and the development of PTSD-like behavior. To this end, we infused SLC6A4 siRNAs into the dorsal raphe nucleus (DRN) to knockdown SERT gene expression after a single prolonged stress (SPS) treatment in rats. Then, we evaluated the effects of SERT gene knockdown on anxiety-related behaviors and extinction of contextual fear memory. We also examined the histological changes and the expression of Beclin-1, LC3-I, and LC3-II in the hippocampus. We found that SPS treatment did not alter anxiety-related behaviors but prolonged the extinction of contextual fear memory, and such a behavioral phenomenon was correlated with increased hippocampal autophagy, decreased 5-HT level, and increased expression of Beclin-1 and LC3-II/LC3-I ratio in the hippocampus. Furthermore, intra-DRN infusion of SLC6A4 siRNAs promoted the extinction of contextual fear memory, prevented hippocampal autophagy, increased 5-HT level, and decreased expression of Beclin-1 and LC3-II/LC3-I ratio. These results indicated that SERT may play a critical role in the pathogenesis of hippocampal autophagy, and is likely involved in the development of PTSD.

Tormentic acid inhibits LPS-induced inflammatory response in human gingival fibroblasts via inhibition of TLR4-mediated NF-κB and MAPK signalling pathway.

OBJECTIVE: Periodontal disease is one of the most prevalent oral diseases, which is associated with inflammation of the tooth-supporting tissues. Tormentic acid (TA), a triterpene isolated from Rosa rugosa, has been reported to exert anti-inflammatory effects. The aim of this study was to investigate the anti-inflammatory effects of TA on lipopolysaccharide (LPS)-stimulated human gingival fibroblasts (HGFs). METHODS: The levels of inflammatory cytokines such as interleukin (IL)-6 and chemokines such as IL-8 were detected by enzyme-linked immunosorbent assay (ELISA). The expression of Toll-like receptor 4 (TLR4), nuclear factor kappa B (NF-κB), IκBα, p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) was determined by Western blotting. RESULTS: The results showed that Porphyromonas gingivalis LPS significantly upregulated the expression of IL-6 and IL-8. TA inhibited the LPS-induced production of IL-6 and IL-8 in a dose-dependent manner. Furthermore, TA inhibited LPS-induced TLR4 expression; NF-κB activation; IκBα degradation; and phosphorylation of ERK, JNK, and P38. CONCLUSION: TA inhibits the LPS-induced inflammatory response in HGFs by suppressing the TLR4-mediated NF-κB and mitogen-activated protein kinase (MAPK) signalling pathway.

The role of RIP1 and RIP3 in the development of aplastic anemia induced by cyclophosphamide and busulphan in mice.

This study aimed to investigate the role of RIP1 and RIP3 in the pathogenesis of aplastic anemia (AA) induced by cyclophosphamide and busulphan in mice. Animals were randomly divided into three groups: the control group, the AA group, and the Nec-1 group. Mouse AA model was established by intraperitoneal injection of cyclophosphamide (40 mg/kg/d) and busulfan (20 mg/kg/d) for 12 days. The Nec-1 group mice received intraperitoneal injection of Nec-1 (2 mg/kg/d) for 12 days prior to intraperitoneal injection of cyclophosphamide (40 mg/kg/d) and busulfan (20 mg/kg/d) for 12 days. The control mice received intraperitoneal injection of equal volume of saline. At 12 h after the last intraperitoneal injection, blood and bone marrow tissues were collected from mice. Peripheral blood cells were analyzed using hematology analyzer and the histological changes of bone marrow tissues were examined using scanning electron microscopy (SEM). The levels of RIP3 and RIP3 in bone marrow were measured using Western blot analysis and the interaction of RIP1 and RIP3 proteins was investigated on the basis of immunoprecipitation analysis. ELISA was used to measure the levels of IL-6, TNF-α, and FLT-3L in bone marrow tissue supernatant. Apoptosis and necrosis of bone marrow cells were analyzed using flow cytometry. Western blot showed that the expression of RIP1 and RIP3 was significantly increases in AA mice compared to the normal controls. Immunoprecipitation detected the pro-necrotic RIP1-RIP3 complex, suggesting that RIP1 and RIP3 mediated necroptosis may involved in the damage of bone marrow cells. Compared to the AA mice, Nec-1 group mice exhibited significantly increase of peripheral blood cells and mononuclear cells in bone marrow tissues and decrease of the apoptosis/necrosis of bone marrow cells. In addition, we observed significant decrease of IL-6, TNF-α, and FLT-3L in bone marrow tissue supernatant in the Nec-1 group mice compared to AA mice. Our results suggest that Nec-1 can prevent the development of AA by inhibiting bone marrow cells necrosis and the production of inflammatory mediators. RIP1 and RIP3-mediated necroptosis may involve in the pathogenesis of AA induced by cyclophosphamide and busulfan in mice.

