[B Cell Activating Transcription Factor Regulates Acute Airway Inflammation in Asthmatic Mice].

OBJECTIVE: To investigate the regulatory effect of B cell activating transcription factor (BATF) on acute airway inflammation and its association with retinoic acid orphan nuclear receptors gammat (RORyt) in asthmatic mice. METHODS: 24 female BALB/c mice were randomly and equally divided into three groups (n 8): normal saline (NS) treated, asthma (AS) control and dexamethasone (DEX) treated. AS mice were sensitized and challenged with OVA to establish murine asthma model. Histological changes in lung tissues of the mice were observed by HE staining. Numbers of white blood cell (WBC), polymorphonuclear leukocyte (PMN) and eosinophils (EOS) in the bronchoalveolar lavage fluid (BALF) of the mice were counted. The concentration of interleukin-17 (IL-17) in BALF was measured by ELISA. Quantitative real-time PCR (RT-PCR) was performed to assess the mRNA expressions of BATF, IL-17 and RORγt in the lung tissues. RESULTS: The HE staining showed a higher level of inflammatory cell infiltration around the bronchi of AS mice compared with those treated with NS, predominantly in the forms of EOS, PMN and lymphocytes. The AS and DEX treated mice had higher levels of EOS, PMN, WBC and mRNA expressions of BATF, IL-17 and RORγt in BALF than those treated with NS (P < 0.05). DEX reduced the levels of EOS, PMN, WBC and IL-17 in BALF significantly (P < 0.05). The mRNA expression of BATF in lung tissues of mice was positively correlated with the expression of IL-17, RORγt and the counts of WBC,EOS and PMN in BALF (P < 0.05). CONCLUSION: Asthmatic mice have increased expressions of BATF, IL-17 and RORγt in bronchial and lung tissues. BATF can, through regulating the secretion of Th17 cells, readjust the airway inflammatory. The regulatory function may take effect through synergy with RORγt .

Aß40 modulates GABA(A) receptor α6 subunit expression and rat cerebellar granule neuron maturation through the ERK/mTOR pathway.

In addition to their neurotoxic role in Alzheimer's disease (AD), ß-amyloid peptides (Aßs) are also known to play physiological roles. Here, we show that recombinant Aß40 significantly increased the outward current of the GABA(A) receptor containing (GABA(A)α6) in rat cerebellar granule neurons (CGNs). The Aß40-mediated increase in GABA(A)α6 current was mediated by an increase in GABA(A)α6 protein expression at the translational rather than the transcriptional level. The exposure of CGNs to Aß40 markedly induced the phosphorylation of ERK (pERK) and mammalian target of rapamycin (pmTOR). The increase in GABA(A)α6 current and expression was attenuated by specific inhibitors of ERK or mTOR, suggesting that the ERK and mTOR signaling pathways are required for the effect of Aß40 on GABA(A)α6 current and expression in CGNs. A pharmacological blockade of the p75 neurotrophin receptor (p75(NTR)), but not the insulin or α7-nAChR receptors, abrogated the effect of Aß40 on GABA(A)α6 protein expression and current. Furthermore, the expression of GABA(A)α6 was lower in CGNs from APP(-/-) mice than in CGNs from wild-type mice. Moreover, the internal granule layer (IGL) in APP(-/-) mice was thinner than the IGL in wild-type mice. The injection of Aß40 into the cerebellum reversed this effect, and the application of p75(NTR) blocking antibody abolished the effects of Aß40 on cerebellum morphology in APP(-/-) mice. Our results suggest that low concentrations of Aß40 play a role in regulating CGN maturation through p75(NTR).

Exposure to extremely low-frequency electromagnetic fields modulates Na+ currents in rat cerebellar granule cells through increase of AA/PGE2 and EP receptor-mediated cAMP/PKA pathway.

