Study on relationship between perinatal hypoxia and placental apoptosis of ICP patients
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《中华医药杂志》英文版
Study on relationship between perinatal hypoxia and placental apoptosis of ICP patients(pdf)
1 Department of Obstetrics and Gynecology,The First Hospital of Yunyang Medical College,Shiyan,Hubei Province 442000,China
2 Department of Obstetrics and Gynecology,The First Hospital of Xian Jiaotong University,Xian,Shaanxi Province 710061,China
Tel:+86-719-6994131
[Abstract] Objective To investigate the probable mechanism of perinatal hypoxia by detecting gene Bax and Bcl2 expression level on placenta. Methods The placental samples were obtained from 30 cases of ICP and 27 cases of normal pregnant women.Immunohistochemistry method was used to detect Bax and Bcl2 expression level on placenta. Results (1)Perinatal hypoxia rate of ICP group was higher than the control,significantly(χ2=1145,P<0005).(2)Bax protein level in syncytiotrophoblast of ICP group was higher than the control (u=3559，P<00005)，and Bcl2 level of ICP was lower than control (u=3461,P<00005),both significantly.(3)While the Bax protein level increase,the perinatal fetus faced high danger of hypoxia(χ2=102239,P<001). Conclusions Bax overexpression on syncytiotyophoblast may be the mechanism of perinatal hypoxia of ICP.
[Key words] perinatal hypoxia;placental apoptosis
INTRODUCTION
The main injury of intrahepatic cholestasis of pregnancy(ICP)is that increase the incidence of fetal distress or asphyxia of newborn,as well as perinatal mortality.However,the exact mechanism is still unknown.Apoptotic regulative proteiBax and Bcl2 present on placenta,and of these two protein determines the placental cells fate.An increase in a ratio of Bax to Bcl2 can alter apoptosis index and placental function.The aim of this study is to explore the mechanism of perinatal hypoxia of ICP by detecting the expression level of Bax and Bcl2 on placenta.
MATERIAL AND METHODS
Patients
Thirty cases of ICP patients and 27 normal pregnancies in our hospital since 2001/12 to 2002/12 were enrolled.The criteria for ICP was according to Chinese Obstetrics and Gynecology compiled by Cao Zeyi.The mean age (292±463 vs.274±27)and gestational peroid (379±165 vs.384±142) of the two group have no significant difference.
Samples
Within 5 min after delivery,1 cm×1 cm×1 cm placental tissues of central part were taken,avoid the infarct area,fixed,embedded and chop into 4 μm slices and reserve for determination.
SP Immunohistochemistry
Rat antihuman Bax monoantibody was bought from Fuzhou Maixin Company;Rabbit antihuman Bcl2 monoantibody bought from Zhongshan Company.The test followed by illustration of the box,critically.Results judged:the cell show brown grain is positive,or else is negative.According to the brown grains expression level in syncytotrophoblast cells,the positive group was divided into three levels,less than 30% and more than 60%,was regarded as (+)and (+++),respectively,between these two level is (++).
Statistics Analysis
Rank sum test and chisquare test were used toanalyse,P<005 was considered statistically significant.
RESULTS
Perinatal Hypoxia Rate of ICP Group and the Control In ICP group,there were 2 asphyxia of newborn and 13 fetal distress,2 of which were rejected for cord entanglement comparing perinatal hypoxia rate (including fetal distress and asphyxia of newborn)of the two groups showed that perinatal hypoxia rate of ICP group was higher than the control significantly (Table 1)(χ2=1145，P<0005). Table 1 Comparison of Perinatal Hypoxia Rate of Two Groups
Expression Level of Bax and Bcl2 in Two Groups
Bax and Bcl2 were showed in both groups.Bcl2 mainly expressed on plasma of syncytiotrophoblast.Bax express in all kinds of placental cells widely,and it predominantly expressed in syncytiotrophoblast.Table 2 shows that Bax expression level in ICP placenta is higher than that in the control (u=3559,P<00005),while Bcl2 expression level is lower than control (u=3641,P<00005),both significantly.
Table 2 Expression Level between Bax and Bcl2 in Two Groups
Relationship between Bax Expression Level and Perinatal Hypoxia Rate
All of the subjects were divided into three groups according to Bax expression level.Group 1:(-)and (+)(n=33),Group 2:(++)(n=15),Group 3:(+++)(n=9).A significant difference of perinatal hypoxia rate was found among these groups (χ2=102239,0005005).
