Study Of The Protect Mechanism Of Fu Guan Injection Ⅰ In Rat's Skeletal Muscle At The Early Stage Of Limb Ischemic Reperfusion Injury

Part 1 The protect mechanism of Fu Guan Injectionâ… in rat's skeletal muscle suffered limb ischemic reperfusion injuryChapter 1 The protect mechanism of Fu Guan Injectionâ… in rat's skeletal muscle oxidative damage in limb ischemic reperfusion injuryObjective: Study the protect mechanism of Fu Guan Injectionâ… in rat's skeletal muscle oxidative damage in limb ischemic reperfusion injury.Method: Dividing 56 SD male rats into 3 groups in random: control group, ischemic reperfusion group(IR group) and Fu Guan injectionâ… intervention group(â… -IR group) There are 8 rats in the control group; 24 rats in the IR group, 24 rats in theâ… -IR group. Dividing the IR group into 3 sub-groups according to the time after reperfusion: IR-2 group means the rats in this group are at the time 2 hours after reperfusion, IR-4 group means the rats in this group are at the time 4 hours after reperfusion, IR-8 group means the rats in this group are at the time 8 hours after reperfusion.â… -IR group was also divided into 3 sub-groups according to the time after reperfusion:â… -IR-2 group means the rats in this group are at the time 2 hours after reperfusion,â… -IR-4 group means the rats in this group are at the time 4 hours after reperfusion,â… -IR-8 group means the rats in this group are at the time 8 hours after reperfusion. The rats in control group were not blocked blood flow in posterior limbs using untied rubber band. The rats in IR group andâ… -IR group were duplicated rats' posterior limbs ischemic reperfusion models by the method suggested by Zhang Lianyuan. No drug was given before sacrifice of rats in control group. 1 ml normal sodium was intraperitoneal injected at the time 12 hours before ischemic, ischemic time and reperfusion time in the IR group. 1 ml Fu Guan Injection I was intraperitoneal injected at the time 12 hours before ischemic, ischemic time and reperfusion time in the IR group. Each group, 1000mg fight gastrocnemius muscle was obtained to tissue homogenate for skeletal muscle's MDA,SOD,MPO test. Some gastrocnemius muscle was obtained to histological section test. Some posterior left gastrocnemius muscle was obtained to dry-humid weight ratio test. Then some blood was obtained from inferior caval vein to blood serum MDA,SOD,MPO,NO test.Results:(1)Results of MDA test: The content of MDA in IR andâ… -IR groups was higher than control group, there were significant difference (P＜0.01). Moreover, the content of MDA was gradual higher with time passed within 8 hours after reperfusion. The content inâ… -IR-2,â… -IR-4,â… -IR-8 was lower than corresponding groups of IR group, there were significant difference between IR group andâ… -IR group (P＜0.01) excluding skeletal muscleâ… -IR-4 group comparing to IR-4 group (P＜0.05).(2) Results of SOD test: The content of SOD in IR andâ… -IR groups was lower than control group, there were significant difference (P＜0.01). Moreover, the content of SOD was gradual lower with time passed within 8 hours after reperfusion. There were significant difference between IR group andâ… -IR group (P＜0.01) excluding serumâ… -IR-2 group comparing to IR-2 group (P＜0.05).(3) Results of PMO test: The content of MPO in IR andâ… -IR groups was higher than control group, there were significant difference(P＜0.01). Moreover, the content of PMO was gradual higher with time passed within 8 hours after reperfusion. The content inâ… -IR-2,â… -IR-4,â… -IR-8 was lower than corresponding groups of IR group, there were significant difference between IR group andâ… -IR group (P＜0.01).(4) Results of skeletal dry-humid weigh ratio test: The skeletal muscle was obviously edema in IR andâ… -IR groups than control group, there were significant difference (P＜0.01). Moreover, the degree of edema was gradual higher with time passed within 8 hours after reperfusion. The degree of edema inâ… -IR-2,â… -IR-4,â… -IR-8 was lower than corresponding groups of IR group, There were significant difference between IR group andâ… -IR group (P＜0.01) excludingâ… -IR-2 group comparing to IR-2 group (P＜0.05).