Large-scale separation of alkaloids from Corydalis decumbens by pH-zone-refining centrifugal partition chromatography and their anticomplement activity.

Centrifugal partition chromatography in the pH-zone-refining mode was successfully applied to the separation of alkaloids from the crude extract of Corydalis decumbens. The experiment was performed with a two-phase solvent system composed of petroleum ether-ethyl acetate-ethanol-water (5:5:3:7, v/v/v/v) where triethylamine (10 mM) was added to the stationary phase and hydrochloric acid (10 mM) to the mobile phase. From 1.6 g of the crude extract, 43 mg protopine, 189 mg (+)-egenine, and 158 mg tetrahydropalmatine were obtained with a purity of 98.2%, 94.6%, and 96.7%, respectively. Tetrahydropalmatine showed an interesting anticomplement effect with CH50 0.11 and AP50 0.25 mg/mL, respectively. In a mechanistic study, tetrahydropalmatine interacted with C1, C3, C4, and C5 components in the complement activation cascade.

Formononetin attenuates psoriasiform inflammation by regulating interferon signaling pathway.

BACKGROUND: Psoriasis is a long-lasting, inflammatory, continuous illness caused through T cells and characterized mainly by abnormal growth and division of keratinocytes. Currently, corticosteroids are the preferred option. However, prolonged use of traditional topical medication can lead to adverse reactions and relapse, presenting a significant therapeutic obstacle. Improved alternative treatment options are urgently required. Formononetin (FMN) is a representative component of isoflavones in Huangqi (HQ) [Astragalus membranaceus (Fisch.) Bge.]. It possesses properties that reduce inflammation, combat oxidation, inhibit tumor growth, and mimic estrogen. Although FMN has been shown to ameliorate skin barrier devastation via regulating keratinocyte apoptosis and proliferation, there are no reports of its effectiveness in treating psoriasis. OBJECTIVE: Through transcriptomics clues and experimental investigation, we aimed to elucidate the fundamental mechanisms underlying FMN's action on psoriasis. MATERIALS AND METHODS: Cell viability was examined using CCK8 assay in this study. The results of analysis of differentially expressed genes (DEGs) between FMN-treated HaCaT cells and normal HaCaT cells using RNA-sequencing (RNA-seq) were presented on volcano plots and heatmap. Enrichment analysis was conducted on DEGs using Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO), and results were validated through RT-qPCR verification. After 12 days of FMN treatment in psoriasis mouse model, we gauged the PASI score and epidermis thickness. A variety of techniques were used to assess FMN's effectiveness on inhibiting inflammation and proliferation related to psoriasis, including RT-qPCR, HE staining, western blot, and immunohistochemistry (IHC). RESULTS: The findings indicated that FMN could suppress the growth of HaCaT cells using CCK8 assay (with IC50 = 40.64 uM) and 20 uM FMN could reduce the level of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) to the greatest extent. FMN-treated HaCaT cells exhibited 985 up-regulated and 855 down-regulated DEGs compared to normal HaCaT cells. GO analysis revealed that DEGs were linked to interferon (IFN) signaling pathway. Furthermore, FMN improved pathological features, which encompassed decreased erythema, scale, and thickness scores of skin lesions in psoriasis mouse model. In vivo experiments confirmed that FMN down-regulated expression of IFN-α, IFN-ß, IFN-γ, decreased secretion of TNF-α and IL-17 inflammatory factors, inhibited expression of IFN-related chemokines included Cxcl9, Cxcl10, Cxcl11 and Cxcr3 and reduced expression of transcription factors p-STAT1, p-STAT3 and IFN regulatory factor 1 (IRF1) in the imiquimod (IMQ) group. CONCLUSIONS: In summary, these results suggested that FMN played an anti-inflammatory and anti-proliferative role in alleviating psoriasis by inhibiting IFN signaling pathway, and FMN could be used as a potential therapeutic agent.

