Content Determination and Chemical Clustering Analysis of Tanshinone and Salvianolic Acid in Salvia spp.

Salvia miltiorrhiza is one of the famous traditional Chinese medicines for treating cardiovascular and cerebrovascular diseases. Tanshinone and phenolic acids are the main active compounds of Salvia miltiorrhiza, whereas the distribution patterns of the two kinds of components are still unclear among Salvia spp. In this work, high-performance liquid chromatography was applied to analyze the distribution patterns of major components in the roots and leaves of 58 Salvia spp. The results showed that the distribution patterns of tanshinone and phenolic acids in Salvia spp. varied significantly. Phenolic acid components such as rosmarinus acid, caffeic acid, and danshensu are widely distributed in the roots and leaves, and phenolic acids in the leaves of Salvia spp. are generally higher than that in roots. Tanshinones are mainly detected in the roots of Salvia przewalskii, Salvia trijuga, Salvia castanea, and Salvia yunnanensis. The content of major components of the different species varied significantly. The content of phenolic acids in most Salvia spp. generally followed the pattern of salvianolic acid B > rosmarinic acid > danshensu > caffeic acid both in the roots and leaves. Tanshinone IIA and cryptotanshinone were the main lipophilic components of Salvia spp. distributed in southwest China. A correlation between the distribution pattern of tanshinone and the genetic relationship of species was indicated in the work. This research systematically reveals the distribution patterns of tanshinone and phenolic acids in Salvia spp., providing a theoretical basis for the development and utilization of medicinal resources of Salvia.

Exploring the potential mechanisms of Danshen against COVID-19 via network pharmacology analysis and molecular docking.

Danshen, a prominent herb in traditional Chinese medicine (TCM), is known for its potential to enhance physiological functions such as blood circulation, immune response, and resolve blood stasis. Despite the effectiveness of COVID-19 vaccination efforts, some individuals still face severe complications post-infection, including pulmonary fibrosis, myocarditis arrhythmias and stroke. This study employs a network pharmacology and molecular docking approach to investigate the potential mechanisms underlying the therapeutic effects of candidate components and targets from Danshen in the treatment of complications in COVID-19. Candidate components and targets from Danshen were extracted from the TCMSP Database, while COVID-19-related targets were obtained from Genecards. Venn diagram analysis identified common targets. A Protein-Protein interaction (PPI) network and gene enrichment analysis elucidated potential therapeutic mechanisms. Molecular docking evaluated interactions between core targets and candidate components, followed by molecular dynamics simulations to assess stability. We identified 59 potential candidate components and 123 targets in Danshen for COVID-19 treatment. PPI analysis revealed 12 core targets, and gene enrichment analysis highlighted modulated pathways. Molecular docking showed favorable interactions, with molecular dynamics simulations indicating high stability of key complexes. Receiver operating characteristic (ROC) curves validated the docking protocol. Our study unveils candidate compounds, core targets, and molecular mechanisms of Danshen in COVID-19 treatment. These findings provide a scientific foundation for further research and potential development of therapeutic drugs.

Germplasm resources and secondary metabolism regulation in Reishi mushroom (Ganoderma lucidum).

Ganoderma lucidum is a valuable medical macrofungus with a myriad of diverse secondary metabolites, in which triterpenoids are the major constituents. This paper introduced the germplasm resources of genus Ganoderma from textual research, its distribution and identification at the molecular level. Also we overviewed G. lucidum in the components, the biological activities and biosynthetic pathways of ganoderic acid, aiming to provide scientific evidence for the development and utilization of G. lucidum germplasm resources and the biosynthesis of ganoderic acid.

Soil applied silicon and manganese combined with foliar application of 5-aminolevulinic acid mediate photosynthetic recovery in Cd-stressed Salvia miltiorrhiza by regulating Cd-transporter genes.

