Crypt-like patterned electrospun nanofibrous membrane and probiotics promote intestinal epithelium models close to tissues.

In vitro intestinal epithelium models have drawn great attention to investigating intestinal biology in recent years. However, the difficulty to maintain the normal physiological status of primary intestinal epithelium in vitro limits the applications. Here, we designed patterned electrospun polylactic acid (PLA) nanofibrous membranes with crypt-like topography and mimic ECM fibrous network to support crypt culture and construct in vitro intestinal epithelium models. The patterned electrospun PLA nanofibrous membranes modified with Matrigels at 0 °C showed high biocompatibility and promoted cell growth and proliferation. The constructed duodenum epithelium models and colon epithelium models on the patterned electrospun PLA nanofibrous membranes expressed the typical differentiation markers of intestinal epithelia and the gene expression levels were close to the original tissues, especially with the help of probiotics. The constructed intestinal epithelium models could be used to assess probiotic adhesion and colonization, which were verified to show significant differences with the Caco-2 cell models due to the different cell types. These findings provide new insights and a better understanding of the roles of biophysical, biochemical, and biological signals in the construction of in vitro intestinal epithelium models as well as the potential applications of these models in the study of host-gut microbes interactions. KEY POINTS: • Patterned electrospun scaffold has crypt-like topography and ECM nanofibrous network. • Matrigels at 0°C modify scaffolds more effectively than at 37°C. • Synergy of biomimic scaffold and probiotics makes in vitro model close to tissue.

Antenatal blockade of N-methyl-D-aspartate receptors by Memantine reduces the susceptibility to diabetes induced by a high-fat diet in rats with intrauterine growth restriction.

Intrauterine growth retardation (IUGR) is closely related to the later development of type 2 diabetes in adulthood. Excessive activation of N-methly-D-aspartate receptors (NMDARs) causes excitatory neurotoxicity, resulting in neuronal injury or death. Inhibition of NMDARs enhances the glucose-stimulated insulin secretion and survival of islet cells in type 2 diabetic mouse and human islets. Here, we examined whether antenatal blockade of NMDARs by Memantine could decrease the risk of diabetes induced by a high-fat (HF) diet at adulthood in IUGR rats. Pregnant SD rats were assigned to four groups: control, IUGR, Memantine, and Memantine + IUGR. The pregnant rats were exposed to hypoxic conditions (FiO2 = 0.105) for 8 h/day (IUGR group) or given a daily Memantine injection (5 mg/kg, i.p.) before hypoxia exposure from embryonic day (E) 14.5 to E 20.5 (Memantine + IUGR). The offspring were fed an HF diet with 60% of the calories from age 4 to 12 weeks. We found that NMDAR mRNAs were expressed in the fetal rat pancreas. An HF diet resulted in a high rate of diabetes at adulthood in the IUGR group. Antenatal Memantine treatment decreased the risk of diabetes at adulthood of rats with IUGR, which was associated with rescued glucose tolerance, increased insulin release, improved the insulin sensitivity, and increased expression of genes related to beta-cell function in the pancreas. Together, our results suggest that antenatal blockade of NMDARs by Memantine in pregnant rats improves fetal development and reduces the susceptibility to diabetes at adulthood in offspring.

[Metabotropic glutamate receptor 8 activation promotes the apoptosis of lung carcinoma A549 cells in vitro].

This study aims to detect the expression of metabotropic glutamate receptors (mGluRs) in lung carcinoma A549 cells, and to investigate the effects of mGluR8 and mGluR4 activation on the growth of A549 cells in vitro. The mRNA expression levels of the 8 subtypes of mGluRs in A549 cells were determined by real-time PCR. Immunohistochemistry was used to analyze the protein expression of mGluR4 and mGluR8 in A549 cells and lung tissue sections obtained from lung adenocarcinoma patients. To observe the effects of mGluR8 and mGluR4 activation on the growth of A549 cells, the cultured cells were treated with (S)-3,4-DCPG (an agonist of mGluR8) and VU0155041 (an agonist of mGluR4), respectively, and then the cell viability was analyzed by CCK-8 kit, the percentage of DNA synthesis was detected by EdU incorporation, and the apoptosis of the cells was measured by hoechst 33258 staining and flow cytometry. The results showed that there were low expressions of mGluR1, mGluR5, mGluR6, mGluR7 mRNA, no expression of mGluR2 and mGluR3 mRNA, and high expressions of mGluR8 and mGluR4 mRNA in A549 cells. Accordingly, there were also mGluR4 and mGluR8 protein expressions in the A549 cells and the lung adenocarcinoma tissue sections. VU0155041 had no effect on the growth of A549 cells, but (S)-3,4-DCPG significantly decreased the cells' growth in a dose-dependent manner and increased the apoptosis of the cells. The results revealed a role of mGluR8 in the growth and apoptosis of A549 cells and suggested a potential target for clinical treatment of lung cancer.