Although the modulation of Ca(2+) channel activity by extremely low-frequency electromagnetic fields (ELF-EMF) has been studied previously, few reports have addressed the effects of such fields on the activity of voltage-activated Na(+) channels (Na(v)). Here, we investigated the effects of ELF-EMF on Na(v) activity in rat cerebellar granule cells (GCs). Our results reveal that exposing cerebellar GCs to ELF-EMF for 10-60 min significantly increased Na(v) currents (I(Na)) by 30-125% in a time- and intensity-dependent manner. The Na(v) channel steady-state activation curve, but not the steady-state inactivation curve, was significantly shifted (by 5.2 mV) towards hyperpolarization by ELF-EMF stimulation. This phenomenon is similar to the effect of intracellular application of arachidonic acid (AA) and prostaglandin E(2) (PGE(2)) on I(Na) in cerebellar GCs. Increases in intracellular AA, PGE(2) and phosphorylated PKA levels in cerebellar GCs were observed following ELF-EMF exposure. Western blottings indicated that the Na(V) 1.2 protein on the cerebellar GCs membrane was increased, the total expression levels of Na(V) 1.2 protein were not affected after exposure to ELF-EMF. Cyclooxygenase inhibitors and PGE(2) receptor (EP) antagonists were able to eliminate this ELF-EMF-induced increase in phosphorylated PKA and I(Na). In addition, ELF-EMF exposure significantly enhanced the activity of PLA(2) in cerebellar GCs but did not affect COX-1 or COX-2 activity. Together, these data demonstrate for the first time that neuronal I(Na) is significantly increased by ELF-EMF exposure via a cPLA2 AA PGE(2) EP receptors PKA signaling pathway.

Neuritin activates insulin receptor pathway to up-regulate Kv4.2-mediated transient outward K+ current in rat cerebellar granule neurons.

Neuritin is a new neurotrophic factor discovered in a screen to identify genes involved in activity-dependent synaptic plasticity. Neuritin also plays multiple roles in the process of neural development and synaptic plasticity. The receptors for binding neuritin and its downstream signaling effectors, however, remain unclear. Here, we report that neuritin specifically increases the densities of transient outward K(+) currents (I(A)) in rat cerebellar granule neurons (CGNs) in a time- and concentration-dependent manner. Neuritin-induced amplification of I(A) is mediated by increased mRNA and protein expression of Kv4.2, the main α-subunit of I(A). Exposure of CGNs to neuritin markedly induces phosphorylation of ERK (pERK), Akt (pAkt), and mammalian target of rapamycin (pmTOR). Neuritin-induced I(A) and increased expression of Kv4.2 are attenuated by ERK, Akt, or mTOR inhibitors. Unexpectedly, pharmacological blockade of insulin receptor, but not the insulin-like growth factor 1 receptor, abrogates the effect of neuritin on I(A) amplification and Kv4.2 induction. Indeed, neuritin activates downstream signaling effectors of the insulin receptor in CGNs and HeLa. Our data reveal, for the first time, an unanticipated role of the insulin receptor in previously unrecognized neuritin-mediated signaling.

[Effect of Budesonide on Smad4, PDGF-A and PAI-1 in a rat model of pulmonary fibrosis].

AIM: To observe the effect of Budesonide (BUD) on TGF-ß1, PDGF-A, Smad4 and PAI-1 in lung tissue in pulmonary fibrosis rats. METHODS: Forty-five adult female Wistar rats were randomly divided into normal solution (NS) group, BUD group and bleomycin (BLM) group. 9 g/L NaCl solution was instilled into the tratracheaes in NS group, and BLM were used in BUD group and BLM group. NS group and BLM group were inhaled with 9 g/L NaCl solution once everyday at day 0-6, and BUD group were used with BUD. Five rats in every group were killed at 7th, 14th 28th day. The appearances of alveolitis and fibrosis were displayed in HE and Masson staining. The expressions of TGF-ß1, PDGF-A, Smad4 and PAI-1 in lung tissue were detected by immunohistochemistry. RESULTS: The extent of the alveolitis in BUD group at 7th, 14th day were significantly lower than that in BLM group(P<0.05). The fibrosis and the expressions of TGF-ß1, PDGF-A, Smad4 and PAI-1 in lung tissues at 7th, 14th, 28th day, BUD group were significantly lower than BLM group(P<0.05). CONCLUSION: After inhaled BUD, the expressions of TGF-ß1, PDGF-A, Smad4 and PAI-1 in lung tissue could be decreased, and the extent of alveolitis and pulmonary fibrosis could be improved in bleomycin-induced pulmonary fibrosis rats.

The antidepressant citalopram inhibits delayed rectifier outward K⁺ current in mouse cortical neurons.

