[Investigate of the etiology and prevention status of liver cirrhosis].

Objective: To investigate the etiology, prevention and treatment status, and their corresponding regional differences of the patients with liver cirrhosis in China, in order to provide scientific basis for the development of diagnosis and control strategies in China. Methods: Clinical data of patients diagnosed with liver cirrhosis for the first time through January 1, 2018 to December 31, 2020 from 50 hospitals in seven different regions of China were collected and analyzed retrospectively, and the difference of etiology, treatment, and their differences in various regions were analyzed. Results: A total of 11 861 cases with liver cirrhosis were included in the study. Thereinto, 5 093 cases (42.94%) were diagnosed as compensated cirrhosis, and 6 768 cases (57.06%) had decompensated cirrhosis. Notably, 8 439 cases (71.15%) were determined as chronic hepatitis B-caused cirrhosis, 1 337 cases (11.27%) were alcoholic liver disease, 963 cases (8.12%) were chronic hepatitis C, 698 cases (5.88%) were autoimmune liver disease, 367 cases (3.09%) were schistosomiasis, 177 cases (1.49%) were nonalcoholic fatty liver, and 743 cases (6.26%) of other types of liver disease. There were significant differences in the incidence of chronic hepatitis B, chronic hepatitis C, alcoholic liver disease, fatty liver, schistosomiasis liver disease, and autoimmune liver disease among the seven regions (P<0.001). Only 1 139 cases (9.60%) underwent endoscopic therapy, thereinto, 718 cases (6.05%) underwent surgical therapy, and 456 cases (3.84%) underwent interventional therapy treatment. In patients with compensated liver cirrhosis, 60 cases (0.51%) underwent non-selective ß receptor blockers(NSBB), including 59 cases (0.50%) underwent propranolol and 1 case (0.01%) underwent carvedilol treatment. In patients with decompensated liver cirrhosis, 310 cases (2.61%) underwent NSBB treatment, including 303 cases (2.55%) underwent propranolol treatment and 7 cases (0.06%) underwent carvedilol treatment. Interestingly, there were significant differences in receiving endoscopic therapy, interventional therapy, NSBB therapy, splenectomy and other surgical treatments among the seven regions (P<0.001). Conclusion: Currently, chronic hepatitis B is the main cause (71.15%) of liver cirrhosis in several regions of China, and alcoholic liver disease has become the second cause (11.27%) of liver cirrhosis in China. The three-level prevention and control of cirrhosis in China should be further strengthened.

Analysis of synonymous codon usage in FAD7 genes from different plant species.

In this study, the codon bias of the FAD7 genes among 10 different plant species was analyzed to identify general patterns of codon usage in the FAD7 genes. Our results showed that U-ended or A-ended codons were preferentially used in FAD7 for dicots, whereas G-ended or C-ended codons were preferentially used in FAD7 for monocots. An ENC-plot showed that some other factors may influence the codon usage of FAD7, except mutation bias in plant species. A correlation analysis between the codon adaptation index and GC or GC3s contents demonstrated that the codon usage bias of the FAD7 gene in plant species could be influenced by the gene expression level. The cluster analysis of relative synonymous codon usage values and phylogenetic trees of protein sequences for FAD7 genes confirm that the codon preference of FAD7 is influenced by genetic relationships. Moreover, Arabidopsis thaliana and Nicotiana tabacum were predicted to be the most appropriate expression hosts for the FAD7 genes from dicots, and Zea mays may be suitable for the expression of the FAD7 genes from monocots. Our results provide useful insights into the evolutionary relationships of plant species.

Novel compounds selectively enhance delta subunit containing GABA A receptors and increase tonic currents in thalamus.