Antiflammin-1 attenuates bleomycin-induced pulmonary fibrosis in mice.

BACKGROUND: Antiflammin-1 (AF-1), a derivative of uteroglobin (UG), is a synthetic nonapeptide with diverse biological functions. In the present study, we investigated whether AF-1 has a protective effect against bleomycin-induced pulmonary fibrosis. METHODS: C57BL/6 mice were injected with bleomycin intratracheally to create an animal model of bleomycin-induced pulmonary fibrosis. On Day 7 and Day 28, we examined the anti-inflammatory effect and antifibrotic effect, respectively, of AF-1 on the bleomycin-treated mice. The effects of AF-1 on the transforming growth factor-beta 1 (TGF-ß1)-induced proliferation of murine lung fibroblasts (NIH3T3) were examined by a bromodeoxycytidine (BrdU) incorporation assay and cell cycle analysis. RESULTS: Severe lung inflammation and fibrosis were observed in the bleomycin-treated mice on Day 7 and Day 28, respectively. Administration of AF-1 significantly reduced the number of neutrophils in the bronchoalveolar lavage fluid (BALF) and the levels of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1ß) in the lung homogenates on Day 7. Histological examination revealed that AF-1 markedly reduced the number of infiltrating cells on Day 7 and attenuated the collagen deposition and destruction of lung architecture on Day 28. The hydroxyproline (HYP) content was significantly decreased in the AF-1-treated mice. In vitro, AF-1 inhibited the TGF-ß1-induced proliferation of NIH3T3 cells, which was mediated by the UG receptor. CONCLUSIONS: AF-1 has anti-inflammatory and antifibrotic actions in bleomycin-induced lung injury. We propose that the antifibrotic effect of AF-1 might be related to its suppression of fibroblast growth in bleomycin-treated lungs and that AF-1 has potential as a new therapeutic tool for pulmonary fibrosis.

[Bioinformatics of mouse uteroglobin binding protein and its polyclonal antibody preparation].

To prepare anti-mouse uteroglobin binding protein (mUGBP) polyclonal antibody, two polypeptides were synthesized based on the bioinformatics analysis of mUGBP, and New Zealand white rabbits were immunized separately with each peptide coupled with keyhole limpet hemocyanin (KLH). The data indicate that a 13-amino acid polypeptide (positions 221st-233rd) was able to generate anti-peptide antibodies. The titer of the antisera detected with ELISA was 1:10(8). The antisera were then purified with immuno-affinity chromatography to obtain antibodies. Western blot analysis of mUGBP expressed as a fusion protein with a green fluorescent protein (GFP) was performed on the cell lysates of COS-1 cells with the purified antisera, suggesting that the antisera specifically recognized UGBP. By immunohistochemistry and indirect immunofluorescence analysis, we examined the expression of UGBP in the lung tissues from a patient undergoing surgical lung resection for a tumor and from normal mouse lung tissue, and found for the first time that UGBP protein was widely expressed in both mouse and human lung tissue with the most abundant expression in bronchial epithelial cells. These results suggest that the antigen epitopes of mUGBP are well predicted by using bioinformatics analysis. We have obtained anti-mUGBP polyclonal antibody, which will be useful for further investigation.

[Role of uteroglobin-binding protein in antiflammin-1 promoting IL-10 expression and secretion in RAW264.7 cells induced by endotoxin].

