Injury effect of recombinant soluble human CD40 ligand on human umbilical vein endothelial cells

AIM: To investigate the damage in human umbilical vein endothelial cells (HUVECs) induced by recombinant soluble human CD40 ligand (rshCD40L). METHODS: The cultured HUVECs were treated with rshCD40L for 12 h. The survival activity of the HUVECs was observed by MTS assay. The expression of E-selectin, intercellular adhesion molecule (ICAM)-1, tissue factor (TF) and tissue factor pathway inhibitor (TFPI) was measured by ELISA. The activity of superoxide dismutase (SOD) and the level of malondialdehyde (MDA) were detected by the methods of thibabituric acid (TBA). RESULTS: Compared with normal group, different concentrations of rshCD40L (0.5, 1, 2, 3 mg/L) had no obvious effect on the survival activity of the HUVECs (P>0.05). rshCD40L at concentration of 0.5 mg/L promoted the secretion of E-selectin, sICAM-1, TF and TFPI in the HUVECs (P<0.01). rshCD40L at concentration of 0.5 mg/L also increased MDA content and reduced the activity of SOD in the HUVECs (P<0.05). CONCLUSION: 0.5~3mg/L rshCD40L has no obvious effect on endothelial cell survival, but already causes endothelial dysfunction by increasing endothelial inflammation and exogenous coagulation reaction, inducing lipid peroxides injury and reducing antioxidant capacity.     

Expression of CD40-CD40L during rat myocardial ischemia-reperfusion injury

AIM:To investigate the expression of CD40 and CD40 ligand(CD40L) during myocardial ischemia/reperfusion injury in rats.METHODS:In the rat modal with myocardial ischemia/reperfusion(MI/R),the changes of CD40 and CD40L expression in blood and myocardial tissues were detected by flow cytometry and immunohistochemistry, respectively. There were seven animal groups in the study, including the normal group (n=3), the ischemia 30 min group(I_{30 min},n=6),the ischemia/reperfusion 1 min,5 min,10 min,20 min and 30 min groups(each group, n=6).RESULTS:The indexes of the expression of CD40 and CD40L in I_{30 min} group were higher than those in control group,(P＜0.05); the indexes of R_{5 min},R_{10 min} groups were higher than those in the I_{30 min} group (P＜0.05);the indexes were the highest in R_{5 min} and then decreased in R_{10 min},R_{20 min},R_{30 min} group. The immunohistochemical results revealed that. CD40-CD40L expression increased on the cardiocyte membraneduring the ischemia/reperfusion injury.CONCLUSION:CD40 and CD40L may participate in the development of myocardial ischemia/reperfusion injury.     

Effect of IL-4, CD40L on RANTES production in murine renal tubular epithelial cells

AIM: To investigate the effect of IL-4, CD40L on RANTES production in murine renal tubular epithelial cells (TEC). METHODS: TEC were obtained from mouse, expression of RANTES and CD40 on TEC were measured. RESULTS: (1) Activation of TEC with IL-4 resulted in significant increase in CD40 expression (P＜0.01).(2) A little RANTES was detectable in supernatants without stimulation. TEC stimulated with either cytokine IL-4 or CD40mAb resulted in strong induction of RANTES production up to 43.61±13.73 or 73.77±4.28(ng/L), respectively. The differences of RANTES between two stimulation groups and that in medium were statistically significant (P<0.01). TEC stimulated with IL-4 and CD40mAb produced more RANTES than that in medium (P<0.01), which was higher than that with single stimulation (P<0.01). (3) TEC stimulated with IL-4 or CD40 activation or combined stimulation of IL-4 and CD40mAb resulted in increase in levels of RANTE mRNA, which were higher than that in medium. CONCLUSION: Co-stimulation of TEC by IL-4 and CD40mAb up-regulated the RANTES production, suggesting the RANTES may participate in the inflammation of TEC.     