Citalopram, a selective serotonin (5-HT) reuptake inhibitor (SSRI) as well as an antidepressant, is thought to exert its effects by increasing synaptic 5-HT levels. However, few studies have addressed the possibility that citalopram has other molecular mechanisms of action. We examined the effects of citalopram on delayed rectifier outward K(+) current (I(K) ) in mouse cortical neurons. Extracellular citalopram reversibly inhibited I(K) in a dose-dependent manner and significantly shifted both steady-state activation and inactivation curves toward hyperpolarization. Neither 5-HT itself nor antagonists of 5-HT and dopamine receptors could abolish citalopram-induced inhibition of I(K) . In addition, intracellular application of GTPγ-S similarly failed to prevent the inhibition of I(K) by citalopram. When applied intracellularly, citalopram had no effect on I(K) and did not influence the reduction of I(K) induced by extracellular citalopram. The effect of citalopram was use dependent, but not frequency dependent, and it did not require channel opening. Electrophysiological recordings in acute cortical slice showed that citalopram significantly reduced the action potential (AP) firing frequency of cortical neurons and increased action potential duration (APD). The selective Kv2.1 subunit blocker Jingzhaotoxin-III (JZTX-III) did not abolish citalopram-induced I(K) inhibition. Transfection of HEK293 cells with Kv2.1 or Kv2.2 constructs indicated that citalopram mainly inhibited Kv2.2 current. We suggest that citalopram-induced inhibition of I(K) in mouse cortical neurons is independent of G-protein-coupled receptors and might exert its antidepressant effects by enhancing presynaptic efficiency. Our results may help to explain some of the unknown therapeutic effects of citalopram.

[Effects of andrographolide on the concentration of cytokines in BALF and the expressions of type I and III collagen mRNA in lung tissue in bleomycin-induced rat pulmonary fibrosis].

AIM: To investigate the effects of andrographolide on the concentration of TNF-α and TGF-ß1 in bronchoalveolar lavage fluid (BALF) and the expressions of type I and III collagen mRNA in Lung tissue in bleomycin (BLM)-induced pulmonary fibrosis in rats. METHODS: 90 healthy SD male rats were randomly divided into 6 groups with 15 rats each group: normal saline (NS) group, BLM group, prednisone (Pred) group and different doses of andrographolide groups (andrographolide group A 62.5 mg/kg, andrographolide group B 125 mg/kg and andrographolide group C 250 mg/kg). BLM was given to BLM group, Pred group and andrographolide group A, B, C by intratracheal instillation, and same volume of NS was given to NS group in the same way. And then NS was given to NS group and BLM group, Pred was given to Pred group and different does of andrographolide were given to andrograoholide group A, B, C by gavage every day. Five rats of each group were killed respectively at day 7, 14, 28 after intratracheal instillation. Alveolitis and fibrosis were observed by HE and Masson staining. Real-time fluorescent quantitative reverse transcription- polymerase chain reaction (FQ RT-PCR) was performed to detect the expressions of type I and III collagen mRNA in lung tissue, and the concentration of TNF-α and TGF-ß1 in BALF was determined by enzyme-linked immunosorbnent assay. Blood urea nitrogen (BUN), creatinine (Cr), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were also examined. RESULTS: (1) The AST, ALT, BUN and Cr in every group had no significant diference(P>0.05). (2) Alveolar septal edema, inflammatory cell infiltration and fibrosis were not found in NS group. In the BLM group, lots of inflammatory cells infiltration were observed in the alveolar at day 7; the alveolitis was still existed, but inflammatory cells were significantly reduced, and the number of the fibroblasts and matrix in alveolar septum were obviously increased at day 14, at the same time, alveolar structure was damaged and alveolar septum widened; the inflammation cells infiltration of the alveolar was relieved, pulmonary fibrosis was increased, and parts of alveolar space was disappeared , severe fibrosis was found at day 28. It was similar between andrographolide group A and BLM group in pathomorphology. A lot of inflammatory cells infiltration and local accumulation were observed at day 7 in andrographolide group B, C and Pred group. However, compared with andrographolide group A and BLM group, the fibrosis at day 14, 28 was significantly reduced.(3) The concentration of TGF-ß1, TNF-α in BALF of NS group was significantly lower than that of Pred group, BLM group, andrographolide group A, B, C at each time point(P<0.05). The concentration of TGF-ß1 and TNF-α in BALF of BLM group at day 7, 14, 28 was higher than that of Pred group, andrographolide group B and andrographolide group C (P<0.05). Compared with BLM group, the concentration of TGF-ß1 and TNF-α in BALF of andrographolide group A had no significant difference. (4) The expression of type I and III collagen mRNA in lung tissue of NS group was significantly lower than that of group Pred, BLM, andrographolide group A, B, C at each time point (P<0.05). The expression of type I and III collagen mRNA in lung tissue of BLM group at day 7, 14, 28 was higher than that of Pred group, andrographolide group B and andrographolide group C (P<0.05). Compared with BLM group, the expression of type I and III collagen mRNA in lung tissue of andrographolide group A had no significant difference. CONCLUSION: In BLM-induced rat pulmonary fibrosis, andrographolide could attenuate alveolitis and fibrosis, decrease mRNA expression of collagen I and III in lung tissue and decrease the concentration of TNF-α and TGF-ß1 in BALF. It had no side effects on liver and kidney function.