The present study investigated the effect of antiflammin-1 (AF-1) on LPS-induced IL-10 secretion from RAW264.7 cells through uteroglobin-binding protein (UGBP). Cultured RAW264.7 cells, a murine monocyte-macrophage cell line, were divided as following: control group, LPS group (1 µg/mL LPS), AF-1 group (100 µmol/L AF-1), LPS+AF-1 group (2 h of 100 µmol/L AF-1 pretreatment before LPS addition), and LPS+AF-1+anti-UGBP group (30 min of anti-UGBP antibody pretreatment before successive treatments with AF-1 and LPS). IL-10 concentration in the supernatants was detected by ELISA assay, and the level of IL-10 mRNA expression in macrophage was detected by using RT-PCR method. The results showed that AF-1 significantly increased LPS-induced IL-10 secretion in RAW264.7 cells in a dose dependent way, and up-regulated its mRNA level. Anti-UGBP antibody pretreatment attenuated the augmented effect of AF-1 on LPS-induced IL-10 secretion and gene expression. These results suggest that AF-1 promotes LPS-induced IL-10 secretion from macrophages, and this effect is mediated by UGBP.

Intestinal models based on biomimetic scaffolds with an ECM micro-architecture and intestinal macro-elasticity: close to intestinal tissue and immune response analysis.

The synergetic biological effect of scaffolds with biomimetic properties including the ECM micro-architecture and intestinal macro-mechanical properties on intestinal models in vitro remains unclear. Here, we investigate the profitable role of biomimetic scaffolds on 3D intestinal epithelium models. Gelatin/bacterial cellulose nanofiber composite scaffolds crosslinked by the Maillard reaction are tuned to mimic the chemical component, nanofibrous network, and crypt architecture of intestinal ECM collagen and the stability and mechanical properties of intestinal tissue. In particular, scaffolds with comparable elasticity and viscoelasticity of intestinal tissue possess the highest biocompatibility and best cell proliferation and differentiation ability, which makes the intestinal epithelium models closest to their counterpart intestinal tissues. The constructed duodenal epithelium models and colon epithelium models are utilized to assess the immunobiotics-host interactions, and both of them can sensitively respond to foreign microorganisms, but the secretion levels of cytokines are intestinal cell specific. The results demonstrate that probiotics alleviate the inflammation and cell apoptosis induced by Escherichia coli, indicating that probiotics can protect the intestinal epithelium from damage by inhibiting the adhesion and invasion of E. coli to intestinal cells. The designed biomimetic scaffolds can serve as powerful tools to construct in vitro intestinal epithelium models, providing a convenient platform to screen intestinal anti-inflammatory components and even to assess other physiological functions of the intestine.

[Activation of mGluRI in neutrophils promotes the adherence of neutrophils to endothelial cells].

L-glutamate (Glu) is an excitatory neurotransmitter in the mammalian central nervous system. Relatively much attention has been paid to functional expression of Glu signaling molecules in peripheral tissues very recently. The present study tested the hypothesis that the activation of group I metabotropic glutamate receptor (mGluRI) in neutrophils stimulated neutrophils adherence to endothelial cells by increasing the surface expression of certain adhesion molecules. Peripheral blood was obtained by venipuncture from healthy donors, and the neutrophils were isolated by Ficoll-Hypaque gradient centrifugation. Neutrophils floating into DMEM/F12 culture medium containing 10% fetal bovine serum were then used immediately. Immunocytochemistry and real-time quantitative RT-PCR were used to detect the expression of mGluRI (mGluR1 and mGluR5) in neutrophils. The adherence of neutrophils to cultured human normal umbilical vein endothelial cells (HUVE-12) was measured by the colorimetric method. Cell surface expression of adhesion molecule CD11a in the neutrophils was determined by flow cytometry. Immunocytochemistry and real-time quantitative RT-PCR showed that mGluR1 and mGluR5 were constitutively expressed in neutrophils. Application of mGluRI agonist S-3,5-dihydroxyphenylglycine (S-DHPG) (1x10(-8)-1x10(-6) mol/L) showed a dose-dependent stimulatory effect on the adherence of neutrophils to HUVE-12 (P<0.05 or P<0.01), with a maximum effect at 1x10(-6) mol/L (P<0.01). Incubations as short as 30 min were sufficient to induce increased adherence after the beginning of S-DHPG treatment. Following time extension (0.5-5 h), S-DHPG (1x10(-6) mol/L) increased the rate of neutrophils adhesion to HUVE-12 with a maximum effect at 0.5 h (P<0.01). However, a time-dependent effect of S-DHPG on the rate of neutrophils adhesion to HUVE-12 was not observed during the experimental period. 1x10(-6) mol/L of S-DHPG also induced an increased surface expression of adhesion molecule CD11a (P<0.01) when neutrophils were preincubated with 1x10(-6) mol/L of S-DHPG for 1 h. Furthermore, the specific mGluRI antagonist (RS)-alpha-methyl-4-carboxyphenylglycine ((+/-)-MCPG, 0.5 mmol/L) significantly abolished the stimulatory effect of S-DHPG (1x10(-6) mol/L) on the adherence of neutrophils to HUVE-12 (P<0.01). These results suggest that the activation of mGluRI in neutrophils results in increased adhesion molecule CD11a expression and thereby promotes the adherence of neutrophils to endothelial cells.