A modified LC-MS/MS method for determination of tetramethylpyrazine in microdialysis samples and calibration of home-made linear probes.

Tetramethylpyrazine (TMP) is one of the most important active ingredients of a Chinese herb Ligusticum wallichii Franchat, which is widely used for the treatment of cardiovascular diseases. Several factors may affect TMP exposure after topical administration, resulting in large variability and demanding further elucidation of drug distribution. This paper describes a new efficient reliable LC-MS/MS assay for the determination of TMP in dermal microdialysate, where TMP was separated on an Agilent C(18) column (3.5 µm, 100 mm × 2.1 mm i.d.) using a mixture of methanol, water and acetic acid (50:50:0.6, v/v/v) at a flow-rate of 0.3 mL/min. The retention time was 1.89 min for TMP and 1.17 min for the internal standard (caffeine). Histological analysis confirmed an inflammatory response to the microdialysis probes and the presence of a collagen capsule. The membrane extraction efficiency (percentage delivered to the tissue space) for TMP was not altered through the implant lifetime. The validation and sample analysis results showed that the method is precise, accurate and well suited to support dermal microdialysis experiments.

Pharmacokinetics of aconitine in rat skin after oral and transdermal gel administrations.

The purpose of this study was to evaluate percutaneous penetration and arrhythmogenic effects of aconitine after transdermal administration, compared with the oral route. Skin penetration of aconitine was tested by a microdialysis technique in rats and in vivo recovery was determined by retrodialysis. After oral and transdermal administration of aconitine, dialysate was sampled at 20 min intervals until the end of the experiment for the determination of concentration of aconitine in skin. Blood samples were collected and analyzed using a validated HPLC-MS/MS method. In addition, we concurrently recorded the electrocardiogram (ECG). The in vivo recovery of aconitine in the skin was calculated to be 39.59%. The C(max) values for aconitine absorbed into the skin after oral and transdermal administration were 1.51 ± 0.53 and 2723.8 ± 848.8 ng/mL, respectively, and within the plasma, 215.86 ± 79.29 and 20.92 ± 3.15 ng/mL. The C(max) value for the plasma concentration of aconitine after oral administration was approximately 10 times higher than with the transdermal route. For oral administration, the ECG revealed various types of arrhythmias at a period of T(max) , which is normal in transdermal gel administration. These results indicate that transdermal aconitine gel is a safe formulation that can deliver the drug in sufficient amounts and safe concentrations to produce therapeutic action in rats.

[Preparation of scopolamine hydrobromide nanoparticles-in-microsphere system].

This study is to prepare scopolamine hydrobromide nanoparticles-in-microsphere system (SH-NiMS) and evaluate its drug release characteristics in vitro. SH nanoparticles were prepared by ionic crosslinking method with tripolyphosphate (TPP) as crosslinker and chitosan as carrier. Orthogonal design was used to optimize the formulation of SH nanoparticles, which took the property of encapsulation efficiency and drug loading as evaluation parameters. With HPMC as carrier, adjusted the parameters of spray drying technique and sprayed the SH nanoparticles in microspheres encaposulated by HPMC was formed and which is called nanoparticles-in-microsphere system (NiMS). SH-NiMS appearances were observed by SEM, structure was obsearved by FT-IR and the release characteristics in vitro were evaluated. The optimized formulation of SH nanoparticles was TPP/CS 1:3 (w/w), HPMC 0.3%, SH 0.2%. The solution peristaltic speed of the spray drying technique was adjusted to 15%, and the temperature of inlet was 110 degrees C. The encapsulation product yeild, drug loading and particle sizes of SH-NiMS were 94.2%, 20.4%, and 1256.5 nm, respectively. The appearances and the structure of SH-NiMS were good. The preparation method of SH-NiMS is stable and reliable to use, which provide a new way to develop new dosage form.

Mannose 6-phosphate-modified bovine serum albumin nanoparticles for controlled and targeted delivery of sodium ferulate for treatment of hepatic fibrosis.

