Baicalin attenuates lipopolysaccharide-induced renal tubular epithelial cell injury by inhibiting the TXNIP/NLRP3 signalling pathway via increasing miR-223-3p expression.

Baicalin has been used in China to treat inflammation-related diseases, such as inflammation-induced acute kidney injury (AKI). However, the specific mechanism of baicalin remains unclear. To observe the protective effects of baicalin on lipopolysaccharide (LPS)-induced inflammatory injury of renal tubular epithelial cells (HK-2 cells) and to explore its protective mechanism. LPS (1 mg/L) was used to induce an HK-2 cell inflammatory injury model in vitro. The cells were divided into seven groups: the normal control group, LPS-induced group, LPS plus 5 µmol/L baicalin treatment group, LPS plus 15 µmol/L baicalin treatment group, LPS plus 25 µmol/L baicalin treatment group, LPS plus 50 µmol/L baicalin treatment group, and LPS plus 75 µmol/L baicalin treatment group. 3-(4,5-dimethyl-2-thiazolyl)-2,5- diphenyl-2-H-tetrazolium bromide (MTT) assay was employed for detecting the relative survival rate of HK-2 cells. Enzyme-linked immunosorbent assay was used for detecting the levels of inflammatory factors, including interleukin-6 (IL-6), IL-1ß, and tumor necrosis factor-α (TNF-α). Moreover, the expression of inducible nitric oxide synthase (iNOS); cyclooxygenase-2 (COX-2); nuclear factor kB65 (NF-κB65); phosphorylated NF-κB inhibitory protein-α (p-IκB-α); NF-κB inhibitory protein (IκB); human thioredoxin interacting protein (TXNIP); and human NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) were determined by Western blot analysis. The expression levels of NLRP3 and TXNIP mRNA and miR-223-3p were determined by RT-PCR. Results found that the relative survival rate of HK-2 cells treated with different baicalin concentrations was significantly increased (P<0.05) and the levels of the inflammatory factors IL-6, IL-1ß, and TNF-α were significantly decreased (P<0.05) compared with those of the LPS-induced group. The expression levels of the inflammatory proteins inducible nitric oxide synthase and cyclooxygenase-2 and the genes expressions of TXNIP and NLRP3 were significantly decreased in the cells (P<0.05), while the expression level of miR-223- 3p was significantly increased (P<0.05). These changes were induced in a dose-dependent manner. The results suggest that baicalin significantly inhibited the expression of inflammation-related proteins and alleviated LPS-induced inflammatory injury in HK-2 cells. The mechanism may be associated with the inhibition of activation of the TXNIP/NLRP3 inflammatory pathway, which might be mediated by increased expression of miR-223-3p. Thus, NLRP3 is a regulatory target of miR-223-3p.

Neonatal mouse-derived engineered cardiac tissue: a novel model system for studying genetic heart disease.

RATIONALE: Cardiomyocytes cultured in a mechanically active 3-dimensional configuration can be used for studies that correlate contractile performance to cellular physiology. Current engineered cardiac tissue (ECT) models use cells derived from either rat or chick hearts. Development of a murine ECT would provide access to many existing models of cardiac disease and open the possibility of performing targeted genetic manipulation with the ability to directly assess contractile and molecular variables. OBJECTIVE: To generate, characterize, and validate mouse ECT with a physiologically relevant model of hypertrophic cardiomyopathy. METHODS AND RESULTS: We generated mechanically integrated ECT using isolated neonatal mouse cardiac cells derived from both wild-type and myosin-binding protein C (cMyBP-C)-null mouse hearts. The murine ECTs produced consistent contractile forces that followed the Frank-Starling law and accepted physiological pacing. cMyBP-C-null ECTs showed characteristic acceleration of contraction kinetics. Adenovirus-mediated expression of human cMyBP-C in murine cMyBP-C-null ECT restored contractile properties to levels indistinguishable from those of wild-type ECT. Importantly, the cardiomyocytes used to construct the cMyBP-C(-/-) ECT had yet to undergo the significant hypertrophic remodeling that occurs in vivo. Thus, this murine ECT model reveals a contractile phenotype that is specific to the genetic mutation rather than to secondary remodeling events. CONCLUSIONS: Data presented here show mouse ECT to be an efficient and cost-effective platform to study the primary effects of genetic manipulation on cardiac contractile function. This model provides a previously unavailable tool to study specific sarcomeric protein mutations in an intact mammalian muscle system.