G-CSF Inhibits Pulmonary Fibrosis by Promoting BMSC Homing to the Lungs via SDF-1/CXCR4 Chemotaxis.

Bone marrow mesenchymal stem cells (BMSCs) have multi-lineage differentiation potential and play an important role in tissue repair. Studies have shown that BMSCs gather at the injured tissue site after granulocyte-colony stimulating factor (G-CSF) administration. In this study, we first investigated whether G-CSF could promote BMSC homing to damaged lung tissue induced by bleomycin (BLM) and then investigated whether SDF-1/CXCR4 chemotaxis might be involved in this process. Next, we further studied the potential inhibitory effect of G-CSF administration in mice with lung fibrosis induced by bleomycin. We examined both the antifibrotic effects of G-CSF in mice with bleomycin-induced pulmonary fibrosis in vivo and its effects on the proliferation, differentiation and chemotactic movement of cells in vitro. Flow cytometry, real-time PCR, transwell and Cell Counting Kit-8 (CCK-8) assays were used in this study. The results showed that both preventative and therapeutic G-CSF administration could significantly inhibit bleomycin-induced pulmonary fibrosis. G-CSF enhanced BMSC migration to lung tissues, but this effect could be alleviated by AMD3100, which blocked the SDF-1/CXCR4 axis. We also found that BMSCs could inhibit fibroblast proliferation and transdifferentiation into myofibroblasts through paracrine actions. In conclusion, G-CSF exerted antifibrotic effects in bleomycin-induced lung fibrosis, in part by promoting BMSC homing to injured lung tissues via SDF-1/CXCR4 chemotaxis.

[Experimental study of acute lung injury induced by glutamate in vivo].

OBJECTIVE: To investigate the possible injury induced by glutamate in the lung. METHODS: The lung wet weight/body weight (LW/BW), lung wet/dry weight (W/D), the content of cells and the total protein (TP) in bronchoalveolar lavage fluid (BALF) were determined together with the micromorphology observation. RESULTS: (1) The LW/BW, W/D, the content of white blood cells, red blood cells and TP in BALF increased in a dose dependent manner 2 hours after the administration of the glutamate (0.50 - 0.75 g/kg). (2) Examination of histological sections showed the presence of lung inflammation charactered by neutrophils recruitment 2 hours after the glutamate administration. (3) The increase of W/D caused by glutamate (0.50 g/kg) was nearly abolished by pre-treatment with MK801 (a specific blocker of NMDA receptor, 0.1 mg/kg) for 30 minutes (P<0.05). CONCLUSION: Glutamate can cause the acute lung injury through the activation of NMDA receptor in vivo.

An excessive increase in glutamate contributes to glucose-toxicity in ß-cells via activation of pancreatic NMDA receptors in rodent diabetes.

In the nervous system, excessive activation of NMDA receptors causes neuronal injury. Although activation of NMDARs has been proposed to contribute to the progress of diabetes, little is known about the effect of excessive long-term activation of NMDARs on ß-cells, especially under the challenge of hyperglycemia. Here we thoroughly investigated whether endogenous glutamate aggravated ß-cell dysfunction under chronic exposure to high-glucose via activation of NMDARs. The glutamate level was increased in plasma of diabetic mice or patients and in the supernatant of ß-cell lines after treatment with high-glucose for 72 h. Decomposing the released glutamate improved GSIS of ß-cells under chronic high-glucose exposure. Long-term treatment of ß-cells with NMDA inhibited cell viability and decreased GSIS. These effects were eliminated by GluN1 knockout. The NMDAR antagonist MK-801 or GluN1 knockout prevented high-glucose-induced dysfunction in ß-cells. MK-801 also decreased the expression of pro-inflammatory cytokines, and inhibited I-κB degradation, ROS generation and NLRP3 inflammasome expression in ß-cells exposed to high-glucose. Furthermore, another NMDAR antagonist, Memantine, improved ß-cells function in diabetic mice. Taken together, these findings indicate that an increase of glutamate may contribute to the development of diabetes through excessive activation of NMDARs in ß-cells, accelerating ß-cells dysfunction and apoptosis induced by hyperglycemia.