Amoxapine inhibits the delayed rectifier outward K+ current in mouse cortical neurons via cAMP/protein kinase A pathways.

Ion channels are known to be modulated by antidepressant drugs, but the molecular mechanisms are not known. We have shown that the antidepressant drug amoxapine suppresses rectifier outward K(+) (I(K)) currents in mouse cortical neurons. At a concentration of 10 to 500 muM, amoxapine reversibly inhibited I(K) in a dose-dependent manner and modulated both steady-state activation and inactivation properties. The application of forskolin or dibutyryl cAMP mimicked the inhibitory effect of amoxapine on I(K) and abolished further inhibition by amoxapine. N-[2-(p-Bromocinnamylamino)ethyl]-5-iso-quinolinesulphonamide (H-89), a protein kinase A (PKA) inhibitor, augmented I(K) amplitudes and completely eliminated amoxapine inhibition of I(K). Amoxapine was also found to significantly increase intracellular cAMP levels. The effects of amoxapine on I(K) were abolished by preincubation with 5-hydroxytryptamine (5-HT) and the antagonists of 5-HT(2) receptor. Moreover, intracellular application of guanosine 5'-[gammathio]-triphosphate increased I(K) amplitudes and prevented amoxapine-induced inhibition. The selective Kv2.1 subunit blocker Jingzhaotoxin-III reduced I(K) amplitudes by 30% and also significantly abolished the inhibitory effect of amoxapine. Together these results suggest that amoxapine inhibits I(K) in mouse cortical neurons by cAMP/PKA-dependent pathway associated with the 5-HT receptor, and suggest that the Kv2.1 alpha-subunit may be the target for this inhibition.

[Therapeutic effects of aerosolized signal transducer and activator of transcription 1 antisense oligonucleotide administered at different time points on bleomycin-induced pulmonary fibrosis: experiment with rats].