Intra-parenchymal injection of tumor necrosis factor-alpha and interleukin 1-beta produces dopamine neuron loss in the rat.

Inflammatory processes are thought to underlie the dopamine (DA) neuron loss seen in Parkinson's disease (PD). However, it is not known if the inflammation precedes that loss, or is a consequence of it. We injected tumor necrosis factor alpha (TNFalpha) and interleukin 1 beta (IL-1beta) into the median forebrain bundle to determine if these pro-inflammatory cytokines could induce DA neuron loss in the substantia nigra (SN) by themselves. The magnitude of the DA cell loss as well as the decreases in striatal DA, were both dose and time to sacrifice dependent. Injecting both cytokines together produced greater cell losses and DA reductions than that seen when the cytokines were injected alone. The DA neuron loss seen was more pronounced in the lateral nigra and its ventral tier and similar to that seen when other toxins are injected. These data suggest that TNFalpha and IL-1beta can induce DA neuron loss by themselves and could produce DA neuron loss independent of other inflammatory events.

Combined toxicity of prenatal bacterial endotoxin exposure and postnatal 6-hydroxydopamine in the adult rat midbrain.

We previously reported that injection of the Gram (-) bacteriotoxin, lipopolysaccharide (LPS), into gravid females at embryonic day 10.5 led to the birth of animals with fewer than normal dopamine (DA) neurons when assessed at postnatal days (P) 10 and 21. To determine if these changes continued into adulthood, we have now assessed animals at P120. As part of the previous studies, we also observed that the pro-inflammatory cytokine tumor necrosis factor alpha (TNFalpha) was elevated in the striatum, suggesting that these animals would be more susceptible to subsequent DA neurotoxin exposure. In order to test this hypothesis, we injected (at P99) 6-hydroxydopamine (6OHDA) or saline into animals exposed to LPS or saline prenatally. The results showed that animals exposed to prenatal LPS or postnatal 6OHDA alone had 33% and 46%, respectively, fewer DA neurons than controls, while the two toxins combined produced a less than additive 62% loss. Alterations in striatal DA were similar to, and significantly correlated with (r(2)=0.833) the DA cell losses. Prenatal LPS produced a 31% increase in striatal TNFalpha, and combined exposure with 6OHDA led to an 82% increase. We conclude that prenatal exposure to LPS produces a long-lived THir cell loss that is accompanied by an inflammatory state that leads to further DA neuron loss following subsequent neurotoxin exposure. The results suggest that individuals exposed to LPS prenatally, as might occur had their mother had bacterial vaginosis, would be at increased risk for Parkinson's disease.

Measurements of the cross-bridge attachment/detachment process within intact sarcomeres by surface plasmon resonance.

We have developed a surface plasmon resonance (SPR) system to monitor the cross-bridge attachment/detachment process within intact sarcomeres from mouse heart muscle. SPR occurs when laser light energy is transferred to surface plasmons that are resonantly excited in a metal (gold) film. This resonance manifests itself as a minimum in the reflection of the incident laser light and occurs at a characteristic angle. The angle of the SPR occurrence depends on the dielectric permittivity of the sample medium adjacent to the gold film. Purified sarcomeric preparations are immobilized onto the gold film in the presence of a relaxing solution. Replacement of the relaxing solution with increasing Ca(2+) concentration solution activates the cross-bridge interaction and produces an increase in the SPR angle. These results imply that the interaction of myosin heads with actin within an intact sarcomere changes the dielectric permittivity of the sarcomeric structure. In addition, we further verify that SPR measurements can detect the changes in the population of the attached cross-bridges with altered concentrations of phosphate, 2,3-butanedione monoxime, or adenosine triphosphate at a fixed calcium concentration, which have been shown to reduce the force and increase the cross-bridge population in attached state. Thus, our data provide the first evidence that the SPR technique allows the monitoring of the cross-bridge attachment/detachment process within intact sarcomeres.

Superiority of mitral valve replacement with preservation of subvalvular structure to conventional replacement in severe rheumatic mitral valve disease: a modified technique and results of one-year follow up.