A rapid, nongenomic action of glucocorticoids in rat B103 neuroblastoma cells.

We report here a new example in which glucocorticoids (GCs) acted in a rapid, nongenomic way. In rat B103 neuroblastoma cells, 5-hydroxytryptamine (5-HT) was found to evoke an immediate rise in intracellular free calcium concentration ([Ca(2+)](i)). Pre-incubation of B103 cells for 5 min with corticosterone (B) or bovine serum albumin-conjugated corticosterone (B-BSA) concentration-dependently (10(-4)-10(-8) M) inhibited the peak increments in [Ca(2+)](i). Cortisol and dexamethasone had a similar effect, while deoxycorticosterone and cholesterol were ineffective. This rapid inhibitory effect of corticosterone could be mimicked by protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and abolished completely by PKC inhibitors Ro31-8220 or GF-109203X. Neither pertussis toxin (PTX) nor nuclear GC receptor (GR) antagonist RU38486 influenced the rapid action of B. Our results suggest that GCs can modulate the 5-HT-induced Ca(2+) response in B103 cells in a membrane-initiated, nongenomic, and PKC-dependent manner.

Evoked intracellular Ca2+ elevations in HT4 neuroblastoma cells.

In this study, membrane depolarization and multiple neurotransmitters (5-HT, acetylcholine, histamine, norepinephrine, epinephrine, glutamate, and ATP) were tested for the ability to elevate the intracellular free Ca2+ concentration ([Ca2+]i) in mouse HT4 neuroblastoma cells. Apart from ATP, none of the treatments gave rise to a detectable Ca2+ response, no matter whether the cells were subjected to temperature-induced neuronal differentiation. Our results provide pharmacological evidence for the co-existence in HT4 cells of both P2X and P2Y receptors, the activation of which by ATP led to Ca2+ influx and Ca2+ release, respectively. The P2Y receptor was found to couple to more than one type of G protein in the signaling pathway, causing the activation of phospholipase C (PLC) and Ca2+ mobilization from intracellular stores. cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) attenuated ATP-evoked [Ca2+]i elevations in different ways. However, no correlation was identified between neuronal differentiation and the ATP-evoked Ca2+ responses in HT4 cells. This work indicates that HT4 cells can serve as a good model to study P2 purinoceptor-associated signaling pathways.

Protective effect of ginsenoside Rg1 on glutamate-induced lung injury.

AIM: To examine the possible protective effect of ginsenoside Rg1, an active component of ginseng, on lung injury caused by glutamate in vivo. METHODS: The lungs of mice receiving glutamate (0.5 g/kg) and/or ginsenoside Rg1 (0.03 g/kg) via intraperitoneal administration were collected. The indexes of lung wet weight/ body weight ratios (LW/BW), lung wet/dry weight ratios (W/D), heart rate (HR), and breathing rate (BR) were determined. The activity of nitric oxide synthase (NOS), xanthine oxidase (XOD), superoxide dismutase (SOD), catalase (CAT), the content of NO, and malondialdehyde in the lung homogenate were measured. RESULTS: Treatment with glutamate for 2 h increased LW/BW, W/D, HR, and BR. These changes were nearly abolished by pretreatment with ginsenoside Rg1 for 30 min before glutamate injection. An analysis of the lung homogenate demonstrated the protective effect as evidenced by the inhibition of NOS (12%) and XOD (50%) inactivity, the enhanced activity of SOD (20%) and CAT (25%). CONCLUSION: Ginsenoside Rg1 has a potential protective role in lung diseases associated with glutamate toxicity.