(5) Results of serum NO test: The content was highest at the time of 2 hours after reperfusion. Moreover, the content was gradual lower with time passed within 8 hours after reperfusion. The content of NO in IR andâ… -IR groups was higher than control group and there were significant difference (P＜0.01). The content inâ… -IR-2,â… -IR-4,â… -IR-8 was lower than corresponding groups of IR group and there were significant difference between IR group andâ… -IR group (P＜0.01).(6) Results of histological section test: Muscle and around tissue edema, muscle fiber fracture and lots of inflammatory cell infiltration at each time in IR andâ… -IR groups. While Fu Guan injectionâ… can decrease inflammatory cell infiltration, muscle or around tissue edema and muscle fiber injury.Conclusion:1. Fu Guan injectionâ… can decrease the production of oxygen free radical;2. Fu Guan injectionâ… can decrease the reducing degree of SOD which can help to get rid of oxygen free radical;3. Fu Guan injectionâ… can decrease PMN emigration amount and aggregation in tissue at he same time, which decrease the degree of inflammation.4. Fu Guan injectionâ… can decrease the content of NO in serum;5. Fu Guan injectionâ… can decrease the tissue edema.Chapter 2 The influence of Fu Guan Injectionâ… in rat's skeletal muscle cell's apoptosis suffered limb ischemic reperfusion injuryObjective: Study the influence of Fu Guan Injectionâ… in rat's skeletal muscle cell's apoptosis suffered limb ischemic reperfusion injury.Method: Making some skeletal muscle sample of part 1 formalin fixation, dehydration, paraffin imbedding for histological section and then adopting the method of SABC for immunohistochemistry staining. Measuring protein grey scale of Bax, Bcl-2 using Motic Image advanced 3.1 image analysis system and then quantitative analyzing immunohistochemistry positive reactive product of Bax, Bcl-2.Result: No obvious positive reactive product of Bax, Bcl-2 was seen in the skeletal muscle of control group while positive reactive product of Bax, Bcl-2 were seen in IR group. During the 8 hours after reperfusion, Bcl-2 in IR group expressed gradual lower with time passed while Bax in IR group expressed gradual higher with time passed. The ratio of Bcl-2 and Bax positive value at the same time was gradual lower. (1) For the grey scale of Bcl-2, there were significant difference between IR group and control group (P＜0.01). (2) For the grey scale of Bax, there were no significant difference between IR group and control group (P＜0.01) except IR-2 group compared to its corresponding control group (P>0.05). (3) The ratio of Bcl-2 and Bax positive value became higher 2 hours after reperfusion and became lower 8 hours after reperfusion, there were significant difference between IR group and control group(P＜0.05). Compared the grey scale of the corresponding time, there were significant difference between IR group andâ… -IR group (P＜0.05) except IR-2 group compared toâ… -IR-2 group.Conclusion: Fu Guan injectionâ… can decrease the expression of Bax to decrease skeletal cell apoptosis, which is help to protect skeletal muscle suffered ischemic reperfusion injury.Part 2: The changes of expression of skeletal muscle's calmodulin in rat's limb ischemic reperfusion injury and Fu Guan Injectionâ… 's contribution to these changesObjective: Study the changes of expression of skeletal muscle's calmodulin in rat's limb ischemic reperfusion injury and Fu Guan Injectionâ… 's contribution to these changes.Method: Dividing 54 rats into 3 groups in random: control group, ischemic reperfusion group(IR group) and Fu Guan injectionâ… intervention group(â… -IR group). There are 6 rats in the control group, 24 rats in the IR group, 24 rats in theâ… -IR group. Dividing the IR group into 4 sub-groups according to the time after reperfusion: IR-0 group means the rats in this group were at the time 0 hour after reperfusion, IR-2 group means the rats in this group are at the time 2 hours after reperfusion, IR-4 group means the rats in this group are at the time 4 hours after reperfusion, IR-8 group means the rats in this group are at the time 8 hours after reperfusion.