OBJECTIVES: The aim was to prepare neoglycoprotein-based nanoparticles for targeted drug delivery to hepatic stellate cells, and to evaluate their characteristics in vitro and in vivo. METHODS: The neoglycoprotein of bovine serum albumin modified with mannose 6-phosphate was synthesised from mannose, and used as wall material to nanoencapsulate the model natural antifibrotic substance sodium ferulate using a desolvation method. The morphology, drug loading capacity, release in vitro and biodistribution in vivo of the nanoparticles were studied. Selectivity of the nanoparticles for hepatic stellate cells was evaluated by immunohistochemical analysis of fibrotic rat liver sections. KEY FINDINGS: The spherical nanoparticles were negatively charged with zeta potential ranging from -2.73 to -35.85 mV, and sizes between 100 and 200 nm with a narrow size distribution. Drug entrapment efficiency of about 90% (w/w) and loading capacity of 20% (w/w) could be achieved. in vitro, the nanoparticles showed an initial rapid continuous release followed by a slower sustained release. After intravenous injection into mice, the nanoparticles showed a slower elimination rate and a much higher drug concentration in liver compared with the sodium ferrate solution, and less distribution to the kidneys and other tissues. Immunohistochemistry indicated that the neoglycoprotein-based nanoparticles were taken up specifically by hepatic stellate cells. CONCLUSIONS: The nanoparticles may be an efficient drug carrier targeting hepatic stellate cells.

Development of a novel HPLC-MS/MS method for the determination of aconitine and its application to in vitro and rat microdialysis samples.

A sensitive and selective LC-MS/MS method was developed and validated for the determination of aconitine in microdialysate and rat plasma. Extraction of plasma sample was conducted by use of 1% trichloracetic acid and acetonitrile solution with 10 ng/mL internal standard (propafenone) spiked. Microdialysates were analyzed without sample purification. After sample preparation, 2 microL were injected and separated with an isocratic mobile phase consisting of acetonitrile:0.1% formic acid (60:40, v/v) at a flow rate of 0.3 mL/min. The Agilent G6410A triple quadrupole LC/MS system was operated under the multiple-reaction monitoring mode (MRM) using the electrospray ionization technique in positive mode. Overall, the assay exhibited good precision and accuracy. The diffusion properties of aconitine investigated in in vitro microdialysis experiments revealed unfavourable concentration dependence avertable by keeping a constant pH 5.77 using isotonic phosphate buffer solution as perfusate. The mean relative recoveries were 48.23% [coefficient of variation (CV 4.47%)] and 55.38% (CV 2.89%) for retrodialysis and recovery experiments, respectively. The in vivo recovery of aconitine was 34.48% (CV 3.05%) and was stable over the 6 h study period. Following characterization of aconitine both in vitro and in vivo microdialysis, the developed setting is suitable for application in pharmacokinetics and pharmacodynamics studies.

Anchoring of ulex europaeus agglutinin to chitosan nanoparticles-in-microparticles and their in vitro binding activity to bovine submaxillary gland mucin.

Focused on the natural biodegradable material of chitosan (CS), this investigation concerned its spray-dried nanoparticles-in-microparticles (NiMPs) modified with ulex europaeus agglutinin (UEA). Chitosan nanoparticles were obtained by ionotropic gelation process with pentasodium tripolyphosphate as gelatinizer. Then UEA lectin was bound onto the CS nanoparticles activated by glutaraldehyde. The conjugated spherical UEA-CS-NiMPs, prepared by spray drying method, exhibited 12-85% coupling efficiency of UEA depending upon the amount of activator glutaraldehyde. And the UEA-grafted particles showed additional higher binding tendency with bovine submaxillary gland mucin as compared to the plain chitosan microparticles. Furthermore, the activity and intrinsic fucose-specificity of UEA were still maintained after the covalent modification. It is thus evident that the UEA anchored CS-NiMPs might be used as a potential drug delivery system targeted to the specific regions of gastrointestinal tract.