Salvia miltiorrhiza is an important medicinal plant that experiences significant growth and biomass losses when cultivated on cadmium (Cd) contaminated soils. High Cd accumulation in plant tissues also increases the risk of metal entry into the food chain. In this study, we proposed that Cd accumulation in S. miltiorrhiza can be restricted through plant growth regulators and nutrient management. Therefore, S. miltiorrhiza seedlings were transplanted into mixed nutrient soil for two weeks, then treated with 30 mg kg-1 CdCl2, 200 mg kg-1 Na2SiO3·9H2O, and 100 mg kg-1 MnSO4, and simultaneously sprayed with 10 mg L-1 ALA on the leaves one week later. This study showed that elevated Cd accumulation significantly reduced plant growth and biomass. This growth inhibition damaged photosynthetic machinery and impaired carbon assimilation. In contrast, 5-aminolevulinic acid (ALA) significantly promoted the biomass of S. miltiorrhiza, and the dry weight of plants treated with ALA combined with manganese (Mn)/silicon (Si) increased by 42% and 55% as compared with Cd+Mn and Cd+Si treatments. Exogenously applied ALA and Si/Mn significantly activated antioxidant enzymes and promoted the growth recovery of S. miltiorrhiza. Further, exogenous ALA also reduced the Cd concentration in S. miltiorrhiza, especially when combined with Si. Compared with the Cd+Si treatment, the Cd+Si+ALA treatment reduced the Cd concentration in roots and leaves by 59% and 60%, respectively. Gene expression analysis suggested that ALA and Si significantly up-regulated genes associated with Cd transport. Other genes related to heavy metal tolerance mechanisms are also regulated to cope with heavy metal stress. These results indicated that the combined action of ALA and Si/Mn could reduce Cd-toxicity by increasing chlorophyll content and changing oxidative stress and can also affect Cd accumulation by regulating gene expression.

Aptamer biorecognition-triggered hairpin switch and nicking enzyme assisted signal amplification for ultrasensitive colorimetric bioassay of kanamycin in milk.

A colorimetric aptasensing strategy for detection of kanamycin was designed based on aptamer biorecognition and signal amplification assisted by nicking enzyme. The aptamer of kanamycin was designed to be contained in the metastable state hairpin DNA. The target DNA as recycling DNA was located in the loop of hairpin DNA. The presence of kanamycin stimulates the continuous actions, including specific recognition of the aptamer to kanamycin, the hybridization between target DNA and signal probe, the cleavage function of nicking enzyme. The actions induced accumulation of numerous free short sequences modified by platinum nanoparticles (PtNPs), which can catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB)-H2O2 to produce a colorimetric response. The aptasensor exhibited good selectivity and sensitivity for kanamycin in milk with a detection limit as low as 0.2 pg·mL-1. In addition, the proposed assay is potentially to be extended for other antibiotics detection in foods by adapting the corresponding aptamer sequence.

DNAzyme-powered DNA walking machine for ultrasensitive fluorescence aptasensing of kanamycin.

A DNAzyme-powered DNA walking machine was constructed to develop the fluorescence aptasensing for sensitive detection of kanamycin. The aptamer for kanamycin is partially hybridized with complementary DNA (cDNA) modified on magnetic beads (MBs). The specific interaction of target and aptamer triggered the cDNA to be free tentatively, which captured walker DNA. Then the autonomous motion of DNA walker on MBs surface was propelled via DNAzyme digestion of recognition sites. The signal probe was separated, and the amplified fluorescence signal was achieved by the accumulation of the signal probe. Kanamycin was used as a model analyte, and the developed assay achieves a detection limit of 0.00039 ng·mL-1 (S/N = 3) within a linear detection range from 0.001 to 2000 ng·mL-1. This aptasensing strategy can be extended for detection of other antibiotics by adapting corresponding target recognition aptamer sequence. Graphical abstract The fluorescence aptasensing for sensitive detection of kanamycin based on DNAzyme-powered DNA walking machine was constructed.

[Effects of different carbon sources on growth and active component contents in Salvia miltiorrhiza and S. castanea f. tomentosa hairy roots].