Inhibition of ATP-induced calcium influx in HT4 cells by glucocorticoids: involvement of protein kinase A.

AIM: In our previous observations, adenosine triphosphate (ATP) was found to evoke immediate elevations in intracellular free calcium concentration ([Ca2+]i) in HT4 neuroblastoma cells of mice. We tried to see if a brief pretreatment of glucocorticoids could inhibit the Ca2+ response and reveal the underlying signaling mechanism. METHODS: Measurement of [Ca2+]i was carried out using the dual-wavelength fluorescence method with Fura-2 as the indicator. RESULTS: Pre-incubation of HT4 cells for 5 min with corticosterone (B) or bovine serum albumin conjugated corticosterone (B-BSA) inhibited the peak [Ca2+]i increments in a concentration-dependent manner. Cortisol and dexamethasone had a similar action, while deoxycorticosterone and cholesterol were ineffective. Both extracellular Ca2+ influx and internal Ca2+ release contributed to ATP-induced [Ca2+]i elevation. The brief treatment with only B attenuated Ca2+ influx. Furthermore, the [Ca2+]i elevation induced by the P2X receptor agonist adenosine 5'-(beta, gamma-methylene) triphosphate (beta, gamma-meATP) was also suppressed. The rapid inhibitory effect of B can be reproduced by forskolin 1 mmol/L and blocked by H89 20 mmol/L. Neither nuclear glucocorticoid receptor antagonist mifepristone nor protein kinase C inhibitors influenced the rapid action of B. CONCLUSION: Our results suggest that glucocorticoids modulate P2X receptor-medicated Ca2+ influx through a membrane-initiated, non-genomic and PKA-dependent pathway in HT4 cells.

[Nongenomic action of corticosterone to calcium rise induced by high-K+ in PC12 cells].

AIM: To analyse the mechanism of corticosterone on the elevation of cytosolic free calcium ([Ca2+]i) induced by high-K+ in pheochromocytoma PC12 cells, METHODS: The [Ca2+]i was real-time checked by fluorescence image system. RESULTS: (1) When the cells were preincubated at 37 degrees C for 5 min in the presence of various concentration corticosterone and stimulated with 55 mmol/L KCl , an inhibitory effect of corticosterone on delta[Ca2+]i was observed in a concentration-dependent manner. (2) When PC12 cells were preincubated with various concentration of B-BSA at 37 degrees C for 5 min and stimulated with 55 mmol/L KCl, an inhibitory effect of B-BSA on delta[Ca2+]i was observed, which is also concentration-dependent manner. (3) The inhibitory effect of corticosterone and B-BSA could not be antagonized by RU38486 at 10(-4) mol/L. (4) cycloheximide could not block the inhibitory effect of corticosterone after pretreating cells at 10(-5) mol/L at 37 degrees C for 3 hours. CONCLUSION: It is obvious that the locus of corticosterone action is on the plasmic membrane. The inhibitory effect of corticosterone is independent of protein synthesis and intracellular glucocorticoid receptor. The effect of corticosterone on [Ca2+]i is nongenomic action in PC12 cells.

Norepinephrine-induced calcium mobilization in C6 glioma cells.

AIM: To investigate the mechanism underlying the norepinephrine-induced elevation in intracellular calcium concentration ([Ca2+]i) in C6 glioma cells. METHODS: Measurement of [Ca2+]i was carried out using the dual-wavelength fluorescence method with fura-2 as the indicator. RESULTS: Norepinephrine was found to induce concentration-dependent increases in [Ca2+ ]i through alpha1-adrenoreceptors. The [Ca2+]i elevations were extracellular-calcium independent and not influenced by the treatment of pertussis toxin. Pretreatments with either U73122 or thapsigargin abolished the subsequent cellular calcium responses to norepinephrine. Preincubation with phorbol 12-myristate 13-acetate (PMA) significantly reduce d the [Ca2+]i elevations, while protein kinase C inhibitors Ro31-8220 or GF-109203X completely blocked the inhibitory action of PMA. However, drugs either activating or inhibiting the function of protein kinase A had no effect on the [Ca2+]i elevations. CONCLUSION: Norepinephrine induces calcium mobilization from internal stores by activation of phospholipase C in C6 cells. The [Ca2+]i elevation is negatively regulated by the activation of protein kinase C.