Preparation and characterization of sodium ferulate entrapped bovine serum albumin nanoparticles for liver targeting.

Sodium ferulate (SF) loaded nanoparticles were prepared by desolvation procedure and subsequent cross-linking of the wall material of bovine serum albumin (BSA). Several factors in the nanoencapsulation process, such as the addition rate of the desolvation agent, composition of BSA and SF solution, amount of the cross-linker glutaraldehyde, were investigated to elucidate their influences on the particle size, zeta potential, drug loading and encapsulation efficiency of the resulted nanoparticles. The obtained spherical nanoparticles were negative charged with zeta potential from -20 to -40 mV, and characterized between 100 and 200 nm with a narrow size distribution. In the condition of introducing 1.0 mL 8% glutareldehyde per mg of BSA, the drug entrapment efficiency (EE) of 80% (w/w) and loading capacity of about 16% (w/w) could be achieved for the cross-linked BSA nanoparticles with SF encapsulated (SF-BSA-NP). And the drug EE was decreased along with the increasing amount of glutareldehyde used for cross-linking. The in vitro drug release properties of SF-BSA-NP behaved with an initial burst effect and then sustained-release stage. To some extent, the drug release rate could be adjusted by cross-linking with different amount of glutaraldehyde. Compared with SF solution, SF-BSA-NP showed a much higher drug distribution into liver and a lower drug concentration in other tissues, after intravenously injected to mice. So, BSA based nanoparticles might be a suitable controlled released carrier for the freely water-soluble drug SF and further hepatic targeted drug delivery.

[Effect of cetirizine hydrochloride on the expression of substance P receptor and cytokines production in human epidermal keratinocytes and dermal fibroblasts].

To investigate the effect of cetirizine hydrochloride on the expression of neurokinin 1 receptor (NK-1R) and cytokines production induced by substance P (SP) in HaCaT cells (a human epidermal keratinocyte cell line) and dermal fibroblasts. The effect of cetirizine on the expression of NK-1R protein was detected by flow cytometry and Western blotting analysis. The modulation of cetirizine on the production of interferon (IFN)-gamma, interleukin (IL)-1beta, IL-6 and IL-8 in HaCaT cells and fibroblasts was measured by ELISA. The results showed that cetirizine significantly inhibited the expression of NK-1R in HaCaT cells and fibroblasts. SP induced the production of IFN-gamma, IL-1beta and IL-8 in both cell types. Cetirizine 1-100 micromol x L(-1) inhibited SP-induced IL-1beta and IL-8 production in HaCaT cells and fibroblasts, while had no effect on the production of IFN-gamma in both cells. Both SP and cetirizine had no effect on the secretion of IL-6 in HaCaT cells and fibroblasts. These findings suggest that cetirizine may be involved in the treatment of SP-induced skin inflammation by inhibiting the expression of substance P receptor and regulation the production of IL-1beta and IL-8 in epidermal keratinocyte and dermal fibroblasts.

Effect of matrine on the expression of substance P receptor and inflammatory cytokines production in human skin keratinocytes and fibroblasts.

Matrine is a kind of alkaloid found in certain Sophora plants, which has been extensively used in China for the treatment of viral hepatitis, cancer, cardiac diseases and skin diseases (such as atopic dermatitis and eczema). It also has been confirmed that substance P (SP) and its receptor (neurokinin-1 receptor, NK-1R) are involved in the pathogenesis of inflammatory skin disorders. So the present study was designed to investigate the effect of matrine on the expression of NK-1R and cytokines production induced by SP in HaCaT cells (a human epidermal keratinocyte cell line) and dermal fibroblasts. In addition, cell viability was also evaluated. The results showed that matrine inhibited the expression of NK-1R in HaCaT cells and fibroblasts. SP induced the production of interleukin (IL)-1beta, IL-8, interferon (IFN)-gamma, and monocyte chemotactic protein (MCP)-1 in both cell types. Matrine 5-100 microg/mL had little effect on cell viability. It inhibited SP-induced IL-1beta, IL-8 and MCP-1 production in HaCaT cells and fibroblasts, while it increased the production of IFN-gamma in HaCaT cells. Both SP and matrine had no effect on the secretion of IL-6. These findings suggest that matrine may have potential treatment function on SP related cutaneous inflammation by inhibition of the expression of substance P receptor and regulation of the production of inflammatory cytokines.