Inhibition in the brain is dominated by the neurotransmitter gamma-aminobutyric acid (GABA); operating through GABA(A) receptors. This form of neural inhibition was presumed to be mediated by synaptic receptors, however recent evidence has highlighted a previously unappreciated role for extrasynaptic GABA(A) receptors in controlling neuronal activity. Synaptic and extrasynaptic GABA(A) receptors exhibit distinct pharmacological and biophysical properties that differentially influence brain physiology and behavior. Here we used a fluorescence-based assay and cell lines expressing recombinant GABA(A) receptors to identify a novel series of benzamide compounds that selectively enhance, or activate alpha4beta3delta GABA(A) receptors (cf. alpha4beta3gamma2 and alpha1beta3gamma2). Utilising electrophysiological methods, we illustrate that one of these compounds, 4-chloro-N-[6,8-dibromo-2-(2-thienyl)imidazo[1,2-a]pyridine-3-yl benzamide (DS1) potently (low nM) enhances GABA-evoked currents mediated by alpha4beta3delta receptors. At similar concentrations DS1 directly activates this receptor and is the most potent known agonist of alpha4beta3delta receptors. 4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridine-3-yl benzamide (DS2) selectively potentiated GABA responses mediated by alpha4beta3delta receptors, but was not an agonist. Recent studies have revealed a tonic form of inhibition in thalamus mediated by the alpha4beta2delta extrasynaptic GABA(A) receptors that may contribute to the regulation of thalamocortical rhythmic activity associated with sleep, wakefulness, vigilance and seizure disorders. In mouse thalamic relay cells DS2 enhanced the tonic current mediated by alpha4beta2delta receptors with no effect on their synaptic GABA(A) receptors. Similarly, in mouse cerebellar granule cells DS2 potentiated the tonic current mediated by alpha6betadelta receptors. DS2 is the first selective positive allosteric modulator of delta-GABA(A) receptors and such compounds potentially offer novel therapeutic opportunities as analgesics and in the treatment of sleep disorders. Furthermore, these drugs may be valuable in elucidating the physiological and pathophysiological roles played by these extrasynaptic GABA(A) receptors.

Basal and touch-evoked fos-like immunoreactivity during experimental inflammation in the rat.

Fos-immunoreactivity can readily be induced in spinal cord neurones by noxious, but to a much more limited extent, by innocuous peripheral stimuli. The present study has investigated whether low intensity stimuli and electrical stimulation of A beta afferents elicit greater c-fos expression during the behavioural sensory hypersensitivity generated by experimental peripheral inflammation. We have examined the time-course of c-fos expression after inflammation produced by either an intra-plantar injection of the irritant turpentine oil or of complete Freund's adjuvant (CFA). In the former case, a significant initial expression in all dorsal horn laminae was followed by a gradual decrease, whereas after CFA injection, an initial expression limited to the superficial laminae subsequently extended into the deep laminae, with a decrease at 24 h and an increase in labelling at later times. Low intensity touch stimuli repeated for 10 min, when applied at 24 h and 48 h after CFA injection, elicited a significant increase in the number of Fos-immunoreactive neurons in both the superficial and deep laminae of the dorsal horn compared to non-inflamed animals. Electrical stimulation of the sciatic nerve 24 h post-CFA injection, at a strength sufficient only to activate A beta-afferents fibres (100 microA, 50 microseconds, 10 min), also elicited a significant increase in labelling relative to the same stimuli applied in control animals, especially in laminae V-VI. The present results demonstrate that low intensity cutaneous stimuli elicit a significantly greater increase in c-fos expression in dorsal horn neurons during peripheral inflammation and that A beta-afferent input contributes to this, a finding that may relate to the allodynia experienced during inflammation.

The expression of bradykinin B(1) receptors on primary sensory neurones that give rise to small caliber sciatic nerve fibres in rats.