Salvia miltiorrhiza(Sm) and Salvia castanea f. tomentosa(Sc) hairy roots were used as experimental materials to study the effects of six different carbon sources, galactose, fructose, lactose, glucose, arabinose and sucrose(control), on fresh weight, dry weight, contents and yields of salvianolic acids and tanshinones. The results showed that galactose was most beneficial to the growth of two kinds of hairy roots, while lactose and arabinose were not conducive to their growth. As for Sm hairy roots, fructose significantly promoted the accumulation of salvianolic acid B, and the content increased by 5.801 times and 10.151 times compared with the control group, respectively. Glucose significantly promoted the accumulation of salvianolic acids. The content and yield of rosmarinic acid were 7.674 times and 9.260 times of that of the control group, and the content and yield of salvianolic acid B were 5.532 times and 6.675 times of the control group. For the hairy roots of Sc, galactose significantly increased the content and yield of rosmarinic acid, reaching 7.820 times and 9.944 times of the control group, respectively. Fructose promoted the increase of the content and yield of cryptotanshinone, reaching 9.242 times and 6.609 times of the control group, respectively. The study confirmed the optimal carbon source for the hairy root culture of Sm and Sc, and provided theoretical guidance for large-scale production of Sm drug-derived components and the utilization of Sc.

A fluorometric aptamer-based assay for ochratoxin A by using exonuclease III-assisted recycling amplification.

A fluorometric assay is described for ochratoxin A (OTA) using an aptamer. The method is based on exonuclease-assisted recycling amplification. The OTA-binding aptamer partially hybridizes with complementary DNA (cDNA) that is released when the aptamer recognizes OTA. Then, cDNA hybridizes with a specifically designed hairpin DNA. Next, short ssDNA and cDNA are, respectively, released by exonuclease III catalyzed hydrolysis of the dsDNA. The cDNA induces the next ring opening and digestion. The short ssDNA captures the sDNA that is labeled with fluorescent FAM and is absorbed on graphene oxide (GO). The green fluorescence of the sDNA/GO system is quenched but is recovered if the sDNA is released from GO. This assay is high sensitive, works in the 5 nM to 200 nM OTA concentration range and has a 0.96 nM lower detection limit. It was applied to the quantitation of OTA in spiked wine and coffee samples. Graphical abstractSchematic of a fluorometric assay based on exonuclease-assisted recycling amplification for quantitative monitoring of OTA without the need of sample separation and multiple washing steps.

Simultaneous detection of five pig viruses associated with enteric disease in pigs using EvaGreen real-time PCR combined with melting curve analysis.

In recent years, a series of porcine diarrhea viruses such as porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), rotaviruses of group A (RVA), rotaviruses of group C (RVC), and porcine circovirus 2 (PCV2) caused enormous economic losses all over the world. While any of these viruses is capable to cause disease alone, there is often concurrent infection with more than one virus on pig farms. In this study, a multiplex real-time PCR method based on EvaGreen fluorescent dye and melting curve analysis was established to simultaneously detect these five viruses in a single closed tube. Five distinct melt peaks were obtained with different melting temperature (Tm) value corresponding to each of the five viruses. This method was highly sensitive to detect and distinguish TGEV, RVA, RVC, PEDV and PCV2 with the limits of detection ranging from 5 to 50 copies/µL. The intra-assay and inter-assay reproducibility were good with coefficient of variation of Tm and cycle threshold values less than 0.32% and 2.86%, respectively. Testing of 90 field samples by the single and multiplex real-time PCR assays demonstrated a concordance of 91.1%. Thus, the EvaGreen multiplex real-time PCR is a rapid, sensitive and low-cost diagnostic tool for differential detection and routine surveillance of TGEV, RVA, RVC, PEDV and PCV2 in pigs.

Detection and differentiation of five diarrhea related pig viruses utilizing a multiplex PCR assay.