[Studies on cutaneous permeation in vitro of Kushen recipe gel].

OBJECTIVE: To establish a suitable dosage form for a traditional anti-anaphylaxis Chinese medicine of Kushen recipe, and investigate the effect of cutaneous permeation in vitro of the recipe. METHOD: Techniques of extracting with ethanol and purifying with absorbent resin to obtain alkaloids from Kushen recipe were adopted, while volatile oil was extracted by steam distillation. The extraction was made to gel. The skin from SD rats' abdomen was used as permeability barriers. Then effects of permeation of the aqueous extraction, the purifying extraction and the gel were compared by Valia-Chien and Franz diffusion cell method. HPLC was utilized to quantitate the alkaloids in permeating liquid. RESULT: In view of the permeation cumulation quantity, the permeation velocity and the lag time of the four kinds of alkaloids, the effect of permeation of purifying extraction was better than the aqueous extraction, and the purifying extraction gel surpassed both the aqueous extraction and the purifying extraction. CONCLUSION: It was certified that the purifying extraction gel had improved the effect of cutaneous permeation of alkaloids, and it is the befitting dosage form for Kushen recipe to treat anaphylaxis disease in skin.

In vivo and in vitro evaluation of essential oils from Ligusticum chuanxiong Hort on the transdermal delivery of flurbiprofen in rabbits.

The present study was designed to evaluate skin permeation enhancement effect of essential oils from Ligusticum chuanxiong Hort (chuanxiong oil) in rabbits and to compare the in vivo absorption and in vitro permeation using flurbiprofen as a model drug. In vivo results demonstrated that chuanxiong oil showed a rapid and marked permeation enhancement effect. The group with 10% oil exhibited the highest value of area under the curve (AUC) of 418+/-124 microg/ml x h, which was 2.43 times the high of control. The AUC value of 3% oil group (245+/-81.6 microg/ml x h) was similar to that of 5% oleic acid group (235+/-74.5 microg/ml x h). Whereas in vitro results indicated the enhancement of chuanxiong oil was relatively weak. The group with 3% oil appeared to the highest flurbiprofen flux (84.9+/-19.3 microg/cm2/h), to some extent lower than 5% oleic acid group (107+/-5.85 microg/cm2/h). At 10% and 15% concentrations, chuanxiong oil even decreased the flux of flurbiprofen compared with the control. Both in vitro results with pretreated skin and flurbiprofen content accumulated in skin indicated the potential mechanism for the in vitro enhancement of chuanxiong oil was the weakened barrier function by improving in the partitioning of flurbiprofen to the stratum corneum. The discrepancy was noted between the in vivo and in vitro results, indicating only about the weakened barrier function was not enough to explain the sharply increment of in vivo absorption of flurbiprofen by chuanxiong oil. The GS-MS results indicated phthalides identified from chuanxiong oil might mainly contribute to enhance in vivo absorption of flurbiprofen because of its large quantities (91.15%).

Chemokine CXCL1 may serve as a potential molecular target for hepatocellular carcinoma.