The bradykinin B(1) receptor has been considered as an important mediator for inflammatory pain. In the present study, we have investigated the fibre types of sciatic nerve primary sensory neurones that express B(1) receptors by retrograde tracing in combination with immunohistochemical staining, or double-immunohistochemical staining. Approximately 12% of the A-fibre dorsal root ganglion neurones, retrogradely labelled from an intra-sciatic nerve injection of fluorescein isothiocyanate-conjugated cholera toxin B subunit, were B(1) receptor-immunoreactive. Over 70% of the small diameter dorsal root ganglion neurones, retrogradely labelled from an intra-sciatic nerve injection of tetramethylrhodamine isothiocyanate-conjugated wheat germ agglutinin, were B(1) receptor-immunoreactive. Over 50% of the (predominantly non-peptidergic) C-fibre dorsal root ganglion neurones, retrogradely labelled from an intra-sciatic nerve injection of fluorescein isothiocyanate-conjugated Bandeiraea simplicifolia isolectin B4, were B(1) receptor-immunoreactive. When calcitonin gene-related peptide, which is contained mainly in small caliber C- and A(delta)-fibre primary afferents, and B(1) receptors were stained with a double-immunofluorescent method, over 80% of the calcitonin gene-related peptide-positive dorsal root ganglion neurones were B(1) receptor-immunoreactive. From these results we suggest that B(1) receptors are predominantly expressed by small diameter primary afferent neurones that give rise to sciatic nerve fibres, which include both peptidergic and non-peptidergic C-fibres and A(delta)-fibres. Since peripheral nociceptive information is primarily transmitted by C- and A(delta)-fibres, B(1) receptors may be involved in the modulation of nociceptive transduction or transmission.

Localization of N-methyl-D-aspartate NR2B subunits on primary sensory neurons that give rise to small-caliber sciatic nerve fibers in rats.

In the present study we have used immunohistochemical staining and retrograde tracing techniques to investigate the relationship between the N-methyl-D-aspartate receptor NR2B subunits and small-diameter primary afferent dorsal root ganglion neurons that give rise to the sciatic nerve fibers. Three days after an intra-sciatic nerve injection of tetramethyl rhodamine isothiocyanate-conjugated wheat germ agglutinin which labels small-diameter primary afferents, many NR2B and wheat germ agglutinin-double-labeled cells ( approximately 70% of wheat germ agglutinin-labeled neurons) were observed in the L5 dorsal root ganglia. Three days after an intra-sciatic nerve injection of fluorescein isothiocyanate-conjugated Bandeiraea simplicifolia agglutinin isolectin B4 which labels predominantly non-peptidergic C-fiber primary afferents, NR2B and Bandeiraea simplicifolia agglutinin isolectin B4 double-labeled neurons ( approximately 90% of Bandeiraea simplicifolia agglutinin isolectin B4-labeled neurons) were also observed in the L5 dorsal root ganglion. Three days after an intra-sciatic nerve injection of fluorescein isothiocyanate-conjugated cholera toxin B subunit, only approximately 40% of cholera toxin B subunit-labeled neurons were NR2B positive and those labeled neurons tended to be small-sized. When calcitonin gene-related peptide and NR2B were labeled by a double immunofluorescent staining technique, we found that the majority of calcitonin gene-related peptide-positive neurons was NR2B immunoreactive (>90% of calcitonin gene-related peptide-positive neurons, and approximately 60% of NR2B-positive neurons) as well. Size frequency analysis also demonstrated that NR2B subunits were predominantly localized on the small and medium-sized neurons. These results suggest that NR2B subunits are predominantly expressed on small diameter primary afferents, and these NR2B containing N-methyl-D-aspartate receptors may play a role in the modulation of neurotransmitter release from primary afferent terminals.

Nerve growth factor contributes to the generation of inflammatory sensory hypersensitivity.

Experimental inflammation produced by an intraplantar injection of complete Freund's adjuvant results in local sensory hypersensitivity and up-regulates the neuropeptides substance P and calcitonin gene related peptide in the primary sensory neurons innervating the inflamed tissue. The inflammation also elevates nerve growth factor levels in the skin. Systemic administration of anti-NGF neutralizing antibodies prevent the behavioral sensitivity, the up-regulation of neuropeptides and the inflammation-induced expression of the immediate early gene c-fos in dorsal horn neurons, without modifying swelling and erythema. Elevation of the neurotrophin NGF in the periphery is a major contributor, therefore, of inflammatory pain.