Porcine viral diarrhea, mainly caused by porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine group A rotaviruses (RVA), porcine group C rotaviruses (RVC) and porcine circovirus 2 (PCV2), is a serious global problem, resulting in substantial economic losses to the swine industry. For fast and reliable diagnosis of the causative agent associated with viral diarrhea in pigs, an inexpensive and easy to perform gel-based multiplex PCR assay was developed in this study to detect and differentiate the different viruses by amplicon size. The assay was able to distinguish between all five viral agents without cross-reacting with other non-target pig viruses. The detection limits of the assay per reaction were 5 copies for PEDV, TGEV, RVC and PCV2 and 50 copies for RVA for the singleplex assays and 50 copies when all five viruses were multiplexed. Sixty-nine field samples were used to validate the developed multiplex assay. The overall prevalence of positive samples was 44.9% (31/69). PCV2 was detected in 37.7% of the samples, PEDV and RVC each in 4.3%, TGEV in 2.9%, and RVA was detected in 1.4% of the samples tested. A total of 5.8% of the samples were co-infected by two or more viruses, and the results of the multiplex assay were in agreement to those obtained by single PCR assays. These findings suggest that the developed cost-effective multiplex assay is specific, sensitive, and will serve as a valuable diagnostic tool for the rapid differential detection of these five viruses and for molecular epidemiological studies and diarrhea disease management.

[Advances in chloroplast expression of recombinant proteins in higher plants].

In recent years, gene engineering is developing rapidly and many recombinant proteins have been expressed. The use of plant bioreactor to express specific pharmaceutical proteins provides a new way for the prevention and treatment of some important diseases in human beings. Nowadays, chloroplast genetic transformation and expression system has become a research hotspot in plant bioreactor. Higher plant chloroplasts have unique advantages in the expression of recombinant proteins due to their special structures and inherited characteristics: such as high expression, site-specific integration, and the maternal inheritance characteristics of exogenous genes. The maternal inheritance of chloroplast is helpful for biological safety of transgene escaping by pollens. Many important pharmaceutical proteins have been successfully expressed in plant chloroplasts. As a chloroplast transformation model of higher plants, tobacco has made significant progress in the expression of pharmaceutical proteins, such as vaccine antigens, antibodies, and other important recombinant proteins. Chloroplast genetic transformation in higher plants also provides new techniques and methods for the study of chloroplast gene expression and regulation mechanisms. In order to provide a new idea for the development of chloroplast expression platform and the expression of important pharmaceutical proteins, this review outlined the progress of chloroplast genetic transformation system in higher plants, including the chloroplast transformation principle, vector construction, expression of recombinant proteins and important pharmaceutical proteins, and the effects of recombinant proteins expression on plant metabolism and traits.

Development and validation of a multiplex conventional PCR assay for simultaneous detection and grouping of porcine bocaviruses.

Porcine bocavirus (PBoV), a newly described porcine parvovirus, has received attention because it can be commonly identified in clinically affected pigs including pigs with post-weaning multisystemic wasting syndrome (PWMS) and pigs with diarrhea. In recent years, novel PBoVs have been identified and were classified into three genogroups, but the ability to detect and classify these novel PBoVs is not comprehensive to date. In this study, a multiplex conventional PCR assay for simultaneous detection and grouping of PBoVs was developed by screening combinations of mixed primer pairs followed by optimization of the PCR conditions. This method exclusively amplifies targeted fragments of 531bp from the VP1 gene of PBoV G1, 291bp from the NP1 gene of PBoV G2, and 384bp from the NP1/VP1 gene of PBoV G3. The assay has a detection limit of 1.0×10(3)copies/µL for PBoV G1 4.5×10(3) for PBoV G2 and 3.8×10(3) for PBoV G3 based on testing mixed purified plasmid constructs containing the specific viral target fragments. The performance of the multiplex PCR assay was comparable to that of the single PCRs which used the same primer pairs. Using the newly established multiplex PCR assay, 227 field samples including faeces, serum and tissue samples from pigs were investigated. All three PBoV genogroups were detected in the clinical samples with a detection rate of 1.3%, 2.6% and 12.3%, respectively for PBoV G1, G2 and G3. Additionally, coinfections with two or more PBoV were detected in 1.7% of the samples investigated. These results indicate the multiplex PCR assay is specific, sensitive and rapid, and can be used for the detection and differentiation of single and multiple infections of the three PBoV genogroups in pigs.

Rapid detection and grouping of porcine bocaviruses by an EvaGreen(®) based multiplex real-time PCR assay using melting curve analysis.