Table 3 Comparison of Perinatal Hypoxia Rate in Different Bax Level
DISCUSSION
Placental Apoptosis and Its Gene RegulationApoptosis is a common physiological phenomenon.Low level of apoptosis on placenta was found at any period of normal pregnancy.Apoptosis play a critical role in placenta growth,development and maintaining normal pregnancy.(1)Apoptosis play a role in placental remodeling.Apoptosis in part of syncytium is contributed to maintain the normal ratio of syncytiotrophoblast and cytiotrophoblast.(2)Apoptosis contributes to placenta abruption at delivery.(3)Apoptosis is the main mechanism of maternalfetal immunotolerance.
Bax and Bcl-2 is a main couple of apoptosis regulator.Bcl-2 locates at chromesome 18q21,containing 3 exons and 2 introns,which code protein with 239 amino acid residues.There are two types of Bcl2 protein,Bcl2 α and Bcl2 β with 26 kD and 21 kD relative molecular quantity respectively.There is a hxdrophobic sequence at carboxyterminal containing 19 amino acid residues,which is the main structure character of Bcl2 protein.Via its hydrophobic carboxyterminal,Bcl2 can insert in lipoidal double layers of membrane.Bcl-2 locates widely in many organelles membrane such as mitochondria,nucleus and endoplasmic reticulum.Bcl2 sites in mitochondria predominantly,especially in mitochondrial permeability transition pore (PTP),which block apoptosis by inhibiting the activation of PTP.
Bax locates in chromesome 19,coding 21 kD protein.Bax protein formed with 192 amino acid residues,21% of which is homogenous with Bcl2.Bax can activate PTP,which in turn facilitate the release of apoptogenic proteins,such as cytochromesC,apoptosis inducing factor and capase,then initiate apoptotic cataract.In physiological conditions,Bax is present predominantly in the cytoplasm.Apoptotic stimuli result in the over expression and reformation of Bax,and which insert in mitochondria membrane leading to alteration of PTP and apoptosis.
Bax/Bcl2 ratio determines cells fate.As an antiapoptotic proteins,Bcl2 inhibit the activation of PTP via form homodimers or heterodimers with Bax,which in turn block the induction of apoptosis by maintaining the location of Bax in cell or block Baxinduced permeability transition.Enhanced Bax/Bcl2 ratio caused by Bax over expression,lead cell to be susceptible and apt to apoptosis,or else,cell survive.
There are many apoptosis regulating factors,such as Bax,Bcl2,Fas/Fasl system on normal pregnant placenta.The expression of Bcl2 is very popular on placenta especially on trophoblast cells;furthermore,the expression level on syncytiotrophoblast is higher than on cytiotrophoblast.This process persists,however,it decreases gradually along with the pregnancy.Syncytiotrophoblast have no ability to proliferate.The increase of Bcl2 expression level can be involved in its survival.
Wang,et al.[3]discovered that expression level of P53 and Bax in cytotrophoblast,syncytiotrophoblast,decidual cell and mediate cell in ICP group was higher than that in the control,but Bcl2 expression level in these cells of ICP group was lower than control.The apoptosis rate increased significantly on overall placental cells.It was shown in this study that Bax expression level of ICP group was significantly higher than the control(P<00005).Bcl2 mainly expresses in cytoplasm of syncytiotrophoblast.Its expression level of ICP group is lower than that of control group significantly.Enhancement of Bax:Bcl-2 can lead to cell apoptosis.There is no Bcl-2 but Bax expression on mediate cell and vessel endothelial cell,which point out these cells are apt to apoptosis.These results were consistent with others findings.
Relationship between Perinatalfetal Hypoxia and Placental Apoptosis of ICP
LI,et al.[4]found that apoptosis level in placenta tissue of ICP was much higher than that in normal placenta.A relevant between perinatal mortality rate and placental apoptosis was thought to exist in ICP.However,they failed to find the change of apoptosis regulate gene.Wang,et al.[3]studied apoptosis of all kinds of placental cell,they found that the number of cells apoptosis increase on placenta of ICP.In that,a closely relationship existed between placental apoptosis and function reduce.
Syncytiotrophoblast play a key role on placental function,the number of apoptosis cells increase in ICP placenta,which may affect the placenta function.For example,syncytiotrophoblast can synthesize and secret estrogen,which improve bloodflow of uteroplacenta by diverse mechanism.When placental cell apoptosis index increases,its hormone synthesis ability reduces,which in turn affects the bloodflow of placental indirectly.Although,to some degree,apoptosis index increased might contribute to nutrition transport via placenta between mother and fetus.When the damage of apoptosisinduced synthesis ability decreases beyond the compensation,placental dysfunction will occur,and the fetus will be in danger.