Expression level of IL-6 secreted by bone marrow stromal cells in mice with aplastic anemia.

Parasecretion of the hematopoietic cytokines is considered as one of the mechanisms account for bone marrow hematopoiesis disorder. In this study, the level of IL-6 secreted by bone marrow stromal cells from a mouse model of aplastic anemia was analyzed. The aplastic anemia mouse model was established with cyclophosphamide in combination with chloramphenicol and (60)Co γ radiation. The impairment of bone marrow hematopoiesis induced by irradiation and chemotherapeutic drugs was subsequently characterized by peripheral blood cell count, pathomorphological changes, and apoptosis rate. Furthermore, the in vitro proliferation of bone marrow stromal cells (BMSC) and the IL-6 secretion levels of BMSC were analyzed. In our model of aplastic anemia, the number of peripheral blood cells and bone marrow cells (BMC) were notably decreased, and the apoptosis rate of BMC increased. Furthermore, the proliferation of BMSC was obviously impeded while the IL-6 secretion levels of BMSC significantly increased. The findings of our study suggested that the IL-6 secretion level may be enhanced to some extent by the induction of aplastic anemia caused by irradiation and chemotherapeutic drugs and that the abnormal level of IL-6 might probably interfere with the stability of the bone marrow hematopoietic microenvironment.

[Aboveground biomass of natural Castanopsis carlesii-Schima superba community in Xiaokeng of Nanling Mountains, South China].

By the method of clear cutting, a measurement was made on the aboveground biomass (AGB) of 24-year old natural Castanopsis carlesii-Schima superba community in an 800 m2 plot in Xiaokeng of Nanling Mountains, South China. The distribution patterns of the total AGB in different forest layers, tree species, and tree layer organs were investigated, and the AGB regression models were constructed. The results showed that when constructing the AGB regression models, more than 12 samples would be feasible. Based on the measured AGB of 265 felled trees, the AGB models of mixed broadleaved species were AGB = 0. 128D(2.372) and AGB = 242.331(D2H)(0.947). The single tree's AGB model of C. carlesii, S. superba, and Cunninghamia lanceolata was also established. The total AGB of the forest community was 115.20 t x hm(-2), of which, the AGB of tree layer, understory layer, liana, and litter layer was 111.25, 1.01, 0.36, and 2.58 t x hm(-2), respectively. The AGB of C. carlesii and S. superba took up 39.1% and 28.7% of the tree layer AGB, respectively. The AGB of tree stem and branch-leaf occupied 81.0% and 19.0% of the tree layer AGB, respectively.

Synergetic effect of yihuo qingyi decoction (see text) and recombinant staphylokinase in treatment of severe acute pancreatitis of rats.

OBJECTIVE: To investigate the effect of recombinant staphylokinase (r-Sak) and the Chinese medicine Yihuo Qingyi Decoction ((see test) Herbal decoction for severe acute pancreatitis) in the treatment of the severe acute pancreatitis (SAP) in rats, and to observe the synergistic effect of the two. METHODS: One hundred and sixty-two adult male SD rats with the body mass of 250-280 g were randomly divided into the following 5 groups: sham operation group (n = 18), control group (n = 36), Yihuo Qingyi Decoction treatment group (n = 36), r-Sak treatment group (n = 36), and Yihuo Qingyi Decoction plus r-Sak treatment group (n = 36). The SAP ratmodel was prepared by retrograde injection of 5% sodium taurocholate into the cholangiopancreatic duct. Two days before modeling, Yihuo Qingyi Decoction was intragastrically administrated, and r-Sak was intraperitoneally injected. The survival rate within 18 h after modeling was determined. The pancreatic blood flow, the weight of ascites, and the serum amylase and lipase were investigated at 6 h, 12 h, and 18kh after modeling, and the pancreatic tissue was examined under light microscopy to see its pathological change. RESULTS: The 18 h survival count of group A, B, C, D and E rats was 9, 2, 6, 7 and 8 respectively. After r-Sak and Yihuo Qingyi Decoction intervention, the serum amylase and lipase and the weight of ascites were significantly decreased, especially in group E.18 h after modeling, the level of the serum amylase and lipase and the weight of ascites in group E was 1 100 +/- 118 U x L(-1), 1 000 +/- 150 U x L(-1) and 13.40 +/- 1.80 g respectively, obviously lower than that of group B (P < 0.05). After SAP was induced, the pancreatic blood flow showed a tendency to decrease, but the decrease extent in the treatment groups was smaller than that in the control group. 18h after modeling, the pancreatic blood flow in group B and group E was 30.16 +/- 8.96 mL x 100 g(-1) x min(-1), and 129.10 +/- 42.58 mL x 100 g(-1) x min respectively, there was significant difference (P < 0.05). The pathological change of the pancreatic tissue was alleviated in the treatment groups. CONCLUSION: Both r-Sak and Yihuo Qingyi Decoction play a beneficial role in the treatment of rat SAP and there is a synergistic effect between the two.