BACKGROUND AND AIM OF THE STUDY: Mitral valve replacement with preservation of the subvalvular apparatus (MRVP) has been proven superior to conventional mitral valve replacement (MVR). We devised a simple modified MVRP method in this prospective, randomized study to investigate the clinical effects and one-year follow up echocardiographic results of MVRP compared with MVR in patients with severe rheumatic mitral insufficiency (MI). METHODS: Sixty-eight patients with severe rheumatic MI with or without stenosis were randomized to MVRP (n = 35) and MVR (n = 33) groups. In MVRP patients, the preserved tissue was pulled back posteriorly to the posterior wall of the left ventricle, then plicated and reaffixed to one-fourth of the annular circumference in the posterior annulus, in order to prevent left ventricular outflow tract (LVOT) obstruction. Clinical data including cumulative ventricular arrhythmias and use of inotropes were collected. Echocardiography examination was performed before surgery, and at five days, three months and one year thereafter. RESULTS: There were no preoperative differences patient data. The cross-clamp time was 2.2 min longer in MVRP patients. The one-month mortality rate after surgery was lower in MVRP patients (2.9% versus 15.2%, p = 0.074). Mechanical ventilation and ICU times were shorter in the MVRP group (17.6 versus 24.8 and 52.5 versus 70.6 h, p = 0.001 and 0.1, respectively). There were fewer ventricular arrhythmias and less need for inotropic support in this group. One year follow up echocardiography data showed better preserved left ventricular ejection fraction (LVEF) and better recovery of heart size after MRVP. There was no indication that preserved valvular tissue interfered with mechanical valve function, or caused LVOT obstruction. CONCLUSION: This modified MVRP technique is simple, effective and without risk of LVOT obstruction. In severe rheumatic MI patients the outcome of MVRP is superior to that of conventional MVR in term's of mortality, postoperative care needs, left ventricular function and heart dimensions.

Pramipexole attenuates the dopaminergic cell loss induced by intraventricular 6-hydroxydopamine.

The D3 preferring dopamine agonist pramipexole has been shown to attenuate the cell loss induced by levodopa in vitro. Pramipexole was herein evaluated in the 6-hydroxydopamine lesion model to determine its in vivo effect. Rats were treated with pramipexole or saline before and after an intracerebroventricular 6-hydroxydopamine injection. In the preliminary study, 6-hydroxydopamine produced a 68% reduction in striatal dopamine and a 62% loss in tyrosine hydroxylase immunoreactive (THir) cell counts in the substantia nigra. Pramipexole treated animals exhibited a 29% and a 27% reduction in striatal dopamine and THir cell counts, respectively. THir cell counts and striatal dopamine were significantly correlated. In the stereological study, 6-hydroxydopamine reduced THir cell counts by 47% in saline treated animals and 26% in pramipexole treated animals. These data demonstrate that pramipexole attenuates the biochemical and THir cell changes normally produced by 6-hydroxydopamine consistent with its neuroprotective actions in vitro.

Effect of verbascoside on decreasing concentration of oxygen free radicals and lipid peroxidation in skeletal muscle.

AIM: To detect the effects of verbascoside on decreasing the concentration of oxygen free radicals (OFR) and lipid peroxidation in skeletal muscle resulting from exhaustive exercise. METHODS: Electron spin resonance (ESR) technique and thiobarbituric acid reaction (TBAR) method were used to detect the concentration of OFR in intact gastrocnemius muscle and the contents of milondialdehyde (MDA) in muscle homogenate. RESULTS: Verbascoside decreased the concentration of OFR (P < 0.05) and the level of lipid peroxidation (P < 0.05) in muscle caused by exercise. CONCLUSION: Verbascoside has the effects of reducing oxidative stress in muscle caused by exhaustive exercise by decreasing the concentration of free radicals and the level of lipid peroxidation.

Changes in membrane fluidity and lipid peroxidation of skeletal muscle mitochondria after exhausting exercise in rats.

We studied the effects of exhausting exercise and exercise training on skeletal muscle mitochondrial membrane fluidity and lipid peroxidation in rats. The first part of the study involved 60 untrained rats divided into six equal groups. Of the total number 10 rats were sedentary and acted as controls. The remaining 50 rats exercised to exhaustion and were sacrificed at 0-h, 24-h, 48-h, 72-h, and 96-h post-exercise. The second part of the study involved 40 rats which were divided into four equal groups. Of these 10 rats were sedentary and acted as controls. The remaining 30 rats underwent 8 weeks of exercise training. They were then subjected to a single period of exhausting exercise and were sacrificed at 0-h, 24-h and 48-h post-exercise. Membrane fluidity was measured using the fluorescence polarization method. Lipid peroxidation was estimated by determining the thiobarbituric acid-reactive substances (TBARS) in mitochondria. In the untrained rats, mitochondrial fluorescence polarization and TBARS contents were significantly increased post-exercise compared with the sedentary controls (P < 0.05). They did not return to near control levels until 96 h and 48 h, respectively. In the trained rats, fluorescence polarization was raised compared with the sedentary controls but this was significantly lower than those measured at the same times of the untrained group post-exercise (P < 0.05). Exhausting exercise decreased membrane fluidity and increased lipid peroxidation in rat skeletal muscle mitochondria. These effects were relieved to some extent by exercise training.

