PK2/PKR1 signaling pathway participates in geniposide protection against diabetic nephropathy in mice / 中国中药杂志

This study aimed to investigate the effects of geniposide(GP) on the expression of prokineticin(PK2) and prokineticin receptor 1(PKR1) in db/db mice with diabetic nephropathy(DN), so as to explore how the PK2 signaling pathway participated in the pathological changes of DN and whether GP exerted the therapeutic effect through this signaling pathway. Male mice were randomly divided into four groups, namely db/m, db/db, db/db+GP, and db/m+GP groups, with five in each group. The mice in the db/db+GP and db/m+GP groups were gavaged with 150 mg·kg~(-1) GP for eight successive weeks. Afterwards, all the mice were sacrificed and the renal tissues were embedded. The morphological changes in glomerulus and renal tubules were observed by Masson and PAS staining. The expression levels of PK2, PKR1, and Wilm's Tumor Protein 1(WT_1) in podocytes were detected by immunohistochemistry, and the protein expression levels of PK2 and PKR1 in mouse kidney by Western blot. The morphological results showed serious glomerular and tubular fibrosis(Masson), high glomerular and tubular injury score(PAS), increased glomerular mesangial matrix, thickened basement membrane, exfoliated brush border of renal tubules, decreased WT_1 in glomerular podocytes, and massive loss of podocytes in the db/db group. After administration with GP, the glomerular and tubular fibrosis was alleviated, accompanied by improved glomerular basement membrane and renal tubule brush edge, and up-regulated WT_1. As revealed by further protein detection, in the db/db group, the expression levels of PK2 and PKR1 and p-Akt/Akt ratio declined, whereas the ratio of Bax/Bcl-2 rose. Ho-wever, PKR2 and p-ERK/ERK ratio did not change significantly. After administration with GP, the PK2 and PKR1 expression was elevated, and p-Akt/Akt ratio was increased. There was no obvious change in PKR2, Bax/Bcl-2 ratio, or p-ERK/ERK ratio. All these have demonstrated that GP improves the renal damage in DN mice, and PK2/PKR1 signaling pathway may be involved in such protection, which has provided reference for clinical treatment of DN with GP.

Prokineticin 2 mediates peripheral and central sensitization of somatic pain / 药学实践杂志

Prokineticin 2 (PK2) is a newly discovered chemokine, which participates in various physiological functions of the body by binding to receptors PKR1 and PKR2. PK signaling pathway is a newly discovered important regulatory pathway for the occurrence and maintenance of pain after tissue injury and nerve injury in recent years. It plays a key role in regulating injury-related nociceptive events and is a potential therapeutic target for many diseases. The activation of PKRs can induce pain sensation and participate in the sensitivity of pain receptors to different stimuli. The PK system (PKs and PKRs) is an important link involved in inflammation and pain transmission in immune cells. PK2 is involved in the regulation of pain perception by activating PKR1 and PKR2 on primary sensory neurons. In rat primary sensory neurons, PK2 also enhances gated ion channel current through the PKC signaling pathway, inhibits GABA-activated currents, and sensitizes purine nucleotide P2 receptor (P2X). This paper reviews the research progress of PK2 in physical pain. We hope to find new drugs for the treatment of inflammatory pain that target the PKs signaling pathway in future studies.

Varenicline modulates autophagy through PKR/STAT3 pathway to improve postoperative cognitive dysfunction / 中国药理学通报