Increased expression of CD40 ligand by peripheral blood mononuclear cells in patients with systemic lupus erythematosus

AIM:To investigate expression and function of CD40 ligand by peripheral blood mononuclear cells (PBMCs) in patients with systemic lupus erythematosus (SLE).METHODS:Expression of CD40 ligand by PBMCs in patients with SLE and control were examined by flow cytometric analysis before and after stimulated by phytohemagglutinin(PHA)and depressed by Dexamethasone(Dex). The correlation between expression of CD40 ligand and SLE activity index(SLEDAI) was analysed in patients with SLE.RESULTS:The expression of CD40 ligand by PBMCs in patients with active SLE was higher than that in patients with inactive SLE and control. Though the expression of CD40 ligang by PBMCs could be stimulated by PHA in three groups, it was the highest in patients with active SLE. Dex depressed the expression of CD40 ligand by PBMCs significantly in patients with SLE, but not in control. There was high positive correlation between expression of CD40 ligand and SLEDAI in patients with active and inactive SLE.CONCLUSION:Increased expression of CD40L by PBMCs in patients with SLE may play an important role in pathogenesis of SLE.     

Effect of 4.25% peritoneal dialysis solution on CD40 expression in rat peritoneal mesothelial cells

AIM:To investigate the effect of 4.25%peritoneal dialysis solution (PDS) on CD40 expression in rat peritoneal mesothelial cells so as to reveal the potential mechanisms by which CD40-CD40 ligand (CD40L) interaction may be involved in the inflammation of peritoneal membrane. METHODS:Rat peritoneal mesothelial cells (MC) were harvested from the peritoneal cavity and maintained under defined in vitro conditions. Expression of CD40 on MC under normal culture or stimulation with 4.25%PDS or 4.25%PDS+IFN-γ was detected by RT-PCR and FACS analyses. After activation of CD40 on MC with CD40 mAb, the expression of intercellular adhesion molecule-1 (ICAM-1) on MC was analyzed by FCAS. RESULTS:MC cultured in vitro expressed CD40 constitutively. 4.25%PDS markedly up-regulated the expression of CD40 mRNA and its protein. The expression of CD40 mRNA and its protein following stimulation with 4.25%PDS+IFN-γ was significantly higher than 4.25%PDS alone. The expression of ICAM-1 on MC was significantly increased after activation of CD40 with CD40mAb.CONCLUSIONS: MC functionally express CD40.The up-regulated CD40 expression on MC fol owing stimulation with 4.25%PDS may play an important role in local peritoneal defense mechanisms and may be involved in the chronic inflammatory process of the peritoneum.     

Significance of soluble CD40 ligand in the vulnerability of coronary atherosclerotic plaque

AIM: To evaluate the significance of serum soluble CD40 ligand (sCD40L) in the vulnerability of coronary atherosclerotic plaque, the relationship between the level of sCD40L and the stenosis degree of the coronary artery by the coronary angiography (CAG), and other inflammatory factors. METHODS: According to WHO diagnostic criterior of coronary heart disease (CHD) and the results of CAG, 84 cases of CHD and 20 cases of non-CHD (NCHD) were included in this study. 84 cases of CHD were divided into three groups: 30 cases in acute myocardial infarction (AMI) group, 30 cases in unstable angina (UA), 24 cases in stable angina (SA). The sera levels of sCD40L in four groups were detected by the enzyme-linked immunosorbent assay (ELISA) and the results were expressed with μg/L. CAG were all conducted in four cases and the results were further evaluated by Jenkins score. ESR and CRP were detected at the same time. RESULTS: The sera levels of sCD40L in four groups were significantly different (P<0.01). The level of sCD40L in AMI group (8.48±4.13) μg/L was higher than that in SA group (4.36±2.68) μg/L, P<0.01 and NCHD group (4.12±1.96) μg/L, P<0.01. The level of sCD40L in UA group (8.72±4.26) μg/L was higher than that in SA group and NCHD group (P<0.01). The level of sCD40L in UA group was slightly higher than that in AMI group, but the difference of two group is not significant (P>0.05). The level of sCD40L in SA group was slightly higher than that in NCHD group, but the difference of two group is not significant (P>0.05). The sera levels of sCD40L in CHD were significantly and positively correlated with Jenkins score (r=0.524, P<0.01). The sera level of sCD40L was positively correlated with the levels of CRP and ESR. CONCLUSION: The sera levels of sCD40L in the patients with various types of CHD are significantly different. The level of sCD40L in the patients with AMI and UA are significantly higher than those in SA and NCHD groups, which may reflect the vulnerability of coronary atherosclerotic plaque. The sera levels of sCD40L is increased with the increasing number of diseased coronary branches and Jenkins score, suggesting that sCD40L promotes atherosclerosis and also can be used as a parameter to predict pathological severity of coronary atherosclerosis. The level of sCD40L is obviously correlated with the levels of CRP and ESR.     