Both the antioxidant and D3 agonist actions of pramipexole mediate its neuroprotective actions in mesencephalic cultures.

Pramipexole (PPX) is a full intrinsic activity, direct-acting dopamine (DA) agonist possessing 7-fold higher affinity for D3 than for D2 receptors. It also is a potent antioxidant. PPX was previously shown to be neuroprotective because it dose dependently attenuated the DA neuron loss produced by levodopa in mesencephalic cultures. Several different drugs with properties similar to PPX were studied here to better understand the mechanism or mechanisms responsible for this neuroprotective effect. The D3-preferring agonist 7-hydroxy-diphenylaminotetralin (7-OH-DPAT) and the D3 antagonist U99194, respectively, increased and decreased the neuroprotective effects of PPX in a dose-dependent fashion. Addition of the selective D2 agonist U95666 or the D2/D3 antagonists domperidone or raclopride did not affect PPX's neuroprotective effect. Interestingly, 7-OH-DPAT by itself did not attenuate the DA neuron loss produced by levodopa. However, when 7-OH-DPAT was combined with a low dose of the antioxidants U101033E or alpha-tocopherol, the toxic effects of levodopa were attenuated. Similar results were observed when the D3-preferring agonist PD128, 907 was studied. In addition, media conditioned by exposure of mesencephalic cultures incubated with all D3-preferring agonists studied was shown to enhance the growth of DA neurons in freshly harvested recipient cultures implicating a D3-mediated trophic activity in the neuroprotective effect. These data suggest that PPX's neuroprotective actions in the levodopa toxicity model are a consequence of its combined actions as a D3 receptor agonist and an antioxidant.

Lengthening of a one-bone forearm. A sequel of neonatal osteomyelitis.

An 8-year-old girl presented with marked shortening of the right forearm due to destruction of both the radius and ulna secondary to neonatal osteomyelitis. A one-bone forearm operation was performed to achieve a stable forearm. Two years later, the one-bone forearm was lengthened for 6 months by callus distraction (callotasis) achieving 12 cm of extra length. The patient was last followed up at the age of 16. The appearance and functional outcome of the right upper limb had been improved and she was independent in all activities of daily living.

Partial purification of a pramipexole-induced trophic activity directed at dopamine neurons in ventral mesencephalic cultures.

We previously demonstrated that media conditioned by exposure to ventral mesencephalic (VM) cultures in the presence of pramipexole (PPX) and other drugs with dopamine (DA) D3 properties, increased the growth and survival of DA neurons in recipient VM cultures. This trophic activity was heat-labile and not present in parietal cortex cultures or cultures pretreated with the DA neuron toxin MPP+. In an effort to begin to identify the protein(s) responsible for this trophic effect, we compared the conditioned media from normal VM cultures, VM cultures incubated with PPX, and VM cultures pretreated with MPP+ and treated with PPX. Neutralization studies using anti-GDNF and anti-BDNF failed to reduce the conditioned media transfer effect, and Millipore Ultrafree centrifugation studies placed the mol.wt. of the activity around 30 kDa. SDS separation revealed three potential bands of interest. A 35-kDa band was present in normal cultures, increased in PPX-incubated cultures, and absent in MPP+-pretreated/PPX-incubated cultures. This conforms to the effect the protein concentrates used to produce these gels had on the growth of DA neurons in VM cultures. Since VM cultures grown in neural basal media, which inhibits the growth of glia, still responded to PPX in a dose-dependent fashion, the trophic activity may be a DA autotrophic factor. However, the gels also revealed two bands at approximately 31 and 55 kDa that were reduced by exposure to PPX and present in MPP+-pretreated cultures. The possibility that these are neuroinhibitory factors that are also regulated by PPX therefore cannot be ruled out.

The conservative management of acute pyogenic iliopsoas abscess in children.

We describe three cases of acute pyogenic abscess of the iliopsoas in children treated conservatively. Two patients had image-guided aspiration and one was managed with antibiotics alone. All made a complete recovery. Acute pyogenic abscess of the iliopsoas in children can be treated effectively and safely with intravenous antibiotics and image-guided aspiration of the abscess.