Vanilloid receptor homologue, VRL1, is expressed by both A- and C-fiber sensory neurons.

The capsaicin receptor (VR1) homologue, VRL1, is thought to be responsible for transducing high-threshold heat responses in Adelta-fiber neurons. In the present study, the expression of VRL1 by A- or C-fiber sensory neurons in rats was investigated by using a VRL1 and 200 kDa neurofilament (NF200, an A-fiber marker) double immunohistochemical staining method. Approximately 46% of VRL-positive neurons were NF200 positive. Though double-labeled neurons tended to be medium to large, many VRL1 single-labeled neurons were large. Dense VRL1 immunoreactivity was also found in laminae I and II of the spinal dorsal horn, where nociceptive Adelta- and C-fibers normally terminate. These results suggest that both C-fiber and Adelta-fiber primary sensory neurons express VRL1, and VRL1 may play an important role in nociception.

Co-localization of 5-HT(1B/1D/1F) receptors and glutamate in trigeminal ganglia in rats.

Anti-migraine triptan drugs are 5-HT(1B/1D) receptor agonists which are thought to block the neurotransmitter/neuropeptide release from sensory nerve terminals and directly constrict blood vessel smooth muscles. In the present study, we have investigated the anatomical basis for a possible modulation of glutamate release from trigeminal ganglion neurons by 5-HT(1B/1D) receptor agonists and by 5-HT1F receptor agonists, using double immunohistochemical staining technique in the rat. The majority of 5-HT1B, 5-HT1D or 5-HT1F receptor positive neurons were also glutamate positive, but both 5-HT1B, 5-HT1D or 5-HT1F receptor single-labeled and glutamate single-labeled neurons were observed. These results suggest that 5-HT(1B/1D/1F) receptor agonists may modulate glutamate release, and that one mechanism of their anti-migraine action could be the blockade of glutamate release.

Basal expression of bradykinin B(1) receptor in the spinal cord in humans and rats.

The bradykinin B(1) receptor has been considered as a receptor induced by tissue injury and inflammation mainly in the peripheral tissues. In the present study, we have investigated whether there is a basal expression in the spinal cord by both reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical staining methods. Southern blotting of the DNA reverse-transcribed from human and rat spinal cord mRNA and amplified by polymerase chain reaction (PCR) showed a substantial basal B(1) receptor expression in both human and rat spinal cord. Immunohistochemical staining demonstrated B(1)-positive neurons in the spinal cord dorsal horn, suggesting that the B(1) receptor is constitutively expressed by spinal neurons.

The progressive tactile hyperalgesia induced by peripheral inflammation is nerve growth factor dependent.

Experimental peripheral inflammation results in cutaneous mechanical hypersensitivity, and repeated low intensity mechanical stimulation of the inflamed skin induces a progressively incrementing hyperalgesia. We have now examined whether the elevation in nerve growth factor (NGF) induced by the inflammation contributes to this progressive hyperalgesia. An i.p. injection of anti-NGF antiserum (5 microliters g-1) 1 h before induction of inflammation by intraplantar complete Freund's adjuvant (CFA) injection and 24 h after, both reduced the basal inflammatory hypersensitivity and significantly attenuated the progressive increase of spontaneous activity, touch-, pinch- and A beta-afferent-evoked responses, and the progressive reduction of the mechanical threshold of biceps femoris/semitendinosus alpha motoneurones normally evoked by repeated (every 5 min) tactile stimulation of the inflamed hindpaw, in decerebrate-spinal rats. NGF contributes, therefore, to the progressive tactile hyperalgesia elicited by repeated touch stimulation of inflamed tissue.

Basal expression of bradykinin B1 receptor in peripheral sensory ganglia in the rat.