Aim To evaluate whether varenicline could regulate autophagy through PKR/STAT3 pathway to improve postoperative cognitive dysfunction. Methods Forty healthy male C57BL/6J mice, aged 18 months and weighing (27. 5 ± 2. 5) g, were randomly divided into four groups (n = 10) j control group (CON group), laparotomy group (LAP group), laparotomy with varenicline administration group (Var + LAP group) and varenicline group (Var group). The mice in Var + LAP group and Var group were treated with varenicline (1 mg kg"1 d"1) one day before operation and lasted until 13th day after operation. The other two groups were treated with equal amount of normal saline instead of varenicline. The model of postoperative cognitive dysfunction was established by laparotomy under sevofiurane anesthesia. The novel object recognition task was performed on 10th ~ 12th day after laparotomy, and the Y-maze test was performed on 14th day after laparotomy to detect the cognitive function of the mice. The expression levels of AT8 and LC3B in hippocampus were detected by Western blot and immunofluorescence staining. The expression levels of p-PKR and p-STAT3 were detected by Western blot, and the interaction between STAT3 and PKR was detected by double labeled immunofluorescence staining. Results Compared with CON group, the error numbers and the latency in LAP group increased, the discrimination index decreased, accompanied with the increased expression levels of AT8 and p-PKR, the decreased expression levels of LC3 B and p-STAT3, and the increased interaction of STAT3 and PKR. Compared with LAP group, the error numbers and the latency in Var + LAP group decreased, and the discrimination index increased, associated with the decreased expression of AT8 and p-PKR, the increased expression of LC3B and p-STAT3, and the decreased interaction of STAT3 and PKR. Conclusions Vareni-cline regulates autophagy and eliminates hyperphospho-rylated tau through PKR/STAT3 pathway to improve postoperative cognitive dysfunction.

Mechanism of Salidroside in Improving Glucose and Lipid Metabolism in Liver of Diabetic Rats / 中国实验方剂学杂志

Objective: To explore the mechanism of salidroside(SAL) in improving glucose and lipid metabolism in liver of diabetic rats. Method: According to blood glucose, 24 male ZDF(fa/fa) rats of 6-7 week old were randomly divided into model group, SAL-low group (0.05 g·kg-1), SAL-high group (0.1 g·kg-1), 8 rats in each group. Another 8 ZDF (fa/+) rats were selected as normal group. All the rats were administered continuously for 6 weeks. After experiment, fasting blood glucose(FBG), triglyceride(TG), total cholesterol(TC), free fatty acids(FFA), superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), fasting insulin(Fins), and homeostasis model assessment insulin resistance(HOMA-IR) were measured. Western blot was used to detect phosphorylated PKR-like ER kinase(p-PERK), NF-E2-relatedfactor2(Nrf2), heme oxidase-1(HO-1) protein expression levels in liver. Result: As compared with normal group, FBG, Fins, HOMA-IR, TC, TG, FFA, and MDA were increased significantly in model group (P PPPPPPPPPPPConclusion: The Salidroside can improve metabolism of glucose and lipid, IR in diabetic rats, and the mechanism may be associated with inhabiting oxidative stress through the up-regulation of p-PERK, Nrf2, and HO-1 protein expression.

Long non-coding RNA HOTAIR promotes UVB-induced apoptosis and inflammatory injury by up-regulation of PKR in keratinocytes

Excessive exposure to ultraviolet (UV) rays can cause damage of the skin and may induce cancer, immunosuppression, photoaging, and inflammation. The long non-coding RNA (lncRNA) HOX antisense intergenic RNA (HOTAIR) is involved in multiple human biological processes. However, its role in UVB-induced keratinocyte injury is unclear. This study was performed to investigate the effects of HOTAIR in UVB-induced apoptosis and inflammatory injury in human keratinocytes (HaCaT cells). Quantitative real-time polymerase chain reaction was performed to analyze the expression levels of HOTAIR, PKR, TNF-α, and IL-6. Cell viability was measured using trypan blue exclusion method and cell apoptosis using flow cytometry and western blot. ELISA was used to measure the concentrations of TNF-α and IL-6. Western blot was used to measure the expression of PKR, apoptosis-related proteins, and PI3K/AKT and NF-κB pathway proteins. UVB induced HaCaT cell injury by inhibiting cell viability and promoting cell apoptosis and expressions of IL-6 and TNF-α. UVB also promoted the expression of HOTAIR. HOTAIR suppression increased cell viability and decreased apoptosis and expression of inflammatory factors in UVB-treated cells. HOTAIR also promoted the expression of PKR. Overexpression of HOTAIR decreased cell viability and increased cell apoptosis and expression of inflammatory factors in UVB-treated cells by upregulating PKR. Overexpression of PKR decreased cell viability and promoted cell apoptosis in UVB-treated cells. Overexpression of PKR activated PI3K/AKT and NF-κB pathways. Our findings identified an essential role of HOTAIR in promoting UVB-induced apoptosis and inflammatory injury by up-regulating PKR in keratinocytes.