â… -IR group was also divided into 4 sub-groups according to the time after reperfusion:â… -IR-0 group means the rats in this group were at the time 0 hour after reperfusion,â… -IR-2 group means the rats in this group are at the time 2 hours after reperfusion,â… -IR-4 group means the rats in this group are at the time 4 hours after reperfusion,â… -IR-8 group means the rats in this group are at the time 8 hours after reperfusion. The rats in control group were not blocked blood flow in posterior limbs using untied rubber band. The rats in IR group andâ… -IR group were duplicated rats' posterior limbs ischemic reperfusion models by the method suggested by Zhang Lianyuan. No drug was given before sacrifice of rats in control group. 1 ml normal sodium was intraperitoneal injected at the time 12 hours before ischemic, ischemic time and reperfusion time in the IR group. 1 ml Fu Guan Injectionâ… was intraperitoneal injected at the time 12 hours before ischemic, ischemic time and reperfusion time in the IR group. Depilating at he same area of right posterior limb at the same time in each group, iodine sterilizing and then discission skin, subcutaneous tissue and deep fascia along the major axis of limb. 1000rag gastrocnemius muscle was obtained to tissue homogenate for RT-PCR test of RyR₁ and SERCA₁Result:(1) RT-PCR results of skeletal muscle type RyR₁mRNA: during the 8 hours before injure caused by ischemic reperfusion, the skeletal muscle type RyR₁mRNA in IR group expressed up-regulation at the time of reperfusion and 2 hours after reperfusion. There were significant difference between IR group and control group (P＜0.01). It recoveried a little at 4 hours after reperfusion. However, the skeletal muscle type RyR₁mRNA in IR group expressed down-regulation at the time 8 hours after reperfusion. There were significant difference between IR group and control group (P＜0.01). Skeletal muscle type RyR₁mRNA expressed up-regulation a little (smaller than IR group ) inâ… -IR group at the time of intervention, there were neither significant difference betweenâ… -IR group and IR group nor significant difference betweenâ… -IR group and control group (P＞0.05); skeletal muscle type RyR₁mRNA expressed up-regulation highest (smaller than IR group ) inâ… -IR group at the time 2 hours after ischemic, there were significant difference betweenâ… -IR group and IR group (P＜0.01); there were no significant difference betweenâ… -IR group and control group (P＞0.05) at the time 4 hours after ischemic; Skeletal muscle type RyR₁mRNA expressed down-regulation (smaller than IR group ) inâ… -IR group at the time 8 hours after ischmic, there were significant difference betweenâ… -IR group and IR group (P＜0.05)(2) RT-PCR results of SERCA₁mRNA: Within 8 hours after reperfusion, SERCA₁mRNA expressed gradual down-regulation with time passed in IR group, there were significant difference between IR group and control group (P＜0.01 ); inâ… -IR group, SERCA₁mRNA also expressed gradual down-regulation (smaller than IR group ) with time passed, there were significant difference betweenâ… -IR group and IR group at the time 0 hour (P＜0.05), 4 hours (P＜0.01) and 8 hours (P＜0.01) after reperfusion.Conclusion:1. Within 8 hours after limb ischemic reperfusion, skeletal muscle type RyR₁mRNA expressed up-regulation then down-regulation, SERCA₁mRNA expressed gradual down-regulation with time passed, this may be The main reason of limb ischemic reperfusion injury.2. Fu Guan injectionâ… can protect skeletal muscle during ischemic reperfusion injury. The reasons for this may be these: (1) two-way regulate expression of RyR₁mRNA; (2) enhance expression of SERCA₁ to decrease calcium overload in kytoplam for protecting cell organ.