The bradykinin B1 receptor is thought to be induced by tissue injury and inflammation. In the present study, we have investigated whether there is a basal expression of B1 receptor in dorsal root ganglion (DRG) and trigeminal ganglion neurons in rats. A substantial number of neurons in both DRGs and trigeminal ganglia were found to be B1-immunoreactive in rats. Both small and medium-sized DRG neurons were B1-immunoreactive, suggesting that they are likely to be Adelta- or C-fibre neurons which are involved in nociceptive transmission. These results support a possible role for B1 receptors in the modulation of nociceptive sensory transmission.

Naloxone blocks the release of opioid peptides in periaqueductal gray and N. accumbens induced by intra-amygdaloid injection of morphine.

The working hypothesis that the periaqueductal gray (PAG), N. accumbens and amygdala were connected serially in a unidirectional loop for antinociception, in which Metenkephalin and beta-endorphin were considered to be two important analgesic neurotransmitters, was examined by simultaneously perfusing the PAG and N. accumbens after microinjection of morphine into the amygdala. Intra-amygdaloid injection of morphine increased the release of enkephalins and beta-endorphin in the PAG and N. accumbens. When the perfusion fluid contained 3 microM of naloxone, the release of enkephalins and beta-endorphin was reduced in both the PAG and the N. accumbens. These results do not support the hypothesis of a unidirectional loop and its putative sequence.

Naloxone blocks opioid peptide release in periaqueductal gray and amygdala elicited by morphine injected into N. accumbens.

Previous studies from this laboratory suggested that the periaqueductal gray (PAG), nucleus accumbens, and amygdala might take part in a serial, unidirectional mesolimbic loop to play their roles in pain modulation. It has been proposed that morphine injected into one of these nuclei would cause the release of opioid peptides in one nucleus after another. This working hypothesis was examined in the present study by perfusing simultaneously the PAG and the amygdala after microinjection of morphine into the N. accumbens. It was found that microinjection of morphine increased the content of immunoreactive enkephalins (ir-ENK) and immunoreactive beta-endorphin (ir-beta-EP) in the perfusate of the PAG and the amygdala. When the perfusion fluid contained 3 microM of naloxone, the increase of ir-ENK and ir-beta-EP was reduced significantly. These results indicate that the three nuclei were not serially connected in a unidirectional loop.

Noxious stimulation accelerated the expression of c-fos protooncogene in cholecystokininergic and dopaminergic neurons in the ventral tegmental area.

Cholecystokininergic (CCK) and dopaminergic systems in the midbrain have been implicated in pain modulation. In the present study, Fos immunoreactivity was used as a marker of neuronal activity to investigate if subcutaneous injection of formalin (a model of noxious stimulation) could activate the CCKergic and dopaminergic neurons in the midbrain. Noxious stimulation increased significantly the numbers of Fos-positive CCKergic and dopaminergic neurons in the ventral tegmental area (VTA), whereas no significant changes in the numbers of Fos-positive CCKergic or dopaminergic neurons were observed in other midbrain nuclei. These results indicate that the VTA may play a role in pain modulation.

Neurochemical studies on the mesolimbic circuitry of antinociception.

Previous studies using the technique of microinjection into brain nuclei indicated that the periaqueductal gray (PAG), nucleus accumbens, habenula and amygdala play an essential role in pain modulation and that these nuclei possibly act through a 'mesolimbic neural loop' to exert an analgesic effect, in which Met-enkephalin (MEK) and beta-endorphin (beta-EP) have been implicated as the two major opioid peptides involved in antinociception. In the present study performed in rabbits, intracranial microinjection was supplemented with push-pull perfusion and radioimmunoassay to determine whether the release of enkephalins (ENK) and beta-EP was increased in these nuclei when the putative neural circuit was activated by morphine administered into one of the nuclei. The results showed: (1) microinjection of morphine into the PAG increased the release of ENK and beta-EP in the N. accumbens, and vice versa; (2) microinjection of morphine into the N. accumbens increased the release of ENK and beta-EP in the amygdala, and vice versa; (3) morphine microinjected into the PAG caused an increase in the release of ENK and beta-EP in the amygdala and vice versa, although the release of ENK in PAG was statistically not significant. These results indicate that PAG, N. accumbens and amygdala are connected in a network served by a positive feedback circuitry.