Several novel porcine bocaviruses (PBoVs) have been identified in pigs in recent years and association of these viruses with respiratory signs or diarrhea has been suggested. In this study, an EvaGreen(®)-based multiplex real-time PCR (EG-mPCR) with melting curve analysis was developed for simultaneous detection and grouping of novel PBoVs into the same genogroups G1, G2 and G3. Each target produced a specific amplicon with a melting peak of 81.3 ± 0.34 °C for PBoV G1, 78.2 ± 0.37 °C for PBoV G2, and 85.0 ± 0.29 °C for PBoV G3. Non-specific reactions were not observed when other pig viruses were used to assess the EG-mPCR assay. The sensitivity of the EG-mPCR assay using purified plasmid constructs containing the specific viral target fragments was 100 copies for PBoV G1, 50 for PBoV G2 and 100 for PBoV G3. The assay is able to detect and distinguish three PBoV groups with intra-assay and inter-assay variations ranging from 0.13 to 1.59%. The newly established EG-mPCR assay was validated with 227 field samples from pigs. PBoV G1, G2 and G3 was detected in 15.0%, 25.1% and 41.9% of the investigated samples and coinfections of two or three PBoV groups were also detected in 25.1% of the cases, indicating that all PBoV groups are prevalent in Chinese pigs. The agreement of the EG-mPCR assay with an EvaGreen-based singleplex real-time PCR (EG-sPCR) assay was 99.1%. This EG-mPCR will serve as a rapid, sensitive, reliable and cost effective alternative for routine surveillance testing of multiple PBoVs in pigs and will enhance our understanding of the epidemiological features and possible also pathogenetic changes associated with these viruses in pigs.

Development of an EvaGreen-based multiplex real-time PCR assay with melting curve analysis for simultaneous detection and differentiation of six viral pathogens of porcine reproductive and respiratory disorder.

Concurrent infection of pigs with two or more pathogens is common in pigs under intensive rearing conditions. Porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), Japanese encephalitis virus (JEV) and pseudorabies virus (PRV) are all associated with reproductive or respiratory disorders or both and can cause significant economic losses in pig production worldwide. An EvaGreen-based multiplex real-time PCR (EG-mPCR) with melting curve analysis was developed in this study for simultaneous detection and differentiation of these six viruses in pigs. This method is able to detect and distinguish PCV2, PPV, PRRSV, CSFV, JEV and PRV with the limits of detection ranging from 100 to 500 copies/µL, high reproducibility, and intra-assay and inter-assay variation ranging from 0.11 to 3.20%. After validation, a total of 118 field samples were tested by the newly developed EG-mPCR. PCV2 was identified in 23%, PPV in 15%, PRRSV in 17% and PRV in 5% of the samples. Concurrent PCV2 and PRRSV infection was detected in 6.7%, PCV2 and PPV in 5% and PPV2 and PRRSV infection was detected in 5% of the cases. The agreement of the EG-mPCR and conventional PCR tests was 99.2%. This EG-mPCR will be a useful, rapid, reliable and cost-effective alternative for routine surveillance testing of viral infections in pigs.

A sensitive multiplex real-time PCR panel for rapid diagnosis of viruses associated with porcine respiratory and reproductive disorders.

The objective of this study was to develop a multiplex real-time PCR panel using TaqMan probes for the detection and differentiation of porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus North American type (PRRSV-NA), pseudorabies virus (PRV), classical swine fever virus (CSFV), porcine parvovirus type 1 (PPV1) and Japanese encephalitis virus (JEV). Specific primer and probe combinations for PCV2, PRRSV, PRV, CSFV, PPV1 and JEV were selected within the conserved region of each viral genome. The multiplex real-time PCR panel which was run in two separate tubes was capable of specific detection of the six selected pig viruses, without cross-reactions with other non-targeted pig viruses. The detection limit of the assays was 10 copies/µL for PCV2, PRV, CSFV and PRRSV and 100 copies/µL for PPV and JEV. The two-tube multiplex real-time PCR panel showed 99.2% concordance with conventional PCR assays on 118 field samples. Overall, the multiplex real-time PCR panel provides a fast, specific, and sensitive diagnostic tool for detection of multiple viral pathogens in pigs and will be useful not only for diagnostics, or ecological, epidemiological and pathogenesis studies, but also for investigating host/virus or virus/virus interactions, particularly during coinfections.