Effect of lipopolysaccharide on apoptosis of human umbilical vein endothelial cells

AIM: To explore the mechanism of lipopolysaccharide (LPS) on vascular endothelial cell(VEC) damage. METHODS: By using cytometry techniques, we studied the effects of LPS on apoptosis of human umbilical vein endothelial cells (HUVECs) in vitro. RESULTS: LPS was able to induce apoptosis of HUVECs in a time-dose-dependent fashion.CONCLUSION:Apoptosis might play a role in LPS-induced damage of vascular endothelial cells.     

Retarding effect of simvastatin on artery remodeling induced by high-salt and high-fat diet in rats

AIM: To investigate the effect of simvastatin intervention on the changes of blood pressure, serum lipid fluctuation and aortic configuration induced by high-sodium and high-fat diet in rats. METHODS: Sixty adult male SD rats were randomly divided into 5 groups (n=12): control (N)group, high salt (S)group, high fat (F) group, high salt+ high fat (SF) group and high salt+high fat + simvastatin (T) group. After fed for 16 weeks, the rats were subject to determine blood pressures and serum concentrations of triglycerides (TG),total cholesterol(TC) and soluble CD40 ligand (sCD40L). The expression of CD40/CD40L in the root of ascending aorta was detected by immunohistochemical method. The thickness of intima media in the ascending aorta as well as the ratio of lumen area/total vascular area were measured and calculated after HE staining. RESULTS: In S group, F group and SF group, systolic blood pressure was significantly higher than that in N group (P<0.01). Systolic blood pressure in T group were slightly higher than that in N group with statistical significance and significantly lower than that in SF group. The serum concentrations of TG and TC in F group and SF group were significantly higher than those in N group and T group (P<0.01), and no significant difference among S group, N group and T group was observed. In S group, F group and SF group, the serum concentrations of sCD40L were higher than that in N group and T group (P<0.05), meanwhile that in SF group was also higher than that in S group and F group (P<0.05). However, no significant difference of sCD40L concentration between S group and F group as well as N group and T group was observed. The expression of CD40/CD40L in the ascending aorta in S group, F group and SF group was higher than that in N group and T group (P<0.05), and that in SF group was also higher than that in S group and F group (P<0.05).No significant difference of CD40/CD40L expression between S group and F group as well as N group and T group was observed. The thickness of intima media in S group, F group and SF group was significantly thicker than that in N group (P<0.01), and no significant difference of the intima media thickness between T group and N group was observed. The ratio of lumen area/total vascular area in S group, F group and SF group was smaller than that in N group (P<0.05), and no significant difference of the ratio between T group and N group was found. CONCLUSION: Feeding high-fat and high-salt diet leads to blood pressure elevation, induces atherosclerosis, increases serum concentration of sCD40L and enhances the expression of CD40/CD40L in arterial tissues. The combination of the stimuli has stronger effect than a single factor. Statins protect the arterial tissues against atherosclerosis by decreasing the level of serum sCD40L and inhibiting the arterial expression of CD40/CD40L.     

Antigen-loaded dendritic cells and CD40L triggers the killing effects of cytotoxic T lymphocytes on K562 cells in vitro

AIM:To investigate the anti-tumor effects of special cytotoxic T lymphocytes (CTLs) activated by dendritic cells (DCs) loaded with antigens and CD40L in vitro.METHODS:Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation from normal human heparinized blood.The adherent cells were cultured with granulocyte-macrophage colony stimulating factor (GM-CSF),interleukin-4 (IL-4),alpha tumor necrosis factor (TNF-α),DCs were co-cultured with frozen-thawed antigen of K562 cells and CD40L,then triggered T cells into specific CTLs.RESULTS:Most suspended cells exhibited distinctive morphological features of DCs which expressed CD40 96%,CD86 97%,CD80 77%,CD1a 69%,and gained the powerful capacity to stimulate proliferation of allogenic lymphocytes.Under the effector∶target ratio of 20∶1,CTLs derived from cultures with DCs and frozen-thawed antigen of K562 cells were showed 71.3% cytotoxicities against K562 cells.CTLs derived from cultures with DCs loaded with frozen-thawed antigen and CD40L were showed 86.9% cytotoxicities against K562 cells.Cytotoxicities by CTLs derived from cultures with unloaded DCs against K562 cells were 37.6% and cytotoxicities by monocytes were 21.1%.Cytotoxicities by CTLs derived from experiment groups were stronger than control groups (P<0.05).CONCLUSIONS:The tumor antigen-pulsed DCs induces efficient and specific anti-tumor immunity,CTLs derived from cultures containing DCs pulsed with CD40L show the strongest cytolytic activities on K562 cells.     