Increased level of Bax expression is one of the early events of cell apoptosis.It was shown in this study,Bax expression level in syncytiotrophoblast of ICP group is significantly higher than that in the control,which indicated that the risk of placenta apoptosis in ICP group was higher than control.Perinatal hypoxia rate of ICP was higher than that of the control significantly.An increased tendency of perinatal hypoxia rate has been shown by further studies,along with Bax expression level increasing.It showed that Bax overexpression was closely related to perinatal hypoxia.An increased apoptosis in placenta caused by Bax over expression results in placental dysfunction,which in turn induce perinatal hypoxia.
Our results indicated that,there was relevance between perinatal hypoxia and Bax/Bcl-2 ratio on syncytiotrophoblast.The higher the ratio is,the more serious of perinatal hypoxia is.Thus,we inferred that a rise of the ratio could result in syncytiotrophoblast apoptosis,which is one of pathology bases of placental function withdrawal of ICP.Further studies will need to determine the reasons and mechanisms of the expression level change in the regulating proteins of placental apoptosis.
REFERENCES
1. Smith SC,baker PN,Sdymords EM.Placental apoptosis in normal human pregnancy.AM J Gynecol,1997,57-65.
2. Pastorino JG,Tafani M,Rothman RJ,Marcinkeviciute A,Hoek JB,Farber JL,Marcineviciute A.Functional consequences of the sustained or transient activation by Bax of the mitochondrial permeability transition pore.J boil Chem,1999,274(44):31734-31739.
3. WA Dongmei,ZHU Qiying,DING Li,et al.Relationship between P53,Bax and Bcl-2 expression and cell apoptosis in intraheptic cholestasis of pregnant.Chin J of Obste and Gynecol,2003,38(1):5-7.
4. LI Hongxia,LENG Jing,YAO Yuyu,et al.Relationship of placental apoptosis and intrahepatic cholestasis of pregnant.Acta Uiversitasis Medicinalis NanJing,2001,(4):312-313.
(Editor Jaque)(ZHAO Yajuan1,YUE Yafei2)
1 Department of Obstetrics and Gynecology,The First Hospital of Yunyang Medical College,Shiyan,Hubei Province 442000,China
2 Department of Obstetrics and Gynecology,The First Hospital of Xian Jiaotong University,Xian,Shaanxi Province 710061,China
Tel:+86-719-6994131
[Abstract] Objective To investigate the probable mechanism of perinatal hypoxia by detecting gene Bax and Bcl2 expression level on placenta. Methods The placental samples were obtained from 30 cases of ICP and 27 cases of normal pregnant women.Immunohistochemistry method was used to detect Bax and Bcl2 expression level on placenta. Results (1)Perinatal hypoxia rate of ICP group was higher than the control,significantly(χ2=1145,P<0005).(2)Bax protein level in syncytiotrophoblast of ICP group was higher than the control (u=3559，P<00005)，and Bcl2 level of ICP was lower than control (u=3461,P<00005),both significantly.(3)While the Bax protein level increase,the perinatal fetus faced high danger of hypoxia(χ2=102239,P<001). Conclusions Bax overexpression on syncytiotyophoblast may be the mechanism of perinatal hypoxia of ICP.
[Key words] perinatal hypoxia;placental apoptosis
INTRODUCTION
The main injury of intrahepatic cholestasis of pregnancy(ICP)is that increase the incidence of fetal distress or asphyxia of newborn,as well as perinatal mortality.However,the exact mechanism is still unknown.Apoptotic regulative proteiBax and Bcl2 present on placenta,and of these two protein determines the placental cells fate.An increase in a ratio of Bax to Bcl2 can alter apoptosis index and placental function.The aim of this study is to explore the mechanism of perinatal hypoxia of ICP by detecting the expression level of Bax and Bcl2 on placenta.
MATERIAL AND METHODS
Patients
Thirty cases of ICP patients and 27 normal pregnancies in our hospital since 2001/12 to 2002/12 were enrolled.The criteria for ICP was according to Chinese Obstetrics and Gynecology compiled by Cao Zeyi.The mean age (292±463 vs.274±27)and gestational peroid (379±165 vs.384±142) of the two group have no significant difference.