Study of IFN-inducible double-stranded RNA dependent protein kinase on antiviral activity of HBV in vitro / 中国药理学通报

Aim To construct and express the eukary-otic expression vector of double-stranded RNA-depend-ent protein kinase (PKR)fusion green fluorescent and analyse its antiviral activity of HBV in vitro.Methods The PKR gene was cloned into an empty expression vector pEGFP-N1 using molecular clone technology. After being confirmed by restriction enzyme digestion and sequencing methods,the recombinant plasmid was named as pEGFP-PKR that was subsequently transfect-ed into HepG2.2.15 cells using LipofectamineTM2000. The expression level of PKR in HepG2.2.15 cells was confirmed by using fluorescent microscopy. Mean-while,HBV DNA and HBsAg/HBeAg were detected by real-time PCR and electrochemiluminescence meth-od,respectively.Results Both restriction enyme di-gestion and sequencing assays showed that the recombi-nant vector pEGFP-PKR was successfully constructed in our study.Fluorescent microscopy observation indi-cated that the fusion protein pEGFP-PKR expressed ef-ficiently in HepG2.2.15 cells.Moreover,compared with the empty vector group,the expression of HBV antigen in supernatants was significantly decreased (P<0.05 ).However,the extracellular HBV DNA ex-pression was not inhibited significantly.Conclusion In vitro,PKR proteion has certain antiviral activity of HBV.

A Novel Type of Non-coding RNA, nc886, Implicated in Tumor Sensing and Suppression

nc886 (=vtRNA2-1, pre-miR-886, or CBL3) is a newly identified non-coding RNA (ncRNA) that represses the activity of protein kinase R (PKR). nc886 is transcribed by RNA polymerase III (Pol III) and is intriguingly the first case of a Pol III gene whose expression is silenced by CpG DNA hypermethylation in several types of cancer. PKR is a sensor protein that recognizes evading viruses and induces apoptosis to eliminate infected cells. Like viral infection, nc886 silencing activates PKR and induces apoptosis. Thus, the significance of the nc886:PKR pathway in cancer is to sense and eliminate pre-malignant cells, which is analogous to PKR's role in cellular innate immunity. Beyond this tumor sensing role, nc886 plays a putative tumor suppressor role as supported by experimental evidence. Collectively, nc886 provides a novel example how epigenetic silencing of a ncRNA contributes to tumorigenesis by controlling the activity of its protein ligand.

Establishment and application of a tandem affinity purification system of innate immune regulatory protein PKR / 中华微生物学和免疫学杂志

Objective To establish a tandem affinity purification(TAP) system of innate immune-regulatory protein PKR and analyze PKR function, for the future screen and identification of novel PKR-interaction proteins. Methods PKR gene was amplified by PCR, and then cloned into a mammalian expression vector pcTAP-A. Recombinant pcTAP-PKR was transfected into PKR knock-down(PKRkd) HeLa cells by LipofectAMINE 2000,and the PKR overexpressed HeLa cells were harvested for mitogen-activated protein kinases(MAPK) activation analysis. Cell extracts of PKR overexpressed cells were purified using TAP kit and examined by Western blot. Results Cal modulin resin(CBP) and streptavidin resin(SBP) tagged PKR was detected in PKRkd HeLa cells as early as 24 h upon transfection with pcTAP-PKR, and its expression decreased at later time points. The overexpression of PKR was autophosphorylated, and thus involved in the regulation of MAPK actviation. After small-scale TAP kit purification, PKR protein was detectable by Western blot. Conclusion We have successfully established a TAP system that over-expresses functional PKR, providing a useful tool for the future study on the identification of PKR interacting proteins.