Effect of CD137-CD137 ligand interaction on expression of NFATc1 in apolipoprotein E-deficient mice

AIM: To observe the effects of CD137-CD137 ligand(CD137L) interaction on the nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) in apolipoprotein E-knockout (ApoE^-/-) mice. METHODS: Atherosclerotic plaque model was produced by perivascular carotid collar placement in ApoE^-/- mice. In vivo, the expression levels of NFATc1 in mouse plaques and lymphocytes were detected by immunohistochemical method and flow cytometry, respectively. In vitro, the expression of NFATc1 at mRNA and protein levels in cultured lymphocytes of ApoE^-/- mice was measured by RT-PCR and flow cytometry, respectively. RESULTS: In vivo, after CD137-CD137L signaling pathway was stimulated, the expression of NFATc1 was significantly increased in the atherosclerotic plaques and lymphocytes. In vitro, the expression of NFATc1 at mRNA and protein levels in cultured leukocytes of ApoE^-/- mice was also significantly increased, with the maximal effect exerted by anti-CD137 monoclonal antibody (mAb) at the concentration of 20 mg/L, and 24 h after stimulation at any concentration (P<0.05). Anti-CD137L mAb significantly inhibited the expression of NFATc1 at mRNA and protein levels in the lymphocytes of ApoE^-/- mice, with the maximal effect exerted by anti-CD137L mAb at the concentration of 20 mg/L, and 24 h after stimulation (P<0.05). CONCLUSION: CD137-CD137L interaction can regulate the expression of NFATc1 in ApoE^-/- mice.     

Effects of peroxynitrite on the endothelial cells

Endothelial cells produce both superoxide and nitric oxide. Nitric oxide and superoxide are known to react rapi dly to formthe stable peroxynitrite anion. Peroxynitrite mediates the oxidation of protein, lipid, deoxyribose and inhibits mitochondrial electron transport. Peroxynitrite may break DNA strands and activate poly(ADP-ribose) synthetase. If the reactionis excessive, it results in a depletion of intracellular NAD⁺ and ATP. There is ultimately cell death.     

Effect of lipopolysaccharide on viability and secretion function of human umbilical vein endothelial cells

AIM: To observe the direct effect of lipopolysaccharide (LPS) on secretion of endothelin-1 (ET-1) and nitric oxide by human umbilical vein endothelial cell and cell viability of the secretor. METHODS: The third passage of human umbilical vein endothelial cells were incubated with different concentrations of LPS (1 g/L, 100 mg/L, 10 mg/L, 1 mg/L, 100 μg/L, 10 μg/L, 1 μg/L) for 6 hours, and the culture supernatants were collected. The concentrations of ET-1 were determined by radioimmunoassay, the concentrations of nitric oxide were determined using Greiss's method. The viabilities of cells were measured by MTT method. RESULTS: The concentration of ET-1 (pg/L) of normal control group was 251.64±10.90. The concentrations of ET-1 (pg/L) of LPS treated groups were 220.85±19.14, 278.67±15.45, 306.40±11.60, 312.87±33.50, 324.38±17.02, 291.49±14.30, 282.11±13.38, respectively (each group compared with normal control group, P<0.05 or P<0.01). The concentration of NO_x (μmol/L) of normal control group was 629.46±13.36. The concentrations of NO_x (μmol/L) of LPS treated groups were 732.58±23.21, 669.87±9.32, 661.24±16.80, 650.33±13.24, 606.59±12.94, 626.75±9.83, 627.61±5.61, respectively (each group compared with normal control group, P<0.05 or P<0.01). The viabilities of endothelial cells of LPS treated groups were 74%, 81%, 86%, 88%,91%, 93%, 93%, respectively. CONCLUSION: LPS of lower concentrations had no significantly lethal effect on human umbilical vein endothelial cells, but enhanced secretion of ET-1 and inhibited NO production. LPS in higher concentrations showed significant lethal effect on human umbilical vein endothelial cells, inhibited secretion of ET-1 and enhanced NO production.     