The purpose of this study was to screen for changes in chemokine and chemokine-related genes that are expressed in hepatocellular carcinoma (HCC) as potential markers of HCC progression. Total RNA was extracted from tumor and peritumor tissues from mice with HCC and analyzed using a PCR microarray comprising 98 genes. Changes in gene expression of threefold or more were screened and subsequently confirmed by immunohistochemical analyses and western blotting. Furthermore, whether chemokine knockdown by RNA interference (RNAi) could significantly suppress tumor growth in vivo was also evaluated. Finally, total serum samples were collected from HCC patients with HBV/cirrhosis (n = 16) or liver cirrhosis (n = 16) and from healthy controls (n = 16). The serum mRNA and protein expression levels of CXCL1 in primary liver cancer patients were detected by qRT-PCR and western blot analysis, respectively. Several genes were up-regulated in tumor tissues during the progression period, including CXCL1, CXCL2, CXCL3, and IL-1ß, while CXCR1 expression was down-regulated. CBRH-7919 cells carrying CXCL1 siRNA resulted in decreased tumor growth in nude mice. The differences in serum CXCL1 mRNA and protein levels among the HCC, hepatic sclerosis (HS), and control groups were significant (P < 0.001). The mRNA and protein levels of CXCL1 in the HCC group were up-regulated compared with the HS group or the control group (P < 0.001). Several chemokine genes were identified that might play important roles in the tumor microenvironment of HCC. These results provide new insights into human HCC and may ultimately facilitate early HCC diagnosis and lead to the discovery of innovative therapeutic approaches for HCC.

[Stereoselectivity of skin carboxylesterase metabolism].

AIM: To study the stereoselectivity of skin carboxylesterase metabolism and its molecular biological foundation for improving drug percutaneous absorption. METHODS: Ketoprofen ethyl ester was used as a model drug, and skin homogenate was applied for studying the stereoselectivity of carboxylesterase metabolism. Human liver L02 cell was used as control of carboxylesterase expression, and RT-PCR was used for studying the expression of carboxylesterase. RESULTS: The main metabolite of ketoprofen ethyl ester in human skin homogenate was R-ketoprofen. Human carboxylesterase-2 was highly expressed in skin and its cells. However, the expression of human carboxylesterase-1 was very weak or not detectable. CONCLUSION: Human carboxylesterase-2 is the main hydrolytic enzyme of prodrugs in percutaneous absorption, and shows metabolic stereoselectivity to prodrugs with chiral esters.

[Percutaneous penetration of ketoprofen and ketoprofen isopropyl ester through a tissue engineering skin reconstructed with HaCaT cells].

AIM: To reconstruct of a tissue engineering skin in vitro for the study of the use of drug percutaneous penetration and metabolism. METHODS: Dermal fibroblasts were embedded in collagen type I. HaCaT cells were seeded on the top of the gel. The skin was generated through air-liquid interface culture. Effects of various culture media on tissues morphology were investigated. Sections of the cultured skin were stained with hematoxylin and eosin and examined under microscope. Permeation and metabolism of ketoprofen and its isopropyl ester through the cultured skin were investigated. RESULTS: HaCaT cells initially developed a multilayer epithelium at the air-liquid interface, but it showed a parakeratotic stratum corneum. Vitamin C enhanced cell proliferation obviously. Vitamin D3 promoted cell differentiation. And estradiol showed little effect on the tissue engineering skin. Ketoprofen isopropyl ester was hydrolyzed into ketoprofen when penetrated through the cultured skin, which resembled in the skin cell homogenates metabolism. CONCLUSION: Cultured at the air-liquid interface, HaCaT cells developed a parakeratotic mutilayer epithelium. Enzyme activity was reserved. This cultured skin could serve as an appropriate model for drug percutaneous metabolism and skin irritation.

[Effect of cetirizine hydrochloride on the expression of substance P in IgE-mediated triphasic cutaneous reaction].