ORF4-protein deficient PCV2 mutants enhance virus-induced apoptosis and show differential expression of mRNAs in vitro.

Porcine circovirus type 2 (PCV2) is the essential infectious agent of PCV associated disease (PCVAD). During previous in vitro studies, 11 RNAs and four viral proteins have been detected in PCV2-infected cells. Open reading frame (ORF) 4 is 180bp in length and has been identified at the transcription and the translation level. It overlaps completely with ORF3, which has a role in virus-induced apoptosis. In this study, start codon mutations (M1-PCV2) or in-frame termination mutations (M2-PCV2) were utilized to construct two ORF4-protein deficient viruses aiming to investigate its role in viral infection. The abilities of M1-PCV2 and M2-PCV2 to replicate, transcribe, express viral proteins, and to cause cellular apoptosis were evaluated. Viral DNA replication curves supported that the ORF4 protein is not essential for viral replication, but inhibits viral replication in the early stage of infection. Comparison of the expression level of ORF3 mRNA among wild-type and ORF4-deficient viruses in infected PK-15 cell demonstrated enhanced ORF3 transcription of both ORF4 mutants suggesting that the ORF4 protein may play an important role by restricting ORF3 transcription thereby preventing virus-induced apoptosis. This is further confirmed by the significantly higher caspase 3 and 8 activities in M1-PCV2 and M2-PCV2 compared to wild-type PCV2. Furthermore, the role of ORF4 in cell apoptosis and a possible interaction with the ORF1 associated Rep protein could perhaps explain the rapid viral growth in the early stage of infection and the higher expression level of ORF1 mRNA in ORF4 protein deficient PCV2 mutants.

Identification and characterization of two novel transcription units of porcine circovirus 2.

Porcine circovirus type 2 (PCV2) infection is associated with porcine circovirus-associated diseases in pigs, which is a serious threat to the swine industry worldwide. To date, only three open reading frames (ORFs) within the PCV2 genome have been reported: ORF1 codes for two replicase proteins (Rep and Rep'), ORF2 for the structural protein (Cap), and ORF3 for a protein implicated in cellular apoptosis. In this study, based on transcription analysis of ORF3 mRNA, a potential ORF4 mRNA was detected and characterized by real-time RT-PCR and rapid amplification of cDNA ends analysis. The results indicate that the ORF4 gene is expressed at the level of transcription in the PCV2-infected cells. In addition, a novel ORF3 associated (ORF3') mRNA was identified during virus replication in PK15 cells. Moreover, a 3' poly(A) addition signal sequence (AUUAAA, nt 258-263) was found 10-30 nucleotides upstream of the cleavage site in the novel ORF4 mRNA in the complementary-strand of the PCV2 genome. Furthermore, alternate trans-splicing was identified in the ORF3' mRNA between orientation diverse transcripts with typical GT-AG donor/acceptor junctions. Similar strategies as in this work can be applied to examine the transcription of other potential ORFs in PCV in the future.

CYP2D6 poor metabolizer genotype and smoking predict severe postoperative pain in female patients on arrival to the recovery room.

UNLABELLED: Recent studies have shown that CYP2D6 acts at critical steps for endogenous morphine biosynthesis. The present study assessed the contribution of CYP2D6 genetic polymorphisms, smoking, and other factors on acute severe postoperative pain (linear analog pain scores ≥8). METHODS: Two hundred thirty-six female patients were found to have adequate information in a previously developed female surgical patient database to be included in this current analysis. Multiple logistic regression analysis was used to assess the predictors for acute severe postoperative pain. DNA had been previously extracted from blood samples in all patients and was genotyped by the Amplichip to determine the specific CYP2D6 genotypes. RESULTS: It was noted that the incidence of acute severe postoperative pain (linear analog pain scores ≥8) was more frequent in patients with the CYP2D6 poor metabolizer (PM) genotype, 71%, compared with 28% in intermediate metabolizers (IMs), 26% in extensive metabolizers (EMs), and 27% in ultrarapid metabolizers (UMs). The overall association between metabolizer groups and severe postoperative pain was significant (P=0.023). PMs were significantly more likely to suffer from severe postoperative pain than IMs, EMs, and UMs (P=0.007, 0.002, and 0.050, respectively). There were no significant differences among IMs, EMs, and UMs. Additionally, it was noted that there was an increased frequency of acute severe postoperative pain in smokers vs nonsmokers (P=0.014). CONCLUSION: This study demonstrated that female patients possessing the PM genotype of CYP2D6 and patients who smoke had a higher incidence of acute severe postoperative pain.