Effect of VEGF over-expressed C6 glioma cells on the expression of Flk-1and Flt-1in cocultured microvascular endothelial cells

AIM: To study the effects of VEGF over-expressed C6 glioma cells on the expression of Flk-1and Flt-1in cocultured microvascular endothelial cells using the rat hepatic cells BRL 3A as control. METHODS: Cocultured systems of rat pulmonary microvascular endothelial cells with C6 and endothelial cells with BRL 3A were established. Immunocytochemical method was used to investigate the expression change of Flk-1and Flt-1protein in cocultured microvascular endothelial cells. The expression of Flt-1and Flk-1mRNA was analyzed by RT-PCR and Northern blot. RESULTS: The microvascular endothelial cells cocultured with C6 showed increased expression of Flk-1and Flt-1protein (P <0.05), while that cocultured with BRL 3A, showed decreased expression of Flt-1and Flk-1protein(P <0.01). The results of RT-PCR and Northern blot showed that the Flk-1, Flt-1mRNA of microvascular endothelial cells were markedly up-regulated after coculture with C6 glioma cells (P <0.01), while the endothelial cells cocultured with BRL 3A had down-regulated expression of Flk-1, Flt-1mRNA (P <0.01). CONCLUSION: VEGF over-expressed C6 glioma cells may markedly up-regulate the expression of Flt-1and Flk-1in cocultured microvascular endothelial cells. These results suggest that this effect could be one of the important mechanisms in glioma angiogenesis in vivo.     

Effect of pulmonary vascular endothelial cells on the development of acute lung injury in rats

AIM: Effect of endothelial cell on the development of acute lung injury and the prevention of dexamethasone in acute lung injury were observed.METHODS:Rats were divided into three groups:1.Control group.2.LPS group:Venous injection with LPS(5mg/kg body weight),execute respectively at 1 h,2 h,6 h and 24 h after LPS injection. 3.dexamethasone group:intraperitoneal injection with dexamethasone ,1 h before LPS injection,execute after 2 hours after LPS injection.RESULTS: Serum NO,TNF-α levels,lung iNOS activity and lung ICAM-1mRNA expression were increased( P <0.05, P <0.01, vs control group),but serum ACE was decreased( P <0.01).Dexamethasone could improve all the changes above mentioned.CONCLUSION:Endothelial cell played a vital role in the development of acute lung injury and dexamethasone could prevent acute lung injury.     

Effect of basic fibroblast growth factor on synthesis of C-type natriuretic peptide in cultured human umbilical vein endothelial cells

AIM: To investigate the effect of basic fibroblast growth factor (bFGF) on C-type natriuretic peptide (CNP) production, release and mRNA expression. METHODS: Human endothelial cell cultured;CNP was mea sured by radioimmunoassay method;CNP mRNA expression was determined by RT-PCR technique.RESULTS: bFGF could augment CNP synthesis in human endothelial cells. Compared with control group,25 ng, 50 ng, 100 ng bFGF increased CNP contents in endothelial cells by 88% (P＜0.05), 95% (P＜0.05), 187% (P＜0.01), respectively.100 ng bFGF also stimulated CNP release from cultured human endothelial cell. In addition, 25 ng, 50 ng and 100 ng bFGF stimulated CNP mRNA expression of cultured human endothelial cells in a dose-dependent manner. CONCLUSION: bFGF might regulate CNP synthesis,release and mRNA expression in cultured umbilical human endothelial cells.     

Effects of amlodipine on apoptosis of vascular endothelial cells induced by oxyeterols

AIM:To investigate the apoptosis of endothelial cells (EC) induced by oxysterols and to examine the effect of amlodipine on the apoptosis induced by oxysterols.METHODS:Light microscope, electron microscope, DNA agarose gel electrophoresis and flow cytometry were used to detect the apoptosis of EC.RESULTS:The characteristic morphological features of apoptosis were observed under light and electron microscope; DNA electrophoresis showed"DNA Ladder"; Flow cytometry showed the sub-G1 peak, apoptotic rate is 32.25% and 23.04% in Triol-treated and 25-OH-treated groups, respectively. While treated EC with amlodipine at the same time, the apoptotic rate decreased significantly.CONCLUSION:Both Triol and 25-OH can induce apoptosis of EC, which can be inhibited by amlodipine.     