A-fibres sprouting from lamina I into lamina II of spinal dorsal horn after peripheral nerve injury in rats.

We have examined the labeling pattern in the spinal dorsal horn by an intra-sciatic nerve injection of cholera toxin B subunit conjugated horseradish peroxidase (HRP) after transection of the posterior cutaneous nerve and inferior gluteal nerve, and found that the cholera toxin B subunit conjugated HRP labeling in lamina I was expanding into lamina II and there was a shrinking gap between lamina I and lamina III. This result suggests that A-fibre sprouting arise after peripheral nerve injury, but mainly from small calibre Adelta-fibres which terminate in lamina I.

Further studies on interactions between periaqueductal gray, nucleus accumbens and habenula in antinociception.

Previous findings from this laboratory with the intracerebral microinjection technique suggested that the periaqueductal gray (PAG), nucleus accumbens, and habenula might constitute a unidirectional loop to play their roles in pain modulation. In the present study we demonstrate that intra-habenular injection of naloxone antagonizes the analgesia elicited by morphine injected into the periaqueductal gray (PAG) and that intra-accumbens injection of naloxone is capable of attenuating the analgesic effects of morphine injected into the habenula. These results indicate that the relationships between these nuclei may be more complex than the putative unidirectional loop.

Tachykinin NK1 receptor antagonist RP67580 attenuates progressive hypersensitivity of flexor reflex during experimental inflammation in rats.

We have now examined whether the tachykinin NK1 receptor is involved in mediating progressive hypersensitivity of spinal flexor motoneurons induced by repeated peripheral stimulation of inflamed tissue in decerebrate-spinal rats. The mechanical threshold of spinal flexor motoneurons was significantly decreased, and the touch- and pinch-evoked responses significantly increased, 48 h after intra-plantar injection of 100 microliters complete Freund's adjuvant. The threshold was further progressively decreased and the touch- and pinch-evoked responses increased over the 80 min testing period. Subcutaneous injection of the tachykinin NK1 receptor antagonist RP67580 (2-[1-imino-2-(2-methoxy phenyl) ethyl]-7,7 diphenyl-4 perhydroisoindolone-(3aR,7aR)) (20 min prior to the beginning of the test) at 1 mg and 10 mg/kg significantly attenuated the progressive decrease of mechanical withdrawal threshold, and the progressive increase of the touch- and pinch-evoked responses. The inactive enantiomer RP68651 (2-[1-imino-2-(2-methoxy phenyl) ethyl]-7,7 diphenyl-4 perhydroisoindolone-(3aS,7aS)) at 1 mg and 10 mg/kg had no significant effect. The present results indicate that substance P and its preferred tachykinin NK1 receptor are involved in mediating progressive hypersensitivity during inflammation.

Resection of sciatic nerve re-triggers central sprouting of A-fibre primary afferents in the rat.

We have investigated whether nerve injury or target deprivation is responsible for the injury induced central sprouting of A-fibres. Cholera toxin B subunit conjugated horseradish peroxidase was used to trace the termination of A-fibre primary afferents. Transection of the sciatic nerve induces central sprouting of sciatic myelinated A-fibre primary afferents into the spinal dorsal horn lamina II, which normally is the termination site of unmyelinated C-fibre primary afferents. The sprouting A-fibre terminals withdrew from lamina II after six to eight months. A second cut to the previously sectioned and ligated sciatic nerve re-triggered the central sprouting of A-fibre primary afferents into the spinal dorsal horn lamina II, suggesting that nerve injury per se rather than the deprivation of target tissues is the cause of central sprouting of A-fibre primary afferents.