Correlation between the distribution of SP and CGRP immunopositive neurons in dorsal root ganglia and the afferent sensation of preputial frenulum.

The aim of this study was to explore the distribution of substance P (SP) and calcitonin gene-related peptide (CGRP) immunoreactive nerve terminals in the penis prepuce and the preputial frenulum. The possible correlation between SP- and CGRP-immunopositive neurons in dorsal root ganglia (DRG) and the afferent sensation of the penile preputial frenulum is also discussed. Immunohistochemistry showed SP- and CGRP-positive nerve terminals in the epidermal basal layer of the prepuce and frenulum in adult human males. The majority of the nerve terminals presented as bundles of different lengths and a few as enlarged nodosities. The density of SP- and CGRP-immunopositive nerve terminals in the preputial frenulum was significantly higher than those in the penis prepuce (P<0.01). Fluoro-Gold (FG) retrograde tracing method was used to trace the origin of nerve terminals in Sprague-Dawley rats. SP and CGRP immunofluorescence labeling was employed to detect the distribution of SP- and CGRP-immunoreactive neurons in DRG. FG retro-labeled neurons were localized in L(6) -DRG and S(1) -DRG. All the FG/SP and FG/CGRP double-labeled neurons were medium or small-sized. One-third of the FG-labeled neurons were SP-immunoreactive, and a half of them CGRP-immunoreactive in L(6) -DRG and S(1) -DRG, respectively. The FG/SP/CGRP-labeled neurons accounted for one fifth of the FG retro-labeled neurons. Taken together, these data suggest that the SP- and CGRP-immunopositive nerve fibers may participate in the transmission of afferent sensation in the preputial frenulum.

The myocardial protective effects of puerarin on STZ-induced diabetic rats.

To investigate the myocardial protective effects of puerarin on streptozotocin (STZ)-induced diabetic rats and the possible mechanism were involved. 45 Sprague-Dawley male rats were randomly divided into 3 groups as diabetic group (intraperitoneally injected STZ 65 mg/kg), puerarin treatment group (intraperitoneally injected STZ 65 mg/kg, and intraperitoneally injected puerarin 100 mg/kg/day for 4 weeks), and control group (intraperitoneally injected saline 6 ml/kg). Four weeks after the model induction, the myocardial changes were observed by H-E stain and Transmission electron microscopy, the alteration of thrombospondin-1 (TSP-1) protein and mRNA expression in the myocardium were also assessed by immunohistochemistry and real-time PCR. The heart function of three groups' rats was tested by Langendorff isolated in vivo heart perfusion. The differences in the data of weight and blood sugar of diabetic between puerarin treatment and normal groups were significant after 4 weeks (P<0.01). Our results demonstrated that diabetic myocardial ultrastructural changes included myofibrillar disarrangements and mitochondria disruption. These damages were significantly less severe in the puerarin treatment group compared with the diabetic group. A significant decrease of TSP-1 expression was observed in the puerarin treated rats' myocardium compared to the diabetic rats (P<0.01). Left ventricular systolic end pressure (LVSEP) and left ventricular developed pressure (LVDP) of puerarin treatment group were also significantly increased compared to diabetic group (P<0.01). Altogether puerarin could improve the left ventricular function of diabetic rats and showed protective effects of myocardium by decreasing the TSP-1 expression in myocardium of diabetic rats.

[Substance P and/or calcitonin gene-related peptide immunoreactive neurons in dorsal root ganglia possibly involved in the transmission of nociception in rat penile frenulum].

OBJECTIVE: To study the relationship between substance P (SP) and/or calcitonin gene-related peptide (CGRP) immunoreactive neurons in dorsal root ganglia (DRG) and the transmission of nociception in the penile frenulum of rats. METHODS: The fluoro-gold (FG) retrograde tracing method was used to trace the origin of nerve terminals in the penile frenulum of rats. And SP and/or CGRP immunofluorescence labeling was employed to detect the distribution of SP and/or CGRP immunoreactive neurons in DRG. RESULTS: FG retrograde tracing showed that the FG retrolabeled neurons were localized in L6-DRG and S1-DRG. SP and/or CGRP immunofluorescence labeling indicated that a large number of DRG neurons were SP- and CGRP-immunoreactive, different in size, bright red and bright green respectively in color, and arranged in rows or spots among nerve bundles. All the FG/SP and FG/CGRP double-labeled neurons were medium or small-sized. One third of the FG-labeled neurons were SP-immunoreactive, and a half of them CGRP-immunoreactive in L6-DRG and S1-DRG respectively. The FG/SP/CGRP-labeled neurons accounted for one fifth of the FG retro labeled neurons. CONCLUSION: SP- and CGRP-immunoreactive neurons in L6-DRG and SI-DRG of rats may be involved in the transmission of nociception in rat penile frenulum.