OBJECTIVE: To investigate the curative effects of inhaling signal transducer and activator of transcription 1 (STAT1) antisense oligonucleotide (ASON) on alveolitis and pulmonary fibrosis and the best administration time. METHODS: Twenty-five adult female Wistar rats were randomly divided into 5 equal groups: BLM group, undergoing intra-tracheal perfusion of BLM so as to establish animal models of alveolitis and pulmonary fibrosis and then inhaling aerosolized normal saline (NS); NS group undergoing intra-tracheal perfusion of NS and then inhaling aerosolized NS; ASON 0 d group, undergoing intra-tracheal perfusion of BLM and then inhaling aerosolized STAT1 ASON 3 ml immediately; ASON 7 d group, undergoing intra-tracheal perfusion of BLM and then inhaling STAT1 ASON 3 ml 7 days later; and ASON 14 d group undergoing intra-tracheal perfusion of BLM and then inhaling aerosolized STAT1 ASON 3 ml 14 days later. Aerosolized inhalation was repeated once every other day for 4 times. Twenty-eight days after intra-tracheal perfusion the rats were sacrificed with their lungs taken out to undergo pathological examination. NS was infused into the right lungs to get bronchoalveolar lavage fluid (BALF). ELISA was used to examine the concentrations of transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha) in the BALF. RESULTS: The pathology result of the lung tissues showed that compared with the BLM and ASON 14 d groups, the alveolitis and pulmonary fibrosis of the ASON 0 d group were obviously milder. The scores of alveolitis and pulmonary fibrosis of the ASON 0 d group were (1.80 +/- 0.84) and (2.60 +/- 0.55) respectively, both significantly lower than those of the BLM group [(2.40 +/- 0.55) and (4.40 +/- 0.55) respectively] and those of the ASON 7 d group [(2.20 +/- 0.45) and (3.00 +/- 0.71) respectively] (all P < 0.05). The scores of pulmonary fibrosis of the ASON 7 d group was significantly lower than those of the BLM and ASON 14 d groups (both P < 0.05). The concentrations of TGF-beta and TNF-alpha in BALF of the ASON 0 d group were (48.11 +/- 3.46) pg/ml and (1.93 +/- 0.14) ng/ml respectively, both significantly lower than those of the BLM group [(57.67 +/- 2.46) pg/ml and (2.45 +/- 0.25) ng/ml respectively, both P < 0.05]. The concentration of TGF-beta in BALF of the ASON 0 d group was significantly lower than those of the ASON 7 d and ASON 14 d groups [(51.42 +/- 3.57) pg/ml and (55.8 3 +/- 1.79) pg/ml respectively, both P < 0.05]. The concentration of TGF-beta in BALF of the ASON7 d group was significantly lower than those of the BLM and ASON 14 d groups (both P < 0.05). CONCLUSIONS: STAT1 ASON administered in the early stage helps depress the pulmonary fibrosis procedure, and the earlier the drug is administrated the better effect would be obtained. Aerosolized STAT1 ASON can be used as a therapeutic method for pulmonary fibrosis.

[Effect of signal transducer and activator of transcription 1 antisense oligodeoxynucleotides on inflammatory mediators, type I and type III collagen mRNA of rat pulmonary fibrosis].

AIM: To investigate the effect of aerosolized signal transducer and activator of transcription 1 (STAT1) antisense oligodeoxynucleotides (ASON) on the expression of inflammatory mediators in bronchoalveolar lavage fluid (BALF) and typeI and typeIII collagen mRNA of the bleomycin-induced rat pulmonary fibrosis. METHODS: 45 adult female Wistar rats were randomly divided into 3 groups: normal saline (NS) group, bleomycin (BLM) group and ASON group. BLM group and ASON group were intratracheally instilled with bleomycin (BLM) while NS group was instilled with NS. NS group and BLM group were aerosolized with NS while ASON group was aerosolized with STAT1 ASON on day 0, 2, 4 and 6 after intratracheal administration. Then each group was divided into 3 subgroups and the rats were sacrificed on day 7, 14 and 28. The concentration of IFN-gamma, TNF-alpha, TGF-beta1 and PDGF-BB in BALF was detected. The lung tissues were removed and HE and Masson staining was performed to observe the extent of alveolitis and fibrosis. The mRNA levels of typeI and typeIII collagen in the lung tissues were measured. RESULTS: Compared with BLM group, the scores of alveolitis and fibrosis in ASON group were remarkably meliorated (P<0.05). Compared with NS group, the concentration of TNF-alpha, TGF-beta1 and PDGF-BB in BALF in BLM group was significantly increased, but it was lower in ASON group than in BLMA group (P<0.05). The concentration of IFN-gamma in BALF was lower in BLM group than in NS group (P<0.05), but it was higher in ASON group than in BLM group (P<0.05). The mRNA levels of typeI and typeIII collagen at various time points in ASON group were significantly lower than those in BLM group (P<0.05). CONCLUSION: The aerosolized STAT1 ASON has anti-fibrosis effect, which may result from the lessened production of typeI and typeIII collagen through reducing the concentration of cytokines in BALF such as TNF-alpha, PDGF-BB and TGF-beta1 and inhibiting the decline of IFN-gamma in BALF.

Aerosolized STAT1 antisense oligodeoxynucleotides decrease the concentrations of inflammatory mediators in bronchoalveolar lavage fluid in bleomycin-induced rat pulmonary fibrosis.