Samples
Within 5 min after delivery,1 cm×1 cm×1 cm placental tissues of central part were taken,avoid the infarct area,fixed,embedded and chop into 4 μm slices and reserve for determination.
SP Immunohistochemistry
Rat antihuman Bax monoantibody was bought from Fuzhou Maixin Company;Rabbit antihuman Bcl2 monoantibody bought from Zhongshan Company.The test followed by illustration of the box,critically.Results judged:the cell show brown grain is positive,or else is negative.According to the brown grains expression level in syncytotrophoblast cells,the positive group was divided into three levels,less than 30% and more than 60%,was regarded as (+)and (+++),respectively,between these two level is (++).
Statistics Analysis
Rank sum test and chisquare test were used toanalyse,P<005 was considered statistically significant.
RESULTS
Perinatal Hypoxia Rate of ICP Group and the Control In ICP group,there were 2 asphyxia of newborn and 13 fetal distress,2 of which were rejected for cord entanglement comparing perinatal hypoxia rate (including fetal distress and asphyxia of newborn)of the two groups showed that perinatal hypoxia rate of ICP group was higher than the control significantly (Table 1)(χ2=1145，P<0005). Table 1 Comparison of Perinatal Hypoxia Rate of Two Groups
Expression Level of Bax and Bcl2 in Two Groups
Bax and Bcl2 were showed in both groups.Bcl2 mainly expressed on plasma of syncytiotrophoblast.Bax express in all kinds of placental cells widely,and it predominantly expressed in syncytiotrophoblast.Table 2 shows that Bax expression level in ICP placenta is higher than that in the control (u=3559,P<00005),while Bcl2 expression level is lower than control (u=3641,P<00005),both significantly.
Table 2 Expression Level between Bax and Bcl2 in Two Groups
Relationship between Bax Expression Level and Perinatal Hypoxia Rate
All of the subjects were divided into three groups according to Bax expression level.Group 1:(-)and (+)(n=33),Group 2:(++)(n=15),Group 3:(+++)(n=9).A significant difference of perinatal hypoxia rate was found among these groups (χ2=102239,0005
Table 3 Comparison of Perinatal Hypoxia Rate in Different Bax Level
DISCUSSION
Placental Apoptosis and Its Gene RegulationApoptosis is a common physiological phenomenon.Low level of apoptosis on placenta was found at any period of normal pregnancy.Apoptosis play a critical role in placenta growth,development and maintaining normal pregnancy.(1)Apoptosis play a role in placental remodeling.Apoptosis in part of syncytium is contributed to maintain the normal ratio of syncytiotrophoblast and cytiotrophoblast.(2)Apoptosis contributes to placenta abruption at delivery.(3)Apoptosis is the main mechanism of maternalfetal immunotolerance.
Bax and Bcl-2 is a main couple of apoptosis regulator.Bcl-2 locates at chromesome 18q21,containing 3 exons and 2 introns,which code protein with 239 amino acid residues.There are two types of Bcl2 protein,Bcl2 α and Bcl2 β with 26 kD and 21 kD relative molecular quantity respectively.There is a hxdrophobic sequence at carboxyterminal containing 19 amino acid residues,which is the main structure character of Bcl2 protein.Via its hydrophobic carboxyterminal,Bcl2 can insert in lipoidal double layers of membrane.Bcl-2 locates widely in many organelles membrane such as mitochondria,nucleus and endoplasmic reticulum.Bcl2 sites in mitochondria predominantly,especially in mitochondrial permeability transition pore (PTP),which block apoptosis by inhibiting the activation of PTP.
Bax locates in chromesome 19,coding 21 kD protein.Bax protein formed with 192 amino acid residues,21% of which is homogenous with Bcl2.Bax can activate PTP,which in turn facilitate the release of apoptogenic proteins,such as cytochromesC,apoptosis inducing factor and capase,then initiate apoptotic cataract.In physiological conditions,Bax is present predominantly in the cytoplasm.Apoptotic stimuli result in the over expression and reformation of Bax,and which insert in mitochondria membrane leading to alteration of PTP and apoptosis.
Bax/Bcl2 ratio determines cells fate.As an antiapoptotic proteins,Bcl2 inhibit the activation of PTP via form homodimers or heterodimers with Bax,which in turn block the induction of apoptosis by maintaining the location of Bax in cell or block Baxinduced permeability transition.Enhanced Bax/Bcl2 ratio caused by Bax over expression,lead cell to be susceptible and apt to apoptosis,or else,cell survive.