Polymorphism in the interleukin-1 receptor antagonist gene is associated with serum interleukin-1 receptor antagonist concentrations and postoperative opioid consumption.

BACKGROUND: The interleukin-1 receptor antagonist (IL-1Ra) is the principal determinant of IL-1ß bioactivity within the IL-1 gene cluster, regulating IL-1α and IL-1ß release. This study was designed to determine whether polymorphisms of the IL-1Ra gene (IL1RN) produce clinically measurable differences in serum IL-1Ra concentrations and opioid consumption in the postoperative period. METHODS: Opioid consumption and pain scores were evaluated in 96 patients undergoing a nephrectomy. DNA was extracted from all patients, and the genotypes of IL1RN were determined by polymerase chain reaction amplification of the variable number of tandem repeats of 86 base pairs in intron 2 of IL1RN. The concentrations of serum IL-1Ra concentrations at baseline and at 24 h postoperatively in 58 subjects were measured. RESULTS: Differences in opioid consumption among the three genotype groups (IL1RN*1 homozygotes and *2 and *3 carriers) were statistically significant in the first and second 12-h postoperative periods (P = 0.010). The IL1RN*2 carrier group consumed 43% (95% CI, 38-48%) less opioids in the first 24 h after surgery than the IL1RN*1 homozygote group (P = 0.003). Differences in the serum IL-1Ra concentration among the three genotype groups were statistically significant at 24 h postoperatively (P = 0.003), with IL1RN*2 carriers having the highest serum IL-1Ra concentrations. CONCLUSIONS: The variable number of tandem repeats in intron 2 of IL1RN may contribute to interindividual variations in opioid consumption in the first 24 h after surgery. Patients homozygous for the IL1RN*1 allele have lower concentrations of IL-1Ra and require higher doses of opioids postoperatively than patients carrying at least one IL1RN*2 allele.

The impact of CYP2D6 genetic polymorphisms on postoperative morphine consumption.

BACKGROUND: Endogenous morphine-like compounds have been identified in humans and are released in response to stress. Human monocytes and granulocytes express the micro opiate receptor, micro3, which is morphine selective but opiate peptide insensitive. Recent studies have shown that CYP2D6 acts at critical steps for endogenous morphine biosynthesis. We theorized that ultrarapid (UM) CYP2D6 metabolizers may have an enhancement of their endogenous pain modulating mechanisms. METHODS: After institutional review board approval, a previously developed surgical patient database was evaluated for information concerning CYP2D6 genotypes and morphine consumption. One hundred forty-two patients were found to have adequate information to be included in this current analysis. The study group was divided, based on morphine consumption, into two subgroups: low morphine consumers (LMC) (< or =10 mg/4 h, N = 80) and high morphine consumers (HMC) (>10 mg/4 h, N = 62). DNA was extracted from blood in all patients and was genotyped by the Amplichip (Roche, Pleasanton, CA) to determine the specific CYP2D6 genotypes. RESULTS: CYP2D6 UM were found to occur more frequently in the LMC group than in the HMC group (8/80 vs 0/62, P = 0.0091). No significant differences were noted for the poor, intermediate, or extensive metabolizers. CONCLUSIONS: Our current results suggest that CYP2D6 UM appear to require less morphine in the acute postoperative period compared with other CYP2D6 metabolizer groups. One possible mechanism for this observation is that CYP2D6 UM may have higher efficiency in synthesizing endogenous morphine compared with other metabolizers, thus increasing endogenous pain modulation and reducing the need for exogenous morphine.