AIM: To investigate the effect of cetirizine hydrochloride on the expression of neuropeptide substance P (SP) in IgE-dependent triphasic cutaneous reaction induced by dinitrofluorobenzene (DNFB) in the ears of BALB/c mice. METHODS: BALB/c mice were passively sensitized by intravenous infection of anti-DNP IgE monoclonal antibody 24 h before DNFB challenge. Skin reaction was elicited by applying DNFB to both sides of each ear of sensitized mice. Mice were treated with cetirizine (1 and 10 mg x kg)-1), ig). The ears were removed for pathohistological examination and immunohistochemical staining of SP at different designated times after challenge. The contents of SP in the skin of mouse ear were determined by radioimmunoassay (RIA). RESULTS: The mice exhibited a triphasic cutaneous reaction with an immediate-phase response (IPR) at 1 h, a late-phase response (LPR) at 24 h and a very late-phase response (vLPR) at 7 days after challenge with DNFB. The expression of SP in different phases increased gradually. Cetirizine (1 and 10 mg x kg(-1)) was shown to significantly inhibit the ear swellings induced by the IPR (P < 0.01), while no obvious effect on the vLPR. The SP contents in ear skin of triphasic cutaneous reaction were decreased by cetirizine. CONCLUSION: SP is considered to be involved in the pathogenesis of allergic dermatitis. Cetirizine hydrochloride can inhibit the expression of SP in IgE-dependent triphasic cutaneous reaction. It might be part of the mechanisms of anti-anaphylaxis of cetirizine.

Targeted silencing of CXCL1 by siRNA inhibits tumor growth and apoptosis in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is an aggressive malignancy and a major cause of cancer-related mortality worldwide. Our previous study shows that chemokine (C-X-C motif) ligand 1 (CXCL1) was upregulated and CXCR1 was downregulated in tumor tissues as compared to peritumor tissues by chemotaxis assay. As the status of CXCL subgroups and their receptors affect progression of HCC, we evaluated potential mechanisms of CXCL1 associated with anticancer effects in HCC based on our previous study. The effects of targeting CXCL1 by RNA interference (RNAi) on the proliferation and apoptosis of CBRH-7919 cells were observed in vitro and in vivo. Additionally, whether CXCL1 knockdown significantly reduce the activity of STAT3, NF-κB and HIF-1 or not were also estimated. RNAi of CXCL1 in the CBRH-7919 cells decreased the growth of tumors in nude mice by inhibited cells proliferation and induced apoptosis. In conclusion, these findings suggest that CXCL1 plays critical roles in the growth and apoptosis of HCC. RNAi of CXCL1 inhibits the growth and apoptosis of tumor cells, which indicates that CXCL1 may be a potential molecular target for use in HCC therapy.

Inflammatory microenvironment and expression of chemokines in hepatocellular carcinoma.

AIM: To study the inflammatory microenvironment and expression of chemokines in hepatocellular carcinoma (HCC) in nude mice. METHODS: CBRH-7919 HCC cells were injected into the subcutaneous region of nude mice. Beginning two weeks after the challenge, tumor growth was measured every week for six weeks. The stromal microenvironment and inflammatory cell infiltration was assessed by immunohistochemistry in paired tumor and adjacent peritumoral samples, and macrophage phenotype was assessed using double-stain immunohistochemistry incorporating expression of an intracellular enzyme. A chemokine PCR array, comprised of 98 genes, was used to screen differential gene expressions, which were validated by Western blotting. Additionally, expression of identified chemokines was knocked-down by RNA interference, and the effect on tumor growth was assessed. RESULTS: Inflammatory cell infiltrates are a key feature of adjacent peritumoral tissues with increased macrophage, neutrophil, and T cell (specifically helper and activated subsets) infiltration. Macrophages within adjacent peritumoral tissues express inducible nitric oxide synthase, suggestive of a proinflammatory phenotype. Fifty-one genes were identified in tumor tissues during the progression period, including 50 that were overexpressed (including CXCL1, CXCL2 and CXCL3) and three that were underexpressed (CXCR1, Ifg and Actb). RNA interference of CXCL1 in the CBRH-7919 cells decreased the growth of tumors in nude mice and inhibited expression of CXCL2, CXCL3 and interleukin-1ß protein. CONCLUSION: These findings suggest that CXCL1 plays a critical role in tumor growth and may serve as a potential molecular target for use in HCC therapy.