There are many apoptosis regulating factors,such as Bax,Bcl2,Fas/Fasl system on normal pregnant placenta.The expression of Bcl2 is very popular on placenta especially on trophoblast cells;furthermore,the expression level on syncytiotrophoblast is higher than on cytiotrophoblast.This process persists,however,it decreases gradually along with the pregnancy.Syncytiotrophoblast have no ability to proliferate.The increase of Bcl2 expression level can be involved in its survival.
Wang,et al.[3]discovered that expression level of P53 and Bax in cytotrophoblast,syncytiotrophoblast,decidual cell and mediate cell in ICP group was higher than that in the control,but Bcl2 expression level in these cells of ICP group was lower than control.The apoptosis rate increased significantly on overall placental cells.It was shown in this study that Bax expression level of ICP group was significantly higher than the control(P<00005).Bcl2 mainly expresses in cytoplasm of syncytiotrophoblast.Its expression level of ICP group is lower than that of control group significantly.Enhancement of Bax:Bcl-2 can lead to cell apoptosis.There is no Bcl-2 but Bax expression on mediate cell and vessel endothelial cell,which point out these cells are apt to apoptosis.These results were consistent with others findings.
Relationship between Perinatalfetal Hypoxia and Placental Apoptosis of ICP
LI,et al.[4]found that apoptosis level in placenta tissue of ICP was much higher than that in normal placenta.A relevant between perinatal mortality rate and placental apoptosis was thought to exist in ICP.However,they failed to find the change of apoptosis regulate gene.Wang,et al.[3]studied apoptosis of all kinds of placental cell,they found that the number of cells apoptosis increase on placenta of ICP.In that,a closely relationship existed between placental apoptosis and function reduce.
Syncytiotrophoblast play a key role on placental function,the number of apoptosis cells increase in ICP placenta,which may affect the placenta function.For example,syncytiotrophoblast can synthesize and secret estrogen,which improve bloodflow of uteroplacenta by diverse mechanism.When placental cell apoptosis index increases,its hormone synthesis ability reduces,which in turn affects the bloodflow of placental indirectly.Although,to some degree,apoptosis index increased might contribute to nutrition transport via placenta between mother and fetus.When the damage of apoptosisinduced synthesis ability decreases beyond the compensation,placental dysfunction will occur,and the fetus will be in danger.
Increased level of Bax expression is one of the early events of cell apoptosis.It was shown in this study,Bax expression level in syncytiotrophoblast of ICP group is significantly higher than that in the control,which indicated that the risk of placenta apoptosis in ICP group was higher than control.Perinatal hypoxia rate of ICP was higher than that of the control significantly.An increased tendency of perinatal hypoxia rate has been shown by further studies,along with Bax expression level increasing.It showed that Bax overexpression was closely related to perinatal hypoxia.An increased apoptosis in placenta caused by Bax over expression results in placental dysfunction,which in turn induce perinatal hypoxia.
Our results indicated that,there was relevance between perinatal hypoxia and Bax/Bcl-2 ratio on syncytiotrophoblast.The higher the ratio is,the more serious of perinatal hypoxia is.Thus,we inferred that a rise of the ratio could result in syncytiotrophoblast apoptosis,which is one of pathology bases of placental function withdrawal of ICP.Further studies will need to determine the reasons and mechanisms of the expression level change in the regulating proteins of placental apoptosis.
REFERENCES
1. Smith SC,baker PN,Sdymords EM.Placental apoptosis in normal human pregnancy.AM J Gynecol,1997,57-65.
2. Pastorino JG,Tafani M,Rothman RJ,Marcinkeviciute A,Hoek JB,Farber JL,Marcineviciute A.Functional consequences of the sustained or transient activation by Bax of the mitochondrial permeability transition pore.J boil Chem,1999,274(44):31734-31739.
3. WA Dongmei,ZHU Qiying,DING Li,et al.Relationship between P53,Bax and Bcl-2 expression and cell apoptosis in intraheptic cholestasis of pregnant.Chin J of Obste and Gynecol,2003,38(1):5-7.
4. LI Hongxia,LENG Jing,YAO Yuyu,et al.Relationship of placental apoptosis and intrahepatic cholestasis of pregnant.Acta Uiversitasis Medicinalis NanJing,2001,(4):312-313.
(Editor Jaque)(ZHAO Yajuan1,YUE Yafei2)