It has been demonstrated that alveolar macrophages (AMs) play a key role in the pathogenesis of pulmonary fibrosis by releasing a variety of cytokines and inflammatory mediators. In addition, abnormal signal transducer and activator of transcription-1 (STAT1) activation in AMs may play a pivotal role in the process of alveolitis and pulmonary fibrosis. In this study, we transfected STAT1 antisense oligodeoxynucleotide (ASON) into rats by aerosolization, and then investigated the effect of STAT1 ASON on inflammatory mediators such as TGF-beta, PDGF and TNF-alpha in bronchoalveolar lavage fluid (BALF) from rats with bleomycin (BLM)-induced rat pulmonary fibrosis. Our results showed that STAT1 ASON by aerosolization could enter into lung tissues and AMs. STAT1 ASON could inhibit mRNA and protein expressions of STAT1 and ICAM-1 in AMs of rat with pulmonary fibrosis, and had no toxic side effect on liver and kidney. Aerosolized STAT1 ASON could ameliorate the alveolitis through inhibiting the secretion of inflammatory mediators in BLM-induced rat pulmonary fibrosis. These results suggest that aerosolized STAT1 ASON might be considered as a promising new strategy in the treatment of pulmonary fibrosis.

[The effect of STAT1 antisense oligonucleotides on lung fibroblast proliferation and hydroxyproline secretion].

OBJECTIVE: To investigate the effect of signal transducer and activator of transcription 1 (STAT1) antisense oligonucleotides on lung fibroblast proliferation and hydroxyproline secretion. METHODS: Ten adult female Wistar rats were randomly divided into two groups: one group was intratracheally instilled with bleomycin (BLM), while another group with 0.9% NaCl solution (NS). After 7 days, the rats were killed by right ventricle of heart exsanguinations under ketamine anaesthesia, and bronchoalveolar lavage (BAL) was performed to obtain alveolar macrophage (AM). AMs from the BLM group were divided into four groups, treated with STAT1 antisense oligonucleotides, STAT1 sense oligonucleotides, dexamethasone and medium alone (control), respectively. AMs and media were collected after culture for 36 h. The mRNA and protein expression of STAT1 and ICAM-1 in AMs were detected by RT-PCR and Cell-ELISA, respectively. The conditioned media were co-cultured with lung fibroblasts for 30 h, and then the cell proliferation and the concentration of hydroxyproline were examined. RESULTS: (1) The STAT1 mRNA expression by AMs in the STAT1 antisense oligonucleotides group (31.8 +/- 3.5) was lower than those of AMs in the STAT1 sense oligonucleotides group (64.2 +/- 4.3), the dexamethasone group (44.1 +/- 4.6) and the control group (65.5 +/- 4.6) (P < 0.05). Moreover, the STAT1 mRNA expression by AMs in the dexamethasone group was also lower than those of AMs in the STAT1 sense oligonucleotides group and the control group (P < 0.05), but the STAT1 mRNA expression by AMs in the STAT1 sense oligonucleotides group was not different from that of the control group (P > 0.05). The STAT1 mRNA expression by AMs in the NS group (14.9 +/- 3.1) was lower than those of AMs in the STAT1 antisense oligonucleotides group, the STAT1 sense oligonucleotides group, the dexamethasone group and the control group (P < 0.05). (2) The mRNA expression of ICAM-1 showed similar changes to the STAT1 mRNA expression by AMs. (3) The STAT1 protein expression by AMs in the STAT1 antisense oligonucleotides group (4.4 +/- 0.6) or in the NS group (3.7 +/- 0.4) was lower than those of AMs in the STAT1 sense oligonucleotides group (7.7 +/- 0.7), the dexamethasone group (5.9 +/- 0.4) and the control group (7.6 +/- 0.6) (P < 0.05); and the STAT1 protein expression by AMs in the dexamethasone group was also lower than those of AMs in the STAT1 sense oligonucleotides group and the control group (P < 0.05), but the STAT1 protein expression by AMs in the STAT1 sense oligonucleotides group was not different from that of the control group (P > 0.05). (4) The changes of ICAM-1 protein expression, lung fibroblast proliferation and hydroxyproline concentration were consistent with the changes of STAT1 protein expression by AMs. CONCLUSIONS: STAT1 antisense oligonucleotides could inhibit the mRNA and the protein expression of STAT1 and ICAM-1 in AMs. STAT1 antisense oligonucleotides also inhibited lung fibroblast proliferation and hydroxyproline secretion.