Changes of CD28, CD56 and CD57 expression on CD8+T cells in the peripheral blood of healthy elderly individuals

AIM:The expression of CD28, CD56 and CD57 on CD8⁺T cells in the peripheral blood of young (age range 20-35) and elderly(age range 60-75) healthy donors were compared to explore the change of the cellular immune function with aging.METHODS:Three-color fluorescent flow cytometry was performed to analyze the differences in percentage of CD8⁺CD28⁺, CD8⁺CD56⁺ and CD8⁺CD57⁺T cells in the peripheral blood between the young and elderly groups.RESULTS:CD8⁺CD28⁺T cells in the peripheral blood of the elderly group was significantly lower than those in the young group, with percentage of 34.07±5.28 and 49.84±7.43,respectively (P＜0.05). Conversely, CD8⁺CD56⁺T cells and CD8⁺CD57⁺T cells in the peripheral blood of the elderly group were significantly higher than those in the young group, and the percentage was 6.60±2.40 vs 2.10±0.35,41.82±6.01 vs 22.89±2.80, respectively(P＜0.05).CONCLUSION:The expression of CD28, CD56 and CD57 on the CD8⁺T cells in the peripheral blood are changed significantly with aging. The decrease in CD28 expression may play an important role in the immunosenescence, while the increase in CD56 and CD57 expression seems to be a compensatory adaptation for the immune dysfunction.     

Effects of aspirin on production of nitric oxide and inducible nitric oxide synthase mRNA expression under inflammatory conditions in human vascular endothelial cells

AIM: To explore the effect of aspirin on inducible nitric oxide synthesis and gene expression under inflammation in endothelial cells. METHODS:Using NADPH, Griess methods and RT-PCR, the activity of isozymes of NO synthase (NOS), nitric oxide (NO) level, and iNOS mRNA expression were examined respectively. Also, the lactate dehydrogenase (LDH) release rate, malondialdehyde (MDA) content and cell viability were measured. RESULTS: Aspirin (3 mmol/L) reduced inducible NO production and NOS activity(P＜0.05), caused a significant decrease in LDH release rate and MDA content with a further increase in cell viability. Aspirin inhibited inducible NO excretion and alleviated the damage caused by NO in a concentration-dependent manner. However,aspirin had no effect on basal NO levels in the absence of stimulation by inflammatory factor. On the other hand, under middle concentration (＜10 mmol/L), aspirin was able to reduce enzymatic activity of NOS and protein expression by increasing the stability of iNOS mRNA. In contrast, at high concentration (20 mol/L), aspirin could decrease the stability of iNOSmRNA. Sodium salicylate and indomethacin did not inhibit inducible NO production. CONCLUSION:Aspirin could significantly inhibit inducible NO production in vascular endothelial cells during inflammation.     

Effect of chronic hypoxia on [Ca2+]iin pulmonary artery endothelial cells and smooth muscle cells under acute hypoxia

AIM AND METHODS: Using Ca²⁺-sensitive fluorescent probe Fura-2,we measured the changes of [Ca²⁺]_iin cultured rat pulmonary artery endothelial cells (PAEC) and porcine pulmonary artery smooth muscle cells (PASMC) from normoxic (NC group) or chronic hypoxic group (CH group) when they were exposed to acute hypoxia. RESULTS: The increase in [Ca²⁺]_iin 6th passage of PASMC caused by acute hypoxia in CH group was significantly lower than that in the same passage of NC group (P<0.05).On the contrary, in PAEC, the acute hypoxia induced increase in _i, which was significantly higher in 5th passage of CH group than that in NC group (P<0.05). CONCLUSION: The decrease of the elevation of [Ca²⁺]_icaused by acute hypoxia in PASMC of CH group indicated that it functioned to lower the constrictive response to hypoxia.The intensive increase in [Ca²⁺]_icaused by acute hypoxia in PAEC of CH group might lead to more relaxing factors derived from PAEC,which results in a decrease in HPV.