Sequencing of E2 and NS5A regions of HCV genotype 3a in Brazilian patients with chronic hepatitis

Hepatitis C virus (HCV) is a major cause of liver disease throughout the world. The NS5A and E2 proteins of HCV genotype 1 were reported to inhibit the double-stranded (ds) RNA-dependent protein kinase (PKR), which is involved in the cellular antiviral response induced by interferon (IFN). The response to IFN therapy is quite different between genotypes, with response rates among patients infected with types 2 and 3 that are two-three-fold higher than in patients infected with type 1. Interestingly, a significant percentage of HCV genotype 3-infected patients do not respond to treatment at all. The aim of this paper was to analyse the sequences of fragments of the E2 and NS5A regions from 33 outpatients infected with genotype 3a, including patients that have responded (SVR) or not responded (NR) to treatment. HCV RNA was extracted and amplified with specific primers for the NS5A and E2 regions and the PCR products were then sequenced. The sequences obtained covered amino acids (aa) 636-708 in E2 and in NS5A [including the IFN sensitivity determining region (ISDR), PKR-binding domain and extended V3 region)]. In the E2 and NS5A regions, we did observe aa changes among patients, but these changes were not statistically significant between the SVR and NR groups. In conclusion, our results suggest that the ISDR domain is not predictive of treatment success in patients infected with HCV genotype 3a.

HIV-1 Infection Causes Intracellular Expression of p53, Which Induces PKR Expression, Followed by Inhibition of HIV-1 Tat Activity

Few papers have reported that the HIV-1 replication was inhibited by p53 in the infected cells. However, the detail mechanism for the p53-medicated HIV-1 suppression has not yet been clearly demonstrated. In our previous report, we addressed that p53-mediated Tat suppression is very likely associated with PKR. In the present study, we found that the amounts of p53 in the HIV-1 infected cells increased over 10 times in the early stages of infection as much as those in normal cells. Particularly noteworthy is that the both exogenous p53 and endogenous p53 enhanced PKR expression in the transformed or treated cells, and the amounts of PKR induced by p53 were almost equivalent to those induced by interferon. In the PKR promoter studies using Ppkr-CAT (CAT reporter system under the control of PKR promoter), CAT activity induced by p53 was stronger than that by interferon, suggesting that the p53-mediated PKR expression might be more efficient than interferon under the control of PKR promoter. Co-immunoprecipitation experiments showed that PKR directly binds to Tat protein. We established eIF-2alpha dominant negative (S51A) Jurkat cells (JK/eIF2alpha-51A) to block the PKR-mediated cell cycle arrest or apoptosis. In the JK/eIF2alpha-51A cells, not only p53 but also PKR inhibited the Tat activity. Taken together, our results demonstrate that the HIV-1 infection induces p53, which enhances PKR expression by promoter activation, followed by the inhibition of the Tat activity, finally resulting in the inhibition of HIV-1 replication. Detail mechanisms for the PKR-mediated Tat inactivation are under investigation.

Construction of double expression retroviral vectors and its effect on phenotype of K562 cells / 第三军医大学学报

Objective To construct double expression retroviral vectors targeting chronic myeloid leukemia (CML) b3a2-type mRNA and investigate its effect on the phenotype of K562 cells. Methods The eGFP coding sequence was inserted into the retroviral vector pMSCV-neo to construct pMSCV/GFP, then H1-RNA pol III-based transcription cassettes was subcloned into it to form pMSCV/GFP-H1-BCR/ABL40AS. Two control vectors pMSCV/GFP-H1-BCR/ABL40S & pMSCV/GFP-H1-BCR/ABL80AS were constructed in addition. All these constructions were identified by restriction enzyme analysis and DNA sequencing. After that, the recombinant vectors were transferred into retrovirus packaging cell line PT67 by using lipofectamine2000, and G418 were used to select stable virus-producing cell lines. Viral titer was determined by infection of NIH3T3 cells sequentially. The cell-growth curve was assayed, cell apoptosis was checked with Annexin V-PE/7AAD double staining and flow cytometry analysis after 24-hour infection, the PKR phosphorylation was assayed by Western blotting. Results The plasmids were successfully constructed. Four cell lines, named as PT67-MSCV/GFP, PT67-40as, PT67-40s and PT67-80as were gained by G418 selection, and virus titers were 6.2?10~ 5 , 5.6?10~ 5 , 4.6?10~ 5 and 6.0?10~ 5 CFU/ml respectively. PT67-40as suspensions could induce K562 cell apoptosis by (22.54?3.19)%, significantly different from PT67-MSCV/GFP or PT67-40s (P

p53-mediated HIV-1 Tat Suppression is Likely to be Assaciated with duble-stranded RNA-dependent Protein Kinase